INDUSTRIAL MICROBIOLOGY

60.451
LAB MANUAL
2003

Lab manual available as a pdf file on website.

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TABLE OF CONTENTS
Title Page
Schedule 3
General Instructions 4
Lab Standard Operations Procedure (SOP) 9
WHMIS 12
EXPERIMENTS
Lab 1:Wine or Beer Making
a) Beer 14
b) Wine 23
Lab 2: Microbial Industrial Fermentation: Citric Acid Production by 29
Aspergillus niger
Part I: Experiment design
Part II: Media preparation and inoculation
Part III: Sample collection and biomass determination.
Part IV: Citric Acid determination
Lab 3: Industrial microbe strain improvement: Enhanced antibiotic 40
production
Part I: Isolation of a “new” antibiotic producing strain from the environment
Part II: Mutagenesis of Streptomyces strains
Part III: Selection of improved strain
APPENDIX
Hydrometer 46
Beer bottle capper operation 46
Hemocytometer (spore concentration determination) 47
Pipetman operation 48
Spectronic 20D operation 50
Floor model centrifuge operation 51
Sample Lab Exam 53
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INDUSTRIAL MICROBIOLOGY LAB SCHEDULE 2003

Date Week # Description

Jan 17 1 Lab 1: (a) Beer Making OR (b)Wine Making (each group does only one project)
Jan 24 2 Lab 2: Microbial industrial fermentation: Citric Acid Production by Aspergillus
niger
Part I: Experiment design
Jan 31 3 Lab 2: Microbial industrial fermentation: Citric Acid Production by Aspergillus
niger
Part II: Media preparation and inoculation
Feb 7 4 Lab 2: Microbial industrial fermentation: Citric Acid Production by Aspergillus
niger
Part III: Sample collection and biomass determination.
Part IV: Citric Acid determination
Feb 14 5 Lab 3: Industrial microbe strain improvement: Enhanced antibiotic production
Part I: Isolation of a “new” antibiotic producing strain from the environment
Part II: Mutagenesis of Streptomyces strains
Feb 21 6 Mid-Term Break
Feb 28 7 Lab 3: Industrial microbe strain improvement: Enhanced antibiotic production
Part III: Selection of improved strain
Mar 7 8 Industrial Microbiology: Data analysis Problem Lab
Mar 14 9 Tour - to be announced
Mar 21 10 Tour - to be announced
Mar 28 11 Question/Answer Lab
April 4 12 Lab Exam

Due dates
Due Date Week # Report or Data
Jan 27 2 need to email Lab 2 materials and methods - edit
Feb 28 7 Lab 2 REPORT: Microbial industrial fermentation: Citric Acid Production by
Aspergillus niger
Mar 4 8 Lab 3 DATA: Industrial microbe strain improvement: Enhanced antibiotic
production
Mar 7a 8 PROBLEM ASSIGNMENT: Industrial Microbiology: Data analysis Problem Lab
Mar 14 9 Lab 3 REPORT: Industrial microbe strain improvement: Enhanced antibiotic
production
Mar 21 10 Lab 1 REPORTb: (a) Beer Making OR (b)Wine Making
a
problem lab due date is tentative...may be due in lab with no extra time given OR an additional week given
b
questions for the beer making or wine making report are identical, ie., you need to answer
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questions that pertain to both beer an wine making

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GENERAL INSTRUCTIONS

Lab instructor: Dr. L. Cameron Office: 414B

Demonstrators: Patrick Chong Lab: 413
Reuben Saba Lab: 417
Lab room: 201 (204) Buller Bldg. All students come to room 201for the first lab.

WEBSITE: www.umanitoba.ca/faculties/science/microbiology/staff/cameron/
OR via University of Manitoba Microbiology Homepage:
https://www.umanitoba.ca/faculties/science/microbiology/course_notes.html
Information available at the website: changes/corrections, additional information, data, marks

REGULATIONS

1. Lab attendance is compulsory.

2. Students must wear a lab coat. There is no smoking, drinking, or eating in the lab.
3. When recommended in the lab protocol, disposable gloves and shield glasses should be
worn. The disposable gloves are provided in the lab.
4. Students are required to supply their own marker for tubes.

EVALUATION
General
1. Lab Mark (Total = 20%):
Lab exam: 13%
Industrial Microbiology data analysis problem lab: 2%
Citric acid lab report: 2%
may want to give marks to initial materials and methods
Project (either beer or wine) report: 2%
Peer evaluation: If not handed in, marks deducted from total lab mark If
peer evaluation is below 60%, marks will be deducted accordingly.
Antibiotic production: 1%
2. Students must pass the lab to complete the course (50%).
3. The lab exam date is included in your lab manual schedule.
Place: lab
Time: 2:30 - 4:00 p.m.
4. Lab reports are to be handed in as stated in schedule by 4:30 pm of that day. Hand in lab
reports through slotted drawer in room 414 ONLY. Instructor and demonstrators do not
accept lab reports. If handing in lab late, 1 mark will be subtracted for each class day late.
Marked lab reports will be returned to students the next week. A late report will not be
accepted after that report has been returned in class.
5. Lab report marks are final unless an obvious error in addition of marks has been made.
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However, if a student feels they have a legitimate complaint, please direct attention to the
lab instructor.
6. Approximately two weeks prior to the lab exam, a brief outline of lab exam format and
information content will be available on the website.

7. You must notify the lab instructor no later than two school days after the missed lab exam
of your intent to write a deferred lab exam. The deferred lab exam must be rescheduled
before the end of this term’s classes. Failure to comply will result in a zero on your lab
exam.
8. Plagiarism (copying another student’s lab report (present or previous year) or
copying published literature without citing is a violation of University regulations.
Refer to the STUDENT DISCIPLINE BY-LAW in your student handbook (rule book)
for action taken for plagiarism.

LAB REPORT PRESENTATION INSTRUCTIONS

1. Include a title page. Include a table of contents. Lab reports MUST BE TYPED. Data
sheets acceptable hand written. Number each page.

2. Lab reports may be done as an individual effort or a group effort by the two students that
carried out the experiment. One report or more reports may be handed in per group. The
decision on the number of reports per group is totally dependent on members of the group.
This decision may be changed any time during the term. Therefore for each lab report the
group has the option to hand in one or two reports exclusive of what has been done before
or after that particular report. Indicate on the cover page of the report if the report is a
group report or an individual report. If handing in an individual report also include lab
partner’s name. Do not put a student’s name on the report who has not assisted with the
report. Marks only given to students whose names are listed on the report.

3. If your group’s data (or part of data) is ‘not workable’ borrow data from another group and
reference. The definition of ‘not workable’ is data that cannot be used for required
calculations. It does not necessarily mean the correct data as long as the calculations can
be performed and data presented as requested.

4. Always include one sample of each type of calculation used when analyzing data. When
presenting sample calculations, explain all volumes and dilutions involved in calculation.

5. Cite reference in text of lab report and record full reference at end of lab report. When
should you cite and reference. The following is a good definition of plagiarism that
explains when you should cite a reference. “The unacknowledged use of another
person’s work, in the form of original ideas, strategies, and research, as well as
another person’s writing, in the form of sentences, phases and innovative
 7

terminology.” (Spatt1, 1983, p.438) This is done by using bracketed reference number that
you used when listing references at end of lab report or by bracketing first authors name
and date. Quote text unless you paraphrase completely in your own words. But remember,
quotes should only be a small part of your work. If you are using the name year system,
list the references alphabetically. Some examples are as follows (McMillan2 1997):

Binder V. Hendriksen C, Kreiner S. 1985. Prognosis in Crohn’s disease - - based on

results from regional patient group from county of Copenhagen. Gut 26:146-50.
Danforth DN, editor. 1982. Obstetrics and gynecology. 4th ed. Philadelphia: Harper and
Row. 1316 p.
Petter JJ. 1965. The lemurs of Madagascar. In: DeVore I, editor. Primate behavior: field
studies of monkeys and apes. New York: Holt, Rinehart and Winston. p 2920319.
If available only on the web:
Kingsolver JC, Srygley RB. Experimental analyses of body size, flight and survival in
pierid butterflies. Evol. Ecol. Res. [serial online] 2000;2:593-612. Available from:
Colgate University online catalog. Accessed 2000 Oct 3.

6. Personal or Professional Electronic sources2:

Cite in-text by putting the following in parentheses, author’s last name or file name (if no
author’s name is available) and publication date or the date of access (if no publication date
is available).
At the end of report list
(i) author or organization
(ii) publication date or date last revised
(iii) title of Web site
(iv) URL site in angle brackets or self underlined as a default link
(v) the date accessed.

Cameron, L. 60.344 Microbial Physiology Lab Information

<http://www.umanitoba.ca/faculties/science/microbiology/staff/cameron/60_344.htm>.
Accessed 2002 April 12.

Table presentation
1. Table number and title (legend) presented above the table body.
2. Number tables using arabic numbers, even if only one table in a report.

1
Spatt, B. (1983). Writing from Sources. New York: St. Martin’s Press.
2
McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston:
Bedford Books: 1997. 197 p. and McMillan, V.E. 2001. Writing Papers in the Biological
Sciences. 3rd ed. Boston: Bedford Books. 123 p.
 8

3. Include enough information in title to completely describe table, eliminating the necessity
to search elsewhere in the lab report to understand information presented in table. Table
title starts with an incomplete sentence. Additional complete sentences may be included to
adequately describe the table (this also applies to figures).
4. If abbreviations are used in table, indicate what abbreviations mean as a footnote. Other
footnotes may be required to clarify material in the table.
5. Like information should be in columns making it easier to view the table.
6. Data in columns should be listed under the centre of each heading. Align decimal points
and dashes. If a number value is less than 1 always include zero before the decimal.
7. Column or Row headings should be complete and self explanatory. A heading is a separate
entity from the title. It cannot be assumed information given in the title is adequate for a
heading. The unit of measurement should only be included in the heading, not in column
data.
8. Group related column headings under larger headings.
9. If information is the same for each column or row do not include but treat as a footnote.
10. Make the table as concise as possible but include all necessary information. For example,
any constant experimental conditions that would change the data presented.
11. Tables should be properly set up with a straight edge. Horizontal lines must be included
but it not necessary to always include vertical lines.

Figure presentation (graphs, diagrams, photographs, films)

12. Figures are to be numbered separate from tables, using arabic numbers. Include figure
number even if only one figure.
13. Figure number and figure legend should be presented below the graph. The figure legend,
like the table, starts with an incomplete sentence describing the graph. For example, do not
repeat just the labels of the x- and y-axis but present in a descriptive manner. Additional
sentences should be included if additional information is required to completely describe
figure, for example, any constant experimental conditions that affect the data presented.
14. All diagrams, photographs, and films are figures and should be completely labelled.
15. For figures of graphs, there is one dependent variable plotted and one or more independent
variables plotted. The dependent variable is a function of the independent variable. It is
accepted practise to plot the independent variable on the x-axis and the dependent variable
on the y-axis. For example the measurement of absorbance (dependent) with increasing
concentration of protein (independent). The size of the graph should fit the plot(s). The
axis should not necessarily start at zero. Place graph completely within graph grid, this
includes axis labels and legend. The overall size of graph should not be too large but
should not be so small that information is obscured. The graph must be completely
labelled (always include units). Use different symbols for each plot (not different coloured
pens) on a graph. If more than one plot, explain symbols in legend or in a key included in
the body of the graph. Graph plots can be drawn in a number of ways (this depends on the
plot): (a) best fit straight line, (b) join each point with a straight line, and (c) use a flexible
curve ruler or french curve. Do not drawn a free hand line.

Note: When writing your lab reports you are frequently requested to present both a table and a
figure for a given set of data, similar to keeping a research journal. This is not the accepted
practice for papers published in journals or books. Usually either a table or a figure is presented
 9

for a given set of data and depending on nature of data, it may only be summarized in the text.
How do you make a choice of data presentation? The aim is to effectively and efficiently
demonstrate what you want to show, for example, correlations, comparisons, pattern, trends, etc
(McMillan 1997)

Reference
McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston: Bedford Books:
1997. 197 p.

References available in the reference binder on reserve in the Science and Technology
Library.
# References available in the reference binder (1 hour reserve in the Science and Technology Library)

Lab 2: Microbial industrial fermentation: Citric Acid Production by Aspergillus niger

1 Hilton, MD. 1999. Small-scale liquid fermentations. In: Demain, AL, Davies, JE., editors. Manual of
Industrial Microbiology and Biotechnology, 2nd edition. Washington: ASM Press. p 49-60.

2 Strobel, RJ., Sullivan, GR. 1999. Experimental design for improvement of fermentations. In: Demain,
AL, Davies, JE., editors. Manual of Industrial Microbiology and Biotechnology, 2nd edition. Washington:
ASM Press. p 80-93

3 Dahod, SK. 1999. Raw materials selection and medium development for industrial fermentation
processes. In: Demain, AL, Davies, JE., editors. Manual of Industrial Microbiology and Biotechnology,
2nd edition. Washington: ASM Press. p 213-220.

4 *Legisa, M., Gradisnik-Grapulin, M. 1995. Sudden substrate dilution induces a higher rate of citric acid
production by Aspergillus niger. Appl. Env. Micro. 61: 2732-2737.

5 *Ruijter, GJG., van de Vondervoort, PJI., Visser, J. 1999. Oxalic acid production by Aspergillus niger:
an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese.
Microbiology. 145: 2569-2576.

Lab 3: Industrial microbe strain improvement: Enhanced antibiotic production

6 Hunter-Cevera, JC, Belt, A. 1999. Isolation of cultures. In: Demain, AL, Davies, JE., editors. Manual of
Industrial Microbiology and Biotechnology, 2nd edition. Washington: ASM Press. p 3 - 13

7 Vinca, VA, Byng G. 1999. Strain improvement by nonrecombinant methods. In: Demain, AL, Davies,
JE., editors. Manual of Industrial Microbiology and Biotechnology, 2nd edition. Washington: ASM Press.
p 103-113.

8 Khetan, A, Hu, W-S. 1999. Metabolic Engineering of Antibiotic Biosynthetic Pathways. In: Demain,
AL, Davies, JE., editors. Manual of Industrial Microbiology and Biotechnology, 2nd edition. Washington:
ASM Press. p 717-739.

Lab 1: Beer and Wine

9 Hough, J.S. 1995. The Biotechnology of Malting and Brewing. Cambridge University Press. p 1-159.
 10

10 Remize, F. Roustan, JL, Sabbayrolles, JM, Barre, P., Dequin , S. 1999. Glycerol overproduction by
engineered Saccharomyces cerevisiae wine yeast strains leads to substantial changes in by-product
formation and to a stimulation of fermentation rate in stationary phase. Appl. Env. Micro. 65:143-149.
*Many scientific journal articles, including the two footnoted above, are available on the internet.
Search using Highwire Library of Sciences and Medicine Search and Browse Tools at
http://highwire.stanford.edu/ © 2001 - 2002 by the Board of Trustees of the Leland Stanford Junior
University. (accessed July 2/02).
 11

LAB STANDARD OPERATIONS PROCEDURE (SOP)

Bench area: Wash bench area before and after use with AIRx109.

Personal safety: You must wear a lab coat. Wear coat only in the lab, transport separately outside of
the lab (in a plastic bag). Wash hands with antibacterial soap before leaving the lab. No eating or
drinking in the lab. Use aseptic technique for transfer of bacteria. This is to protect yourself as much
as to ensure the purity of your culture. Protect hands with gloves and eyes with glasses when needed.
The gloves provided in the lab are to be disposed of after use.

Biohazards: Know biosafety risk groups. Handle all cultures as potential pathogens. Never mouth
pipette. Always use a pro-pipette. If you spill a culture, cover the spill with paper towels. Pour
AIRx109 over the towels to saturate. Gather up soaked towels and discard. Wipe area to dryness with
fresh paper towels. Wash hands with soap and water. Place cultures on discard trolley. All cultures
are autoclaved before disposing. Dispose of eppendorf tubesa in petri plate containers. Dispose of
pipetman tipsa in clear plastic lined basins along with glass or plastic Pasteur pipets, broken glassware,
glass slides, brittle plastic objects, metal objectsa (not needles or blades). Bacteria dilutions may to be
poured down the sink and the tubes rinsed before placing on the discard trolley. Rinse sink with lots
of water.
a
due to the multi-use nature of the teaching lab, all eppendorf tubes, pipetman tips, Pasteur pipets,
brittle plastic or metal objects will be treated the same as similar items contaminated with
microorganisms.
Wear gloves when handling level 2 microorganisms (Aspergillus niger is level 2). All cuts should be
covered with bandages (available in first aid in the lab). Be aware that whenever you flame your loop
aerosols are created.

Glassware (unbroken): Remove tape and pen markings (use alcohol) from glassware before placing
on discard trolley. Used glassware should be rinsed and placed on the discard trolley. Rinsed test
tubes should be placed in tray provided on the discard trolley. Used glass pipettes should be placed in
pipette holders.

Petri plate culture and non-sharps solid culture material disposal: use covered plastic containers
lined with clear plastic bags for contaminated petri dishes or any bacteria contaminated solid non-
sharps material (eppendorf tubes, API strips, antibiotic strips, microtitration plates, etc)

Hazardous material disposal: Examples: radioactive material, ethidium bromide, sovents, etc. The
lab demonstrator will instruct proper disposal methods for labs that contain hazardous materials.
These materials must be disposed of in appropriately labelled containers and disposed via the safety
office. Use fumehood when recommended. A MSDS binder available in lab gives information on all
hazardous materials used in the lab. Use extreme care with flammable solvents. Alcohol used to
flame spread rod should never be positioned within 40 cm of flame. Never put a very hot spread rod
into a beaker of alcohol. The alcohol may catch fire. Many of the immunochemicals are preserved in
0.1% Na azide...handle with gloved hands. Handle caustic (acids and bases) solutions with care. Never
discard an acid or base greater than one molar down the sink. Discard in labelled glass containers
provided. Use lots of water when discard caustic solutions (< 1M). These materials are disposed of
through the university safety office. Never pour solvents down the sink (eg. phenol, ether,
chloroform, etc). Discard in labelled containers provided.
 12

Sharps disposal: Dispose of all sharps (needles, syringes, razors, scalpel blades) in specified
container. Dispose of syringe with needle attached - do not take apart. Do not replace the needle cap
before disposing (high frequency of accidents occur when replacing cap). Sharp’s containers are
autoclaved before disposing. .

Broken glass disposal: Dispose of broken glass in labelled plastic containers lined with clear
plastic. Transferred to boxes before discarding.

Know location: Exits, fire extinguisher, eye wash, sink shower, and first aid kit. This information
is given in the first pre-lab.

Equipment operation: Know how to operate equipment before use. DO NOT use equipment
unless you know exactly how to operate the equipment. The demonstrator is always available to
assist. Please follow instructions in appendix for proper clean up of Spectronic 20D. Ensure the
spec tubes are thoroughly washed and rinsed with distilled water before replacing in rack upside
down as you (hopefully) found the tubes.

Leave your bench area clean All equipment and supplies should be returned to original location.
 13

LABORATORY BIOSAFETY GUIDE

Aspergillus niger is a level 2 microorganism. It is important that you follow standard operation
procedures, SOP (see above). When handling environmental samples you must assume they
contain level 2 microorganisms.

The University of Manitoba Biosafety Guide (Feb 2000) and Health Canada Laboratory Biosafety
Guidelines booklets are available in your lab. Biosafety information is also available at the Health
Canada websites:
Guidelines: http://www.hc-sc.gc.ca/hpb/lcdc/biosafty/docs/index.html
MSDS (infectious agents): http://www.hc-sc.gc.ca/hpb/lcdc/biosafty/msds/index.html
There is no listing of level 1 agents in the guidelines or MSDS pamphlets
Risk group 1 bacteria are low individual and community risk and are unlikely to cause disease in
healthy workers.
Risk group 2 bacteria are moderate individual risk and limited community risk. Bacteria in this
group can cause human or animal disease but are unlikely to infect healthy laboratory workers.
Effective treatment is available. Risk of spreading is limited.

CONTAINMENT LEVEL 1 (UM biosafety guide p. 11)

16. microbiology lab with washable walls, countertops and hand wash sink
17. established safe laboratory practices (hand washing and disinfection of countertops)
18. general WHMIS safety training
19. UM lab registration

CONTAINMENT LEVEL 2 (UM biosafety guide p.11)

20. all of level 1 specifications
21. biosafety permit
22. biological safety cabinet (not required)
23. biohazard signage
24. a written standard operations procedure
25. MSDS for the infectious agent
 14

WHMIS
The Workplace Hazardous Materials Information System (WHMIS) is a system for safe
management of hazardous materials. WHMIS is legislated by both the federal and provincial
governments.
Under WHMIS legislation, laboratories are considered to be a workplace, and students are
workers. By law, all workers must be familiar with the basic elements of the WHMIS system.

The WHMIS program includes:

1. Cautionary labels on containers of controlled products. Consumer products, explosives,
cosmetics, drugs and foods, radioactive materials, and pest control products are regulated
separately, under different legislation.
2. Provision of a Material Safety Data Sheet (MSDS) for each controlled product.
3. A worker education program

1. A. SUPPLIER LABELS
Controlled products must have a label of prescribed design which includes the following
information:
PRODUCT IDENTIFIER - trade name or chemical name
SUPPLIER IDENTIFIER - supplier's name and address
MSDS REFERENCE - usually, "See MSDS supplied"
HAZARD SYMBOL - (see illustration on next page)
RISK PHRASES - describes nature of hazards
PRECAUTIONARY MEASURES
FIRST AID MEASURES

B. WORKPLACE LABELS
All material dispensed in a workplace container must be labelled with the Product Name,
Precautionary Measures (simplified) and Reference to Availability of MSDS.

2. MSDS
Individual course MSDS are located in a binder in your lab (Room 201 binder located in 204).
The main MSDS binders are located in the Microbiology preparation room, 307/309 Buller.
MSDS are also available on the local area computer network (see your demonstrator, if necessary).
The MSDS will provide: relevant technical information on the substance, chemical hazard data,
control measures, accident prevention information, handling, storage and disposal procedures, and
emergency procedures to follow in the event of an accident.

3. SAFETY
The Laboratory Supervisor will provide information on the location and use of safety equipment,
and emergency procedures.
 15

WHMIS DIAGRAM
 16

LAB 1a WINE MAKING

(all groups are responsible for wine making for lab exam)

INTRODUCTION
In Manitoba there is only one commercial winery, D.D, Leobard Cellars, located in St.
Boniface. D.D. Leobard’s specialize in berry wines and coolers using many local berries such as
strawberries, blueberries, and saskatoons (testing stage).
In your lab each group will use a traditional grape wine kit to ensure reasonable quality
product. Wine quality is dependent on the grapes (type, age of vine, soil and weather) and yeast
strain. In addition, it is very important how the must (grape juice) is fermented. It is important to
follow instructions carefully. How fermentation occurs determines the alcohol content, sweetness,
acidity and presence of chemicals that add flavour and aroma. Grape juice contains 18-22% sugar,
much higher than other fruits and berries. Both glucose and fructose occur naturally in grapes.
Since yeast preferentially ferments glucose any sweetness attributed to the wine is most likely due
to the presence of fructose. Acidity of wine should be between pH 3.2 and 3.6 (ie., 0.5 to 0.8%
acid by volume) for good fermentation. If the pH is too low, fermentation will be slow. Grape
juice contains mostly tartaric acid. Malic acid may be present if wines are grown in cooler
climates. If malic acid is present a strain of yeast that ferments malic acid is used. Flavor and
aroma (bouquet for aged wines) are dependent on the variety of fruit which in turn give the name
to the wine. Some well known wine grapes are Cabernet Sauvignon (red), Pinot Noir (red),
Chardonnay (white), Riesling (white) and Sauvignon Blanc (white). It is often entertaining how
wines are described. For example, Chardonay “smoky, austere, steel-like. Bursts with intense
flavours capped with a long full finish.” Or Pinot Noir, “very fruity, yet spicy character velvety
flavour that is met with a fullness of aroma resembling black currants (actually n-octanol and 2-
methoxy-3-isobutylpyrazine). Aging uncovers further complexity.”(1) Grapes contain tannin
(phenolic compound) which give wine an astringent taste. In small quantities, tannins give wine
the full bodied taste.
How is the Vintners Reserve wine kit made? The grapes purchased each year from
vineyards must meet variety type standards to ensure the kit remains the same from year to year.
The grapes go to a winery where the grapes are crushed and sulfite is added. White grapes and red
grapes are processed differently. White grapes are pressed, enzymes are added to remove pectins,
and bentonite is added. After the mixture is stirred it is rapidly cooled to facilitate precipitation of
grape solids and prevent deterioration. The supernatant is passed through a filter and sulfite
concentration adjusted. The resulting grape juice (must) is ready to be concentrated if required or
packaged in the kit. Red grapes are processed slightly different to extract color, aroma and flavour
from the grape skin. The red grapes are crushed, sulfited, chilled and pectinoglycolytic enzymes
are added for 2 or 3 days to breakdown the cellulose membrane and extract tannins of the grape
skin. The must is pressed. The pressed skins are reprocessed with enzymes and pressed again to
extract components that are added to the juice. After pressing, the red grape juice is processed
similar to white grapes.
Brew King Vintners Reserve (1) is the wine kit supplied in lab. Each year the variety of
wine may vary, therefore instructions vary. If ingredients vary from procedure listed below,
follow the instructions given in the kit. However, the procedure presented below has been
changed to obtain a good finished product (2 extra syphoning steps) - these extra steps should be
included. The kit makes 23 liters of table wine. A filtering apparatus is used to give a good
finished product.
References
(1) Leener’s Brew Works http://www.leeners.com/vinreserve.html (accessed July 3/02)
 17

MATERIALS

Vintners Reserve - Wine Kit:

-grape concentrate, preserved with sulfur dioxide, may also contain di ammonium
phosphate, liquid invert and/or fructose (corn) syrup, citric acid, malic acid, tartaric acid.
-packet of yeast
-packet of bentonite, packet of potassium metabisulphite
-packet of potassium sorbate
-packet of gelatin or isoglass.
-may contain oak chips
-may include an F-pack (concentrated flavor added at start of conditioning step - gives
wine a full body)
23 liter primary fermenter pail (x1)
23 liter secondary glass fermenter carboy (fitted with air-lock) (x2)
20 to 30 - 750 or 1000 ml wine bottles (please locate yourself, some are available in the lab)
syphon
large plastic spoon
hydrometer (refer to appendix for instructions)
straight corks to fit wine bottles (30)
wine corker
filter apparatus (2 course filters)
long wire brush for cleaning equipment

PROCEDURE
STUDENTS WORK IN PAIRS
After today the remainder of the beer or wine making lab is to be completed on your own time.
The lab is open weekdays (7:30 am to 5:00 pm, if you want to come later please discuss ahead of
time with the instructor). Refer to lab room schedule posted on each lab door, to determine when
the lab is available. If assistance is required, obtain help from teaching assistant.

Primary Fermentation
Week 1

1. Clean primary fermenter (plastic pail) and spoon with a dilute solution of sodium bisulfite
(~5 ml/liter). Rinse thoroughly. Use only clean sanitized utensils throughout procedure.

2. Open the kit box lid and remove additive packets.

3. Add 2 liters of warm/hot tap water to the bucket. Let water run about three minutes before
 18

using. Using the long handled spoon vigorously stir while slowly sprinkling the contents
of bentonite packet over the surface. Continue to stir until the bentonite is dispersed (about
30-60 sec). Break up clumps with spoon.

4. Carefully remove cap of grape concentrate. It spills very easily. Pour contents into bucket.
Fill concentrate bag with warm water and pour into pail.

5. Optional depending on wine type: If a packet of berries or bark is included in your kit,
now is the time to add it into the bucket.

6. Top up bucket with distilled water* to just above the 23 liter mark (indented ring around
top of pale) and stir well (1 minute). It is very important that the grape concentrate is
completely dispersed.
*tap water is acceptable

7. Transfer ~100 ml to a graduated cylinder. Check and record the specific gravity. Record
expected range for your type of wine. The initial and final specific gravity varies
depending on the type of wine. Record room temperature. Record the temperature of the
diluted grape concentrate. The temperature should be between 18 - 24oC. If it is slightly
below 18oC, still add yeast. Record the pH of the diluted grape concentrate.

8. Sprinkle yeast on wine solution surface.

9. Cover pail with a plastic bag, tape to secure and place out of direct sunlight. Incubate at
room temperature (18 - 24 oC) for 7 - 10 days. The time varies depending on room
temperature. The fermentation should be complete - no vigorous bubbling or movement.
Record specific gravity, pH and temperature of wine.

Secondary Fermentation

10. Move primary fermentation to table top. Syphon the contents of your primary fermenter
into a clean glass 23 liter carboy. The syphon should be carefully placed in primary
fermenter with the hook or capped side down. The hook allows transfer of wine leaving
the sediment in the primary fermenter. The water aspirator can be used to start the siphon.
Do not use the vacuum line. Top up carboy with distilled water (optional), attach air-lock
(see diagram). Wash air-lock with a dilute solution of sulfite, rinse. You may use water or
dilute sulfite solution when setting up air-lock.
 19

11. Incubate at room temperature for 10 days.

12. Important additional step not included in wine kit instruction manual: Move wine
carboy to table top. Syphon into a clean pail. Wash original glass carboy. Then siphon
wine back into original carboy. This step is to remove sediment before carrying out
stabilization step.

Stabilizing

13. Add the contents of packet #2 (potassium metabisulphite and potassium sorbate) to 125 ml
warm water and stir to dissolve. Depending on the type of wine, gelatin or isinglass (both
bind impurities such as yeast, proteins and lipids, and settle out of solution) may also be
added at this time. Stir vigorously with long spoon for 2 min to drive of CO2. Add
isinglass. Stir vigorously for 2 min. Some wines come with a F-pack that needs to be also
added. Follow instructions with wine kit for the addition of F-pack. You may need to
remove some wine to make room for the F-pack contents. Level of the wine should be
within 5 to 13 cm of carboy neck, if lower, add distilled water. Attach clean air-lock.

14. Incubate for 15 days. Record clarity of wine, pH and specific gravity. Proceed to the
bottling step after the wine is completely clear.

Bottling

15. Bring a liter beaker of distilled water (500 ml) to a boil. Remove from heat and add

corksa (require 30 corks). Submerge corks in water by weighing down with a smaller
beaker containing water. Let sit for minimum of 30 min.
a
corks are a natural product made from the bark of cork oak tree
 20

16. Syphon wine to a clean pail (important additional step not given in wine kit instruction
manual). You want to incorporate as little air into the wine as possible so when syphoning
wine, make sure the end of syphon is at bottom of new container or set up so the liquid will
run down the side of the pail.

17. Filter wine with electric filtering apparatus, see figure. Filtering clarifies and increases
shelf life of the wine by removing particulate matter and most of the yeast.
a) Soak filters (3) in distilled water for 5 min. Make sure the filters are well separated
during soaking.
b) Attach the small diameter tubing to outlet A (figure ) located under the drip tray. This is
the run off from the filters. Place in a clean bucket or bottle. Discard the initial water that
comes out of the filter. The remainder of the runoff (if any) may be re-filtered and bottled.
c) Loosen the two black hand wheels. Remove the central plates. This is where the filters
are placed. Remove one filter pad from the water and place in the filter body with the
course side facing you as shown in figure). The filter pads have holes to allow the filter to
fit tightly only one way, and all aligned. Press down on the pad to ensure the sides of the
pad are sitting on th two side bolts and the pad is straight along the top. Next place one of
the central filtering plates followed by the second filter pad, another central filtering plate
and end with the last filter pad.. Make sure all are even along the top.
d) Tighten the black hand wheels. There must be a tight seal on the pads.
e) Attach the longer slightly larger tubing to outlet C (figure 4). This tubing is used to fill
your wine bottles.
f) Insert the intake hose to outlet B. Place the other end of hose (contains a stainless steel
wire and a anti-sediment tip) in the bucket containing your freshly syphoned wine. Make
sure the short hose is attached tightly and outlet put in a discard bucket (some leakage may
occur).
g) Plug in the filter and turn on the switch located at rear of filter.
h) Fill each wine bottle to about 1 cm below cork.

f) Cleaning: Remove short tubing from pump. Remove tubing from A outlet and attach to
pump with the end placed in a pail for discard. Run about 3 liters of distilled water through
the pump to clean. Unplug. Remove filter pads and discard. Clean filter housing area,
drip tray and all tubing first with warm water. Do not allow any water to get into the motor
section. Dry apparatus and return to box.

18. Drain corks. A floor model hand powered wine corker (red) is available in the lab. Ask
demonstrator for assistance if required.

19. After corking wine, leave the bottles upright for 3 days to allow drying of the inserted cork.
Place the bottles on their side to age.

20. Wine should be acceptable to drink in 1 month (not when you bottle), although best flavour
occurs with aging (at least 6 months). Some varieties peak after one year in the bottle.
 21

Glossary of additives

Saccharomyces cerevisiae: yeast added to dilute grape juice to produce an alcoholic beverage by
converting sugar (mostly glucose) to ethanol and carbon dioxide.
Bentonite: natural clay (aluminosilicate) fining agent. Bentonite both clarifies and stabilizes
wine. As a point of interest, bentonite was at one time harvested at Mount Nebo in southern
Manitoba. Bentonite prevents haze formation in wine by adsorbing proteins and other particles that
produce haze. Bentonite settle completely out of solution. It may seem unusual to add bentonite
right at the start of wine fermentation as it works on the principle of attaching to a molecule and
settling out of solution even before fermentation starts. This in fact does happen in the first 48
hours. However, bentonite is soon back into circulation due to yeast fermentation of sugar to
produce alcohol and carbon dioxide. Carbon dioxide bubble forms on the bentonite (nucleation)
causing it to rise to surface and release carbon dioxide, again sinks adsorbing molecules. The
process is repeated until fermentation slows.
 22

Gelatin: used to clarify wine. Gelatin, positively charged protein, acts by binding all negatively
charged molecules (including the yeast cells) that create a haze or off-taste. The molecules along
with the gelatin settle out of the wine. As the gelatin settles out of solutions it also acts as a sieve
entrapping other molecules.

Isinglass: isolated from the swim bladders of certain fish. Contains about 80% gelatin. Acts as a
clarifying agent in a similar manner to gelatin. It is a long, mostly positively charged, molecule
that binds negatively charged yeast, proteins and lips. Since collagen is a coil of three long
polypeptides it acts as a sieve as it sinks to the bottom of the container entrapping many molecules.

Oak bark chips: flavor/full body to wine

Sulfites: used for cleaning all equipment. Antimicrobial by prevent growth of microorganisms.

Potassium metabisulfite: It is source of sulfite to (1) prevent the growth of yeast, mould and
acetobacter and (2) prevent oxidation of wine, ie. stabilizes the wine - no change in taste,
appearance.

Potassium sorbate: antimicrobial preservative that works in concert with potassium metabisulfite.
It is important that both packets be added to increase the shelf life of the wine (only 1 or 2 months
if not added). If only the potassium sorbate packet is added (not the potassium metabisulfite)
malolactic bacteria convert sorbate to hexadienol, a rotting smelling molecule. If you leave out
one packet both must be left out. However, this is not advised. It is commonly thought that sulfides
in wine are what gives some people headaches. More likely, it is the presence bio-amines formed
by malolactic bacteria.

Glossary of Wine Terms (Not required for lab exam)

breathe/aerate: after opening, believed to allow off-odors to dissipate or soften young wines
balance: when alcohol, sugar, acid and tannin are balanced, no one component is distinctive
body: impression of wine in your mouth - light, medium and full analogous to skim milk, whole
milk and cream.
Some words used to describe wine. What do they really mean?
crisp - clean, tart (higher in acid) wines, opposite of soft
soft - alcohol and sugar dominate acid and tannin
creamy/buttery -describe texture and flavor of wines (eg. Chardonay) that have undergone a
second malolactic fermentation, rich smooth mouth feel, full body.
smoky - aroma and flavor imparted by oak barrel fermentation and aging
supple - full texture in the mouth, not harsh
nutty - hint of nuts in wine, eg hazelnut, almonds, etc
malolactic fermentation - natural secondary fermentation, softens total acid by converting malic
acid to lactic acid.
 23

Wine Record Sheet

Wine Type: _____________________________
Group #:_________
Group Members:______________________________________________________________

Procedure Datea (record) Requested Data Comments (optional)

Primary Day 1: Room temperature: Record expected specific gravity
Fermentation Grape juice of your wine type:
temperature:
specific gravity:
pH:
Secondary Day 7 to 12: wine
Fermentation temperature:
specific gravity:
pH:
Stabilizing ~Day 25-32: wine Record expected finished specific
clarity: gravity of your wine type:
pH:
specific gravity:
Clarity on ~Day 32 to 37: Clarity:
bottling

Bottled wine 1 month (if Flavor:

possible) after
bottling

Aroma:
a
day may changed, just correct to correlate to your actual schedule
Compare the quality of your wine to a similar commercial wine:
 24

Wine Lab Report (MUST BE TYPED, record sheet acceptable hand written.)

Data presentation and analysis

1. Include a copy of completed wine record sheet.

2. Relate specific gravity readings taken to (i) Saccharomyces cerevisiae fermentation and (ii)
expected flavor of wine. Include an explanation of specific gravity reading meaning.

3. Comment on the acceptability of your wine pH. Did it change during fermentation? Explain
why/why not.

Questions
Wine
1. Discuss the variables that allows the production of so many different types of wine.

2. “The usual glycerol concentration in wine ranges from 4 to 9 g/liter.” 3

a) What environmental factors influence the concentration of glycerol in wine?
b) Is it good or bad to have glycerol in wine? Explain your answer.
c) Does the Saccharomyces cerevisiae strain engineered by Remize et al (1999), Table III3,
meet acceptable levels of products? Explain you answer for each product. Include in you
answer how the yeast strain is modified, ie., explain pVT100-U-ZEO as compared to
pVT100-U-ZEO-GPDI.

Beer
1. On occasions problems occur when making beer from a kit. Below are listed a number of
these problems. Give two possible causes for each. Explain your answer if not self-
explanatory.
(a) flat beer
(b) poor head retention (foam on top of beer)
(c) exploding bottles

2. Many types of beer are brewed with distinctive flavors. Explain why this is possible by
comparing Coopers lager and Coopers ale.

3. Why is there no alcohol produced in bread making? Answer must include a comparison of
yeast metabolic equations for bread and beer making.

3
Remize, F. Roustan, JL, Sabbayrolles, JM, Barre, P., Dequin , S. 1999. Glycerol overproduction by
engineered Saccharomyces cerevisiae wine yeast strains leads to substantial changes in by-product formation and to
a stimulation of fermentation rate in stationary phase. Appl. Env. Micro. 65:143-149.
 25

LAB 1b BEER MAKING

(all groups are responsible for beer making for lab exam)

INTRODUCTION
The Coopers beer kit (1) has been selected for the Industrial Microbiology lab. Cooper’s
yeast strain is the most tolerant of variable warm temperatures that occur in your lab. The Cooper’s
beer kit contains only wort and yeast. The wort is made from natural ingredients; water, hops and
malt. In the lab, you will add dextrose as an additional nutritional source for the yeast,
Saccharomyces cerevisiae, which increases the alcohol content without adding any additional
flavour. Coopers uses two varieties of barley that grows in the warm dry regions of Australia. The
hops, Pride of Ringwood variety, are grown in either Australia or Tasmania. The yeast strain is
Coopers own strain that they have had since their inception about 90 years ago.
So what is wort? The process starts with barley. Barley is the grain of choice since there are
few technical problems when compared to other grains. Barley has a high starch and a low protein
content. Malt: barley seeds, that has been steeped, are allowed to germinate initiating enzymatic
degradation of endosperm. Dominion Malting in Winnipeg is a world renown source of malt for
breweries. Much of the barley is grown here in Manitoba. Fort Garry brewery, which you will
possibly tour later in term, uses two varieties (light and dark) of Dominion Malt barley which they
blend to develop different flavoured brews. The first step in the process of making malt is steeping.
The barley grains are re-hydrated by cyclic soaking in water and air. Next the moist seeds are spread
out and allowed to germinate (1). Germination activates enzymes, among them amylases that
breakdown (15oC) starch (amylose and amylopectin) to polyglucose molecules. The germination
time depends on the degree of modification (breakdown) of the barley kernels required. Next, the
germinate barley is kilned. This is a drying process with air circulation to halt germination while
preserving the integrity of the amylases. The grain is initially dried at 38oC until it has reached 5-8%
moisture. At this moisture content, the amylases are stable at high temperatures (80-100oC)(1). This
temperature is used to achieved desired grain color (caramelisation) and moisture content. The malt is
crushed (milled) to produce a “gritty flour” or grist (1). Water is added and mashing of malt takes
place. Enzymes, especially amylases degrade the mash to soluble carbohydrates and other
components until only the husks and small particles of the grain remain. The sweet liquid (wort) is
separated from the spent grain (used as cattle feed). The wort is further processed by boiling in the
presence of hops. Boiling serves many purposes, (i) stop enzyme activity, (ii) sterilization, (iii)
coagulation of proteins and tannins, (iv) lowers pH, (v) distillation of volatile substances, (vi) wort
concentration and (v) color (caramelisation of sugars) production and (vi) flavor production (nutty,
burnt, toffee) (2) . The hops add a bitter taste to the beer. After boiling the wort, it is clarified,
separated from the hops and cooled. The resulting concentrated wort is ready to be packaged.
The wort consists of many components, Table 1 (2). Yeast uses these nutrient to
Table 1. Typical wort components.
Component Quantity (g/l) Component Quantity (g/l)

fructose 2.1 non-fermentable sugars 23.9

glucose 9.1 nitrogen and amino acids 2.75

sucrose 2.3 phenolic compounds 0.25

maltose 52.4 iso " acid 0.038

maltotriose 12.8 calcium ions 0.065

grow and produce energy, ethanol and carbon dioxide.

In the brewery, beer is filtered and carbonated before bottling. In the lab you do not have the
 26

facilities. Clarified beer is siphoned, small amount of dextrose added, bottled and capped. Since
siphoning does not remove all yeast cells, the dextrose is converted to ethanol and carbon dioxide
(carbonation). Soon the nutrients are depleted and the yeast cells settle to the bottom of the bottle.

References
(1) Coopers Brewing Kits http://www.coopers.com.au/homebrew/d.htm (accessed, July 04/02)
(2) Hough, J.S. 1995. The Biotechnology of Malting and Brewing. Cambridge University Press. p
1-159. (available in reference binder)

MATERIALS
Cooper’s Beer kit (wort, dry Saccharomyces cerevisiae packet )
dextrose
1 23 liter beer making kit including packet of yeast.
4-6 dozen beer bottles - please locate yourself (some are available in lab)
bottle capper/caps
23 liter polyethylene bucket (primary fermenter)
23 liter plastic rectangular carboy OR glass carboy fitted with air-lock (secondary fermenter)
syphon
hydrometer (refer to appendix for instructions)
large plastic spoon
long wire brush for cleaning equipment

PROCEDURE
After today the remainder of the beer or wine making lab is to be completed on your own time. The
lab is open weekdays (7:30 am to 5:00 pm, if you want to come later please discuss ahead of time
with the instructor). Refer to lab room schedule posted on each lab door, to determine when the lab is
available. If assistance is required, obtain help from teaching assistant.
STUDENTS WORK IN PAIRS
Instructions vary for the different types of beer. Compare your kit instructions to those given below
and adapt if necessary.

Primary Fermentation

Week 1

1. Clean primary fermenter (polyethylene bucket) with a dilute solution of sodium bisulfite (5
ml/liter). Rinse thoroughly. It is extremely important that the sodium bisulfite is completely
removed. Use only clean utensils throughout procedure.

2. Empty contents of can (malt extract) into polyethylene bucket. Add 2.5 liters boiling water. It
is best to boil water in two containers to reducing boiling time. Both tall burners and heater
 27

stirrers are available in the lab. Stir until the malt is completely dissolved.

3. Top up with distilled water to just above the 23 liter mark (indented ring around top of pale or
markings on pail) and stir well. Slowly stir in 500 ml to 750 ml dextrose adding a little at a
time (beer store grade only). If adding more than 500 ml dextrose it is important that each
stage of fermentation goes to completion or beer will be too frothy to drink and chance of
exploding bottles. Your starting temperature should now be between 18oC - 24oC. Record
temperature of wort. Read specific gravity of wort using hydrometer and record. Since each
kit varies record the expected specific gravity.

4. Sprinkle yeast on surface and stir well. Cover pail with a plastic bag and tape with masking to
secure. Place pail out of direct sunlight. Incubate at room temperature (~21oC).

Secondary Fermentation
5. The time to syphon beer to secondary fermenter (rectangular plastic carboy) will vary, but
usually takes 12 days. The fermentation time depends on room temperature. Beer made at
lower room temperatures has less by-products and tends to taste better. The fermentation must
be complete before syphoning - rapid movement in fermentation pail should be finished.
Read and record specific gravity of beer. Record expected specific gravity for you beer type.

6. Move the primary fermenter to a table. Be careful not to disturb the sediment. The syphon
should be carefully placed in primary fermenter. The bottom of the siphon is hooked or
partially covered to allows transfer of beer leaving the sediment in the primary fermenter.
Syphon beer carefully to avoid picking up sediment. The syphon can be started by using the
water aspirator. Do not use the vacuum line.

7. Some air space at top of carboy is okay, although carboy can be topped up with distilled
water. Fit top with air lock. Place carboy out of direct sunlight or cover. Incubate at room
temperature for 12 days (conditioning).

8. Make sure the siphon is clean. Check specific gravity by siphoning off beer into a graduated
cylinder which allows the hydrometer to float in the beer when the cylinder is full. Read the
specific gravity in the cylinder. The specific gravity should now be between 1.004 and 1.008
again depending on the type of beer. Record beer temperature. Leave beer a further two days
then check specific gravity again. If the same reading is noted then your beer may be bottled,
if not leave a further two days and check again, repeat until the specific gravity reading is
constant.

Bottling
9. Wash beer bottles thoroughly, first by hand using a bottle brush, then by dish washer (if
available) to ensure that they are clean. Rinsing with sodium bisulfite solution is not
necessary if using a dish washer. If washing bottles with sodium bisulfite solution, be sure to
rinse bottles thoroughly with distilled water before filling with beer.

10. Carefully move beer carboy to a table. Syphon beer from carboy into clean primary pale,
again being careful not to pick up sediment.
 28

11. Slowly add 180 ml dextrose to beer as gently stirring. Stir beer until dextrose is completely
dissolved.

12. Syphon beer into cleaned beer bottles filling to within 1.5 cm from top, and cap with sanitized
crown caps. Easiest to pinch off syphon with fingers between beer bottles. Cap beer bottles.
If required ask demonstrator for assistance with the bottle capper.

13. Store beer in a warm place for 5 to 7 days, then move to a cooler place for storage, always
keep your beer covered (bright light gives beer an off taste). Beer may be tested in 2 weeks,
but best flavour develops after 2 months.
Note: When tasting beer, carefully decant off beer into a glass so as not to disturb the
sediment on the bottom of the beer. Why is there a sediment?
 29

Beer Record Sheet

Wine Type: _____________________________
Group #:_________
Group Members:______________________________________________________________

Procedure Datea (record) Requested Data Comments (optional)

Primary Day 1: Room temperature: Expected specific gravity of beer
Fermentation type:
Wort temperature:

Wort specific gravity:

Start ~Day 12: Wort temperature:

Secondary
Fermentation Specific gravity:

End ~Day 24 to Wort temperature: Expected specific gravity of beer

Secondary 26: type:
Fermentation Specific gravity:

Specific gravity after 2 more

days:

Bottled beer 1 month (if Flavor:

possible) after
bottling
Clarity:

a
day may changed, just correct to correlate to your actual schedule

Compare the quality of your beer to a similar commercial beer:

 30

Beer Lab Report (MUST BE TYPED, record sheet acceptable hand written)

Data presentation and analysis

1. Include a copy of completed beer record sheet.

2. Relate specific gravity readings taken to (i) Saccharomyces cerevisiae fermentation. Include
an explanation of specific gravity reading meaning.

3. Discuss the relevance of incubation temperature at all stages of your beer production.

Questions
Wine
1. Discuss the variables that allows the production of so many different types of wine.

2. “The usual glycerol concentration in wine ranges from 4 to 9 g/liter.” 4

a) What environmental factors influence the concentration of glycerol in wine?
b) Is it good or bad to have glycerol in wine? Explain your answer.
c) Does the Saccharomyces cerevisiae strain engineered by Remize et al (1999), Table III3,
meet acceptable levels of products? Explain you answer for each product. Include in you
answer how the yeast strain is modified, ie., explain pVT100-U-ZEO as compared to
pVT100-U-ZEO-GPDI.

Beer
1. On occasions problems occur when making beer from a kit. Below are listed a number of
these problems. Give two possible causes for each. Explain your answer if not self-
explanatory.
(a) flat beer
(b) poor head retention (foam on top of beer)
(c) exploding bottles

2. Many types of beer are brewed with distinctive flavors. Explain why this is possible by
comparing lager and ale beer.

3. Why is there no alcohol produced in bread making? Answer must include a comparison of
yeast metabolic equations for bread and beer making.

4
Remize, F. Roustan, JL, Sabbayrolles, JM, Barre, P., Dequin , S. 1999. Glycerol overproduction by
engineered Saccharomyces cerevisiae wine yeast strains leads to substantial changes in by-product formation and to
a stimulation of fermentation rate in stationary phase. Appl. Env. Micro. 65:143-149.
 31

LAB 2 MICROBIAL INDUSTRIAL LIQUID FERMENTATION: Citric Acid

Production by Aspergillus niger

OBJECT
The object of this experiment is to (i) optimize citric acid production by Aspergillus niger by
investigating environmental parameter separately, (ii) learn the fundamentals of experiment design
and (iii) present findings.

INTRODUCTION
In your lab you will carry out small-scale liquid fermentation for the production of citric acid.
Small-scale liquid fermentation allows testing of experimental parameters that lead to increased
product production essential before step-up to large scale fermentation. In any fermentation it is
important to understand what limits growth of the organism and subsequently what limits product (1).
In your lab you will use standard Erlenmeyer flask with metal cap
stoppers and floor rotary shakers to maximize oxygen transfer rate.
Temperature is controlled by air incubator room. In small fermentation, a CH 2COOH
sample should only be removed once (1). If time points are required, a
series of identical flasks should be set up and sample taken only once from
each flask.
Citric acid production by Aspergillus niger is a world wide
HOCC00H
industry, 400,000 tons produced annually (2). Citric acid, sold as sodium
citrate, calcium citrate, or potassium citrate, is an safe edible acidifier.
There are many reasons why citric acid is so widely used, for example, it
is soluble, has low toxicity, chelates cations, pH adjustor, environmentally H2CCOOH
safe and has a good taste. It is used as a food preservative, anticoagulant,
found in many medicines, ore leaching, detergent ingredient, chemical
industry, etc (7). Citric acid
Citrate acid excretion in large amounts by A. niger only occurs when cell growth is not
optimal. Roehr et al Legisa and Gradisnik-Grapulin showed that high levels of citric acid
accumulated in the absence of manganese, high initial sucrose concentration and aeration (3). The
absence of zinc also seems to be important for accumulation of citric acid (4). Low pH is important
for the accumulation. Ruijter et al demonstrated the an A. niger mutant (did not produce oxalic
acid),lacking glucose oxidase and oxaloacetate acetylhydrolase, grew at pH 5 in the presence of
manganese was able to accumulate citric acid (5). It is apparent, the ability of A. niger to excrete
citrate depends on many variables, both genetic and environmental. Some strains are good citrate
accumulators, while other strains do not accumulate at all. Citrate accumulation property may be
induced by mutation.
In the industrial microbiology lab, you will design, carry out and report on the effect of
various environmental factors on citrate acid production. Environmental conditions to be
investigated are absence of manganese, sucrose concentration, sucrose dilution, pH, temperature,
aeration (oxygen transfer rate), and spore inoculum density.

Citric Acid Determination (6): Citric acid concentration may be determined accurately and
conveniently by coupling two reactions catalysed by the enzymes citratase (citrate lyase) and malic
dehydrogenase. As the products of the citratase reaction inhibit it, a large excess of malic
dehydrogenase must be used. This will force the citratase reaction to the right and bring it rapidly to
completion (the equilibrium of the malic dehydrogenase reaction lies far to the right). The reaction is
followed on spectrophotometer by observing the decrease in absorbance at 340 nm (due to NADH,
NAD+ does not absorb). When there is no further decrease in absorbance, it is assumed that all the
 32

citrate has reacted. This is equivalent to the amount of citrate originally present in the cuvette.

Keq = 0.64

citrate oxaloacetic acid + acetate

++
CITRATASE, Mg

+
oxaloacetate + NADH malate + NAD
MALIC DEHYDROGENASE

+
citrate + NADH malate + acetate + NAD

References
(1) Hilton, MD. 1999. Small-scale liquid fermentations. In: Demain, AL, Davies, JE., editors. Manual
of Industrial Microbiology and Biotechnology, 2nd edition. Washington: ASM Press. p 49-60.
(2) Legisa, M., Gradisnik-Grapulin, M. 1995. Sudden substrate dilution induces a higher rate of citric
acid production by Aspergillus niger. Appl. Env. Micro. 61: 2732-2737.
(3) Roehr, M, Kubicek, CP, Kominek, J. 1992. Industrial acids and other small moleculs. In: Bennett,
JW, Klich, MA, editors. Aspergillus-biology and industrial applications. Stoneham: Butterworth-
Heinemann. p 91-131.(not in reference binder)
(4) Wold, WSM, Suzuki, I. The citric acid fermentation by Aspergillus niger: regulation by zinc of
growth and acidogenesis. Can. J. Microbiol. 22: 1083-1092. (not in reference binder)
(5) Ruijter, GJG., van de Vondervoort, PJI., Visser, J. 1999. Oxalic acid production by Aspergillus
niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of
manganese. Microbiology. 145: 2569-2576.
(6) Seiffer, S, S Seymour, B Novie, and E Munteoyler, Archives of Biochemistry vol. 25 (1950),
p.191.
(7) http://www.apctt.org/database/to6092.html
(8) Strobel, RJ., Sullivan, GR. 1999. Experimental design for improvement of fermentations. In:
Demain, AL, Davies, JE., editors. Manual of Industrial Microbiology and Biotechnology, 2nd edition.
Washington: ASM Press. p 80-93
 33

MATERIALS
supplies available (unless a special request you must limit your supplies used number available
to the following list) per group
standard 250 ml erlenmeyer flask with metal caps (cannot vary) 10
pipetmen: P200 and P1000/tips 1
digital scale
pH meter
28oC & 35oC incubator room with floor rotary shaker
50 ml sterile 0.5% Tween 80 (prepared in double distilled water) in MDBa 1
sporulated culture of Aspergillus niger 1
sterile large screw capped tube to transfer A. niger spore suspension 1
all supplies necessary for preparation of BASIC medium
if other media components are not available on the shelves, request of
instructor
Week 4 supplies available/group (keep all supplies on ice):
0.1 M phosphate buffer, pH 7.5 200 ml
1.0 mM NADH: prepared by dissolving 75.3 mg in 100 ml cold 0.1 M 25 ml
phosphate buffer, pH 7.5.
20 mM MgCl2.6H2O 25 ml
malic dehydrogenase: prepared by dissolving 2250 units of enzyme in 50 ml 25 ml
phosphate buffer.
25 ml
citratase (citrate lyase): prepared by dissolving 100 units of enzyme in 47 ml
phosphate buffer.
0.5 mM citrate: prepared by dissolving 14.7 mg of trisodium citrate (MW = 50 ml
2.94.1) in 100 ml phosphate buffer..
a
MDB = milk dilution bottle

BASIC Medium:
1.3 g NH4NO3, 0.5 g KH2PO4, and 0.13 g MgSO4.7H2O in glass distilled water. Adjust pH to 2.0
with 1 N HCl. Bring volume to 250 ml with double distilled water. Dispense 25 ml into each 250 ml
Erlenmyer flask fitted with a metal cap (number varies depending on your experiment). Autoclave
for 10 min. For each salts flask, dissolve 7.5 g sucrosea in a final volume 25 ml double distilled water.
Pour into a milk dilution bottle (MDB). Loosely cap. Autoclaveb. When solutions have cooled to
below 50oC, aseptically pour sucrose into salts flask.
a
high yields of citric acid production are obtained with 14%-22% sucrose
b
do not over autoclave
Add ONLY if experiment requires added trace elements: add at final concentration of 1.0 mg/l
manganese chloride: dissolved 10 mg MnCl2.4H2O in a final volume 100 ml double distilled water.
Dispense in MDB. Autoclave. After cooling aseptically add 0.5 ml to each flask required.
 34

Clearly label all flasks to be autoclave with all your group names (last names in full) as all media
autoclaved together.

Week 2

Experiment Group Assignment

group # Object: To optimize citric acid production by A. niger by varying designated
environmental parameter:
1 dilution effect on high initial sucrose (for information see Legisa and Gradisnik-
Grapulin reference (2)).
2 media composition: sucrose concentration
3 media composition: nitrogen concentration
4 media composition: manganese concentration
5 media composition: complex carbon source(s)
6 temperature
7 oxygen transfer rate (aeration)
8 pH
9 time (growth phase) - must be completed in 96 h
10 spore inoculum density (must measure spore density using a hemocytometer - see
appendix and instructor to get hemocytometer and location of microscope)
11 dilution effect on high initial sucrose (for information see Legisa and Gradisnik-
Grapulin reference (2)).
12 media composition: manganese concentration
13 oxygen transfer rate (aeration)
14 time (growth phase) - must be completed in 96 h
All groups must use references available in the reference binder on reserve in the Science and
Technology library.
 35

Procedure
1. Acid washing is not required by any groups. Use double distilled water available in lab for all
media preparation.

2. Aspergillus niger cultures are available in student cold box

3. Even though most groups are doing a different experiment, all experiments must be carried
out as scheduled in lab manual.

4. All groups measure biomass and citric acid production regardless of parameter varied.

5. STUDENTS WORK IN PAIRS (same group as beer or wine making)

Week 2

Part I: Experiment Design

6. Experiment planning: Allotted time - 1 hour ONLY. See experiment group assignment for
experiment to be done by your group. Come to lab well prepared and bring required
references. Ask as many questions as possible of the instructor and teaching assistants. Make
sure you read the complete experiment before coming to lab as it will make designing your
experiment much easier.

7. Guidelines for experiment:

-each flask should only be sampled once
-no zero time sample required, control should be sampled at the same time but not inoculated
with A. niger
-all groups use BASIC medium and vary specified environmental requirement
-when making solutions, remember to always slowly add solid chemical to distilled water
while stirring to prevent fromation of insoluble clumps
-duplicate flasks of each condition should be inoculated to ensure no problems, eg.
contamination, loss, spilled, etc.. But, only one flask required for sampling.
-from reference material you should find what the parameter optimum condition for citric acid
production - make sure parameter range include expected optimum

8. Oral Presentation: An outline of experiment PROCEDURE must be presented to the class (5

min including presentation and discussion). Overhead sheets/pens supplied if required.
Presentation will be given in a lecture room. Come prepared to ask questions and give
suggestions to other groups. Allotted time 1 ½ hour ONLY. If you use overhead(s) for your
procedure presentation, do not write everything just brief introduction for each point. A good
guideline is for each overhead is 7 line with maximum 7 words per line. Read from overhead
screen not overhead. But remember to talk to class not screen.
 36

9. Procedure Report: A detailed Materials and Methods report must be emailed to

le_cameron@umanitoba.ca no later than 4:30 pm Monday (attach as a Word or Wordperfect
file). Include every detail. If you include any procedures found in your lab manual, do not
repeat, just cite noting any changes.
(1) materials
-include a list of all equipment and supplies required for experiment. Include number of each
supply required.
-footnote all supplies that are not available for this lab (see list). This is very important as any
additional supplies must be ordered from the prep room ahead of time.
(2) methods
-numbered steps only
-include all experimental details (similar to experiment procedures in the your lab manual). I
need to know all the details.
Marked procedure will be returned in lab Friday. If possible a corrected version will be
returned by email before Friday. Make sure to include all group member email addresses on
the cover of the report.

Week 3

Part II: Media Preparation and Inoculation

Friday
Each group should use the standard medium (see above) and incubation conditions other than the
environmental parameter varied. Remember to measure and record all constant parameters.

Standard environmental parameters:

temperature: 28-30oC
sampling time: ~96 h (4 days)
pH: 2.0
rate of aeration: standard floor model shaker speed

1. Prepare BASIC medium as above or vary depending on your group’s experiment. Remember
the basics of media preparation. Use double distilled water for all media preparation. Use
volume less than required to dissolved media components. Water should be added to a beaker
larger than maximum volume required. Add a stirring bar and place on stirrer. Start stirring.
Slowly add media component. Stir until dissolved before adding the next component. Use
HCl to adjust pH. Bring to final volume with distilled water.

2. Using masking tape, label all flasks in detail (complete names of your group - no initials).
This ensures that no other group takes your flasks. Put media on trolley for teaching
assistants to autoclave.
 37

Monday

3. All autoclaved media will be in the lab Monday. Remember to add appropriate components
to you salts media before inoculation.

4. Aspergillus niger slants are located in the student’s cold box (room 202 off room 201).
Prepare a spore suspension by adding approximately 10 ml sterile 0.5% Tween 80 per slant.
Tween 80 is a detergent which serves as a wetting agent to produce a homogeneous
suspension of highly hydrophobic spores. Aseptically loosen the spores very carefully with a
wire loop (do not disturb the surface of the slant); make sure the Tween 80 contacts the entire
slant surface. Make sure the cap is secure and shake vigorously for 30 sec. If necessary,
vortex the suspension to break up the clumps. Aseptically pour spore suspension to a sterile
large screw capped tube.

5. Unless you are varying the spore inoculum, add 0.5 ml Tween 80 spore suspension to each
flask (104 to 105 spores/ml) except control.

6. Incubate the flasks (properly labelled) in 28oC incubator room on the large rotary shaker at
150 -200 rpm (room 115 Buller). Remember to record all constants. Check thermometer on
wall, do not assume 28oC.

7. No samples are collected for the majority of groups until Friday (4 days, ~96h). However, if
your group is investigating growth phase (time), you need to remove a flask each day and
immediately process (separate supernatant from A. niger biomass before freezing at -20oC -
see below for protocol).

Week 4 (Friday)

Part III: Sample collection and biomass determination

8. Process samples: Pour each sample (all liquid and fungal growth) into sterile centrifuge tubes.
Probably the best idea is to divide each sample between two centrifuge tubes (40 ml plastic
open top). centrifuge at 10000 rpm for 5 min. Pour supernatant into a labelled screw capped
culture tube (not sterile). Put on ice. (For the group sampling with time: freeze samples, both
fungal biomass in weigh dish and supernatant. Make sure you do not put more than 15 ml in
each tube before freezing as the tubes may break upon freezing.

9. Weight and record weight of an aluminum weigh dish. Use a spatula to scrap pellet(s) into a
labelled aluminum weigh dish. Make sure you add complete fungal pellet collected from one
flask (50 ml). Put on tray for drying. The samples will be dried for 22 h at 100oC.
Monday: The tray containing the samples will be available in the lab. As soon as possible
weigh and record the weight.
 38

Part IV: Citric Acid Determination

Each group must prepare a standard curve and measure each sample in triplicate

1. Blank the spec 20 with 5 ml 0.1 M phosphate buffer, pH 7.5, not the reaction mixture.
Remember NADH aborbs at 340 nm and must not be added when blanking the spec20.
2. Reaction Mixture: Keep all solutions on ice, especially the enzymes. Once you remove the
NADH solution from the fridge, cover with foil as NADH is light sensitive.
In a spec 20D cuvette, carefully pipette the following:
20 mM magnesium chloride 0.5 ml
malic dehydrogenase 0.5 ml
citratase 0.5 ml
NADH 0.5 ml
0.1 M phosphate buffer, pH 7.5 to 5.0 ml (including sample volume)
It is best to do one reaction at a time.
3. Change in absorbance measurement: Add all components of reaction mixture except
sample (either your experiment sample or known citrate standard). Mix the cuvette well by
inverting several times (capped with parafilm).
For each measurement your need two spec 20D readings, before and after you add the
sample.
Absorbance reading 1: First take a spec 20D absorbance reading of reaction mixture at
340 nm.
Absorbance reading 2: Next, add 0.5 ml sample(or stock citrate), mix, wait 1 min, then
take spec 20D absorbance. The 1 min wait allows the enzyme reaction to go to
completion.
4. Standard Curve (change in absorbance at 340 nm vs mM citrate): The standard curve for
citrate is made by following the total change in absorbance at 340 nm using 1000, 750, 500,
250 and 100 :l of 0.5 mM citrate standard solution. Maintain the total volume in the cuvette
at 5.0 ml by varying the amount of phosphate buffer added each time. Prepare samples for
standard curve in duplicate.
5. Experiment samples: Each sample must be measured in triplicate. You may need to dilute
but first carry out experiment with undiluted sample then determine if a dilution is required.
Check supply list for amount of each solution available.
6. Wash cuvette tubes and return to plastic covered tube rack beside spec20D.
 39

LAB REPORT (MUST BE TYPED - Both a hard copy and a Word or Wordperfect file emailed to
le_cameron@umanitoba.ca no later than 4:30 pm on requested data. The file is important. I need to
post all reports on the website to allow groups to see the results of all experiments - required for lab
exam.

Include a Title
• The title should identify major finding(s).
• Be concise.
• Avoid abbreviations.
• Include taxonomic names if relevant.

Introduction
• Write in paragraph format.
• Explain why you did the experiment (hypothesis).
• Include information that gives background to your experimental findings.
• Cite and reference all necessary information.
• Write from general to specific.
• End by summarizing results in one sentence.

Materials and Methods

• Present in paragraph form using a separate paragraph for materials and each different method.
• Include enough information that would allow a peer to repeat the experiment.
• Organize information.
• Name all equipment used, reagents added, media used, environmental conditions, etc.
• If materials or methods are found in lab manual, do not include, just cite
• Be concise, do not include unnecessary details.
• Do not include results.
• Use materials and methods section of Applied and Environmental Microbiology journal as an
example.

Data Presentation (Present results as requested)

1. Biomass Determination:
a) Present a table of A. niger biomass determination. Record biomass as dry weight (g/l).
b) Include sample calculation.

2. Citric Acid Standard Curve:

a) Tabulate standard curve data. Include a column of mM citrate concentration and a sample
calculation.
b) Plot a standard curve of change in absorbance at 340 nm versus citrate concentration
(mM).
 40

3. Sample citric acid determination:

a) Determine citric acid concentration (mM) of samples. Include a sample calculation.
b) Determine average and standard deviation of citric acid triplicate samples using Microsoft
Excel spreadsheet (any spreadsheet is acceptable but remember to cite). Include a printout of
data entry , triplicate citric acid (mM), average and standard deviation. Make sure the
spreadsheet is clearly labelled.

4. Plot citrate (mM) and biomass (mg/ml) as a multiple bar graph (all data on one graph). If you
are sampling over time plot Plot citrate (mM) produced (mM citrate in sample minus mM
citrate in control) and biomass (mg/ml) vis time Tabulate data used for plot.
At the top of each bar place a standard deviation (SD) bar (see sample graph - standard
deviation bar length is equal to + and - standard deviation, ie 2x SD).
Comment: Combined information presented in a bar chart quickly allows the reader to clearly
understand the comparison of information

Microsoft Excel PASTE FUNCTION procedure

(i) Enter triplicate data on an Microsoft Excel spreadsheet or any spread sheet is acceptable but must
reference. Be sure to include all necessary headings.
(ii) Determine AVERAGE. Highlight cell where you want to record average. Select paste function
button, then statistics, then AVERAGE. Or use pull down menu - select Insert, function, statistics,
then AVERAGE. A pop-up menu appear. Using your mouse right click the first or last1 cell of data
set. Hold down button and scroll down to the last or first cell in the data set. Release button. Click
OK on pop-up menu. The average value appears in your selected cell. Repeat for remaining data sets.
1
Sometimes it is best to start with last cell then box on top of data disappears, then reappears.
(iii) Determine STDEV (standard deviation). Highlight cell where you want to record standard
deviation. Select paste function button, then statistics, then STDEV. Or use pull down menu - select
Insert, function, statistics, then STDEV. A pop-up menu appear. Using your mouse right click the
first or last cell of data set. Hold down button and scroll down to the last or first cell in the data set.
Release button. Click OK on pop-up menu. The standard deviation value appears in your selected
cell. Repeat for remaining data sets.

Discussion
 41

• Write from specific to general.

• Write with confidence.
• Be clear and organized.

• Analyze results citing figures or tables and referring to your data.

• Explain what your results mean (if more than one explanation is possible, select the most
likely and explain why.
• Do your results support your hypothesis or literature results.
• State experiment limitations.
• End with one or two sentences summarizes your results as related to the big picture.

Questions

1. Comment on the role of Aspergillus niger intracellular glycerol levels with reference to citric
acid production.
2. Why study oxalic acid production by A. niger relative to citric acid production?
3. How would you liked to change experiment with reference to your environmental parameter
to enhance citric acid production but were unable to due to equipment and supplies limitations
in the lab. Discuss only the environmental condition you investigated.
4. Explain why a sample should only be removed once from a small fermentation flask.
5. In your experiment, only “a one-factor-at-a-time” (8) changed while all other factors were
held constant to limit the size of the experiment. Briefly outline a better approach to the
improvement of citric acid fermentation.

Acknowledgements
State what each project member contributed to the experiment and report.

References
Include a list of cited reference as outlined in general introduction.
 42

LAB 3: INDUSTRIAL MICROBE STRAIN IMPROVEMENT: Enhanced antibiotic production

Introduction
In the Aspergillus niger citric acid lab, you investigated enhanced product production by
changing environmental parameter. In this lab you will demonstrate the importance of the microbe
strain in the over production of desired product. This is because the improvement of product
production by varying environment parameters is limited by microorganism metabolic rate-limiting
pathways (1). Antibiotic production by Streptomyces has been selected due to the easy of product
assay.
A member of the Actinomycetes group, Streptomyces, a gram positive filamentous bacteria,
produce 70% of all fermentative bioactive products (2). Some of the well known antibiotics
produced by Streptomyces are streptomycin, tetracyline, and vancomycin. Antibiotics are produced
via secondary metabolism. Since secondary metabolism is more specialized, many types of
antibiotics exist as each species may produce a different set of antibiotics. In your lab your will use a
traditional mutagenic method, UV illumination, to enhance antibiotic production. However, it is
becoming more and more difficult to isolate new antibiotics by traditional strain mutagenesis. DNA
technology, which is beyond the scope of this lab, is the method of choice for strain enhancement and
the isolation of new antibiotics. Classical mutagenesis and screening does have some advantages.
For example, product may be increased by mutagenesis with very little knowledge of the physiology
or genetics of the microorganism required. This translates into minimal start up time (1). Often both
classical methods and DNA technology are used to improve product production. Screening for the
overproducing mutant is the most labor and time consuming step. This is the step that determines the
success of the operation. Screening may be either manual or automated. Manual screening is
dependent on the number of staff available. It does have the advantage that there is lower initial cost
start up. For example, if a company has the capability to examine 500 mutants a week with an
expected gain of a “good” mutant of 1:10000, a new strain may be found every 10 to 20 weeks (1).
Each “good” mutant is used as the parent for the next round of mutagenesis. Mutants are initially
screen on agar plates to illuminate most of the bacteria, only over producing mutants go onto liquid
culture. An experienced researcher soon learns to rapidly screen mutant colonies noting other
important factors such as growth rate and appearance.
In addition, you will attempt to isolate a new enhanced antibiotic producing strain from the
environment. To increase the chances of finding a desired product it is best to consider product
characteristics, process development and use of “eco-physiological methods” for isolation and
screening (3). That is, know the bacteria physiology, go to the best source that provide conditions
that enhance that particular group of bacteria. Actinomycetes are found in soil and compost.
Desiccated soil that has been incubated in the presence calcium carbonate to enrich for actinomycetes
(3). The growth medium, casamino peptone, is specially designed to enrich for antinomycetes.
Streptomyces are oxidative chemoorganotrophs that are able to degrade a wide range of carbon
sources. The optimum pH range is 6.5-8.0 and the optimum growth temperature is 25 -35oC.
Streptomyces form discrete raised hard colonies. Older colonies develop aerial mycelia appear either
granular or powdery. Many species are pigmented and some produce diffusible pigments.
A rapid assay for screening for antibiotic production (5) allows screening of numerous
organisms on a minimum number of plates. Straw size plugs of colonies to be tested are inserted onto
a Czapek Medium agar plate that has been spread with test bacterium. Since many of the antibiotics
secreted by Streptomycetes are active against gram negative bacteria, E. coli has been selected.

References
(1) Vinca, VA, Byng G. 1999. Strain improvement by nonrecombinant methods. In: Demain, AL,
Davies, JE., editors. Manual of Industrial Microbiology and Biotechnology, 2nd edition. Washington:
ASM Press. p 103-113.
 43

(2) http://www.galilaeus.fi/research.php#i (accessed July 12/02)

3 Hunter-Cevera, JC, Belt, A. 1999. Isolation of cultures. In: Demain, AL, Davies, JE., editors.
Manual of Industrial Microbiology and Biotechnology, 2nd edition. Washington: ASM Press.
p 3 - 13
(4) Atlas, RM. 1995. Handbook of Media for Environmental Microbiology. Boca Raton: CRC Press,
Inc. p 90.
(5) http://www.accessexcellence.org/AE/AEC/AEF/1994/barnard_isolation.html

Materials
Casamino Peptone (Czapek Medium)(4)
Composition per liter: 30 g sucrose, 15 agar, 2 g peptone,, 1 g casamino acids, 1 g K2 HPO4, 0.5 g
KCl, 0.5 g MgSO4, 7H2O, 0.01 g FeSO4.7H2O. Add all components to final volume 1 liter distilled
water. Mix thoroughly. Gently heat to boiling. Aliquot. Autoclave at 15 psi for 15 min at 121oC.
Pour Petri plates. Cycloheximide added to inhibit fungal growth. Aseptically add 10 ml/liter of stock
0.5% (w/v) cycloheximide.

Procedure

Week 5

Part I: Isolation of a “new” antibiotic producing strain from the environment

1. Enrichment of Streptomyces: Prior to the start of the lab, garden soil was collected, incubated
in a container with an equal amount of 0.5 g/l CaCO3. The container lid was lined with filter
paper and loosely placed over the container. Incubated at 28oC for 10 days. The soil was
spread out on trays and dried at 37oC for several days to dessicate.

STUDENT LAB STARTS HERE

2. Serial dilutions of soil: Add 10 g of your soil sample to 90 ml sterile saline (10-1 dilution).
Shake vigorously for 2 min. Transfer 10 ml of the 10-1 dilution to 90 ml saline solution (10-2
dilution). Mix by shaking. Transfer 0.5 ml of the 10-2 dilution to 4.5 ml saline solution in 5"
metal capped test tubes (10-3 dilution). Mix by shaking. Transfer 0.5 ml of the 10-3 dilution to
4.5 ml saline solution (10-4 dilution). Vortex. Transfer 0.5 ml of the 10-4 dilution to 4.5 ml
saline solution (10-5 dilution). Vortex. (prepare your own 4.5 ml saline tubes using saline in
the flask)

3. Dilution plating: Spread plate 0.1 ml of 10-2, 10-3, 10-4 and 10-5 dilution of soil sample on
Czapek agar plates containing cycloheximide. You must use the plates containing
cycloheximide.
a) Aseptically transfer 0.1 ml of dilution to center surface of agar plate. Never lift the lid of agar plate
completely off the plate or place on a bench surface. Best method is with the lid tilted above the plate.
b) Dip hockey stick spreader in bottle of alcohol.
 44

c) Flame the spreader until alcohol ignites. Immediately remove spreader from flame and wait until alcohol
completely burns off. Cool slightly.
d) Open lid keeping it tilted over the plate and touch spreader to surface of plate that does not contain culture
drop. If spreader is still hot, it will kill bacteria. Still holding the lid tilted over plate, move the plate around
spreading bacteria evenly over agar surface. (Use turntable to rotate plate if available or use spread
microorganisms evenly over the surface of the plate.)
Masking tape plates together and place in trays provided. Label plates well as it will be two
weeks before returning to this experiment. Plates will be incubated at 28oC for three days
then transferred to 4oC until the next lab.

Part II: Mutagenesis of Streptomyces strains

1. Prior to the start of the lab a culture of Streptomyces griseus (department stock collection) and
recent Streptomyces sp. isolate from the environment (soil) was grown to log phase in Czapek
broth. Centrifuged and resuspended in 0.1 M MgSO4 buffer. Exposed to UV for 60 sec.
Covered with foil and put on ice.
Need UV treatment in detail protocol....5 ml etc
STUDENT LAB STARTS HERE

2. Each groups should repeat only two of the following dilution and plating experiments:
(even group numbers do (i) and (ii) while odd group numbers do (iii) and (iv).
(i) Streptomyces griseus- no UV exposure control
(ii) Streptomyces griseus - UV exposed
(iii) Streptomyces (soil) - no UV exposure
(iv) Streptomyces (soil) -UV exposed

3. Serial dilutions: Transfer 0.5 ml of the culture to 4.5 ml saline solution in 5" metal capped
test tubes (10-1 dilution). Mix by shaking. Transfer 0.5 ml of the 10-1 dilution to 4.5 ml saline
solution (10-2 dilution). Vortex. Repeat until 10-3 dilution for UV treated and 10-5 dilution for
untreated. (prepare your own 4.5 ml saline tubes using saline in the flask)

4. Dilution plating: For untreated spread plate 0.1 ml of 10-2, 10-3, 10-4 and 10-5 dilution of soil
sample on Czapek agar plates. For UV treated spread plate 0.1 ml of 10-0, 10-1, 10-2 and 10-3
dilution of soil sample on Czapek agar plates. Do not use plates containing cycloheximide.

5. Masking tape plates together and place in trays provided. Label plates well as it will be two
weeks before returning to this experiment. Plates will be incubated at 28oC for three days then
transferred to 4oC until the next lab.
 45

Week 7

Part III: Selection of improved strain

1. Spread plate six Czapek agar + cycloheximide plates with 200 :l E. coli culture. Prepare a
legend for each plate. Make sure plates are completely labelled.
Plug placement:
(a) “new” antibiotic from the environment ... if possible plate 27 morphologically different
colony plugs on three Czapek plates. Select only actinomycetes. Colony description: discrete
raised, hard, granular or powdery colonies. Often the colonies are pigmented and some
produce diffusible pigments. Label plugs one through 27.
(b) +/- UV treated Streptomyces griseus or Streptomyces (soil) - plate plug colonies as shown
in diagram. Repeat twice - total of three Czapek plates. Plate one - label control 1 and label
UV mutants 2 through 9, plate two label control 10 and UV mutants 11 through18, and plate
three label control 19 and UV mutants 20 through 27.

2. Colony placement technique: Use sterile straws to remove a colony. This is done by inserting

a sterile straw over the colony right to the bottom of the agar. Remove plug and place on agar
plate. If you have difficulty removing the plug use a sterile toothpick to help remove plug.

3. Incubate all plates for 3 days at 28oC. The TA will check the plates after 1 or 2 days to make
sure the zone of inhibition is not too large. If that is the case the plates will be placed at 4oC
(student cold box).

4. Monday (Day 3): Record zone diameter of all samples. Hand in a COPY of data sheet (next
page). Or data sheet is available on website in lab manual (pdf file). Data is due by 4:30 pm
Tuesday, Mar 4. Class data will be compiled and available on the website as soon as possible.
 46

GROUP DATA SHEET Due Tuesday March 4, 2003

Lab 3: Industrial microbe strain improvement: Enhanced Antibiotic Production
Group #:________________ Date:______________________
Group Names (include last names):

Czapek agar + cycloheximide spread with bacterium, ______________________________.

(a) “new” antibiotic from the environment: data from three plates
isolate # inhibition zone isolate # inhibition zone isolate # inhibition zone
diametera diametera diametera
1 10 19
2 11 20
3 12 21
4 13 22
5 14 23
6 15 24
7 16 25
8 17 26
9 18 27
(b) +/- UV treated Streptomyces griseus or soil Streptomyces - plate plug colonies as shown in
diagram. Repeat twice - total of three Czapek plates.

Circle your group’s organism: Streptomyces griseus Streptomyces (soil)

isolate # inhibition zone isolate # inhibition zone isolate # inhibition zone

diametera diametera diametera
1 10 19
(control) (control) (control)
2 11 20
3 12 21
4 13 22
5 14 23
6 15 24
7 16 25
8 17 26
9 18 27
a
diameter includes plug, measure in mm (straw diameter ~6 mm equivalent to no inhibition zone)
 47

Lab Report (MUST BE TYPED, data sheet acceptable hand written)

Data Presentation and Analysis

1. Include a copy of your group’s data sheet as an appendix. Footnote all environmental
parameters.

2. Present a copy of class data. Present as table(s). Just add table #, title and footnotes to
printout from website. Footnote your data.

3. a) What ratio of new antibiotic producing isolates should be further evaluated? Criteria
>15 mm effective producer of antibiotic.

4. a) Is the Streptomyces soil isolate a more effective antibiotic producer than the stock
culture of Streptomyces griseus? Is this expected? Explain why/why not.

b) Did mutagenesis increase antibiotic production? How many new enhansed antibiotic
producing strains were isolated by the class?

5. Explain why E. coli was chosen as the host bacterium for antibiotic production assay.

6. What are the next experiments that should be done to characterize the effective producers
of antibiotic?

Questions

1. What advantage does manual screening have over automated screening?

2. In your lab, you concentrated on increased antibiotic improvement. What other feature(s)
of strain improve are important in antibiotic production?

3. List six possible mechanisms of increased antibiotic production via metabolic engineering.
 48

APPENDIX

HYDROMETER
A hydrometer measures the weight of a liquid in relation to water. All solutions are measured
against waer that has a SPECIFIC GRAVITY of 1.000 g/ml. As you add sugar or other soluble
solids, the numbers after the decimal point will increase, ie., 1.010 to 1.020 up to 1.100. Ethanol
has a specific gravity of 0.8 g/ml. To use the hydrometer put the liquid in glass cylinder. Spin
hydrometer to dislodge air bubbles. At eye level read the figures on the stem of the hydrometer
where the surface of the liquid cuts across the stem.

BEER BOTTLE CAPPER OPERATION

1. As illustrated in figure 4, bring lever to position 1 (unlocked). Lever must be in vertical
position.
2. Place the bottle cap (26 mm) against the magnetic support of the crimping cup (B).
3. Place the beer bottle on the stand.
4. Slide unit A down until bottle cap reaches bottle neck. (That is, crimping cup 4 to 5 mm
from from top of bottle neck.)
5. Depress the lever to position 3 (seals cap on bottle).
6. After sealing of each bottle, bring lever back to position 2, thus allowing the sliding unit
to be kept in locked position.
 49

HEMOCYTOMETER INSTRUCTIONS (estimation of spore count)

1. Each hemocytometer holds two samples, inlet either side. Just before using rinse slide and
cover in EtOH. Remove and off the hemocytometer and coverslip with a kimwipe.
2. Transfer ~10 :l of solution to V-shaped grove of the hemocytometer*, cover lengthwise
with cover slip and allow sample to settle for 1 minute then count. Place on the
microscope. Microscopically count spores in grids using 40x objective count, see figure.
Count spores in squares only. If cells overlap rows, make sure you do not read the same
cell twice. See figure below for diagram of centre part of ruled slide consisting of a 5 by 5
double lined grid. There is a smaller single line grid inside each double lined square but
is used only to help you count. Count the number of cells in rows 1, 2, 4 and 5 (omit row
3). Each row consists of 5 doubled lined squares. Using the formula (average of two
rows divided by 10 is the number of million cells per ml) an estimate of the spore number
can be determined. Interpreted as the total counts of rows 1, 2, 4 and 5 divided by 20 times
106. For example: row 1 = 22 spores, row 2 = 28 spores, row 4 = 24 spores and row 5 =
18 spores. The total is 92 spores. Therefore, 92/20 x 106 = 4.6 x 106 spores/ml.
3. After using hemocytometer, rinse slide and cover slip in ethanol. Dry.
 50

PIPETMAN OPERATION

In your lab, you have available three different pipetmen depending on the lab. If you look at the
top of the plunger it states the size of the pipetman. P20 measures accurately from 2 :l to 20 :l.
P200 measures accurately from 20 :l to 200 :l. P1000 measures accurately from 100 :l to 1000
:l. Never turn the pipetman above the maximum volume; 20 :l for P20, 200 :l for P200, and
1000 :l for P1000 as this breaks the pipetman. The scale on the pipettor is read different for each
type - refer to Figure 5 for an example of how to read the scale.

(Excerpted from Gilson pipetman operation manual.)

1. Setting the volume: The required volume is set on the digital volumeter by turning the
knurled adjustment ring (Figure 6-2A). When the volumetric setting is increased, it is
necessary to go about 1/3 of a turn above the desired setting and then come back to the
exact value. When the volumetric setting is decreased the desired value may be selected
directly. The volumeter display is read from top to bottom in :l for P20 and P200 and ml
for P1000 (Figure 6-2).
2. Place a disposable tip on the shaft of the Pipetman. Press on firmly with a slight twisting
motion to ensure an airtight seal. Depress the push-button to the first positive stop (Fig. 6-
3A). While holding the Pipetman vertical, immerse the tip 2-4 mm into the sample liquid.
Release the push-button slowly to draw up the sample (Fig. 6-3B). Wait 1 to 2 seconds,
then withdraw the tip from the sample.
3. To dispense the sample, place the tip end at a 10-45o angle against the inside wall of the
vessel and depress the push-button SMOOTHLY to the first stop (Fig 6-3C). Wait 1 to 2
seconds and then depress the push-button completely to expel any residual liquid (Fig. 6-
3D). With the push-button fully depressed, carefully withdraw the Pipetman, sliding the
tip along the inside wall of the tube. Release the push-button. Remove the used tip by
depressing the tip ejector button (Figure 6-1F).
 51

pipetman operation diagram

 52

SPECTRONIC 20D OPERATION

The spectronic 20D is a single beam spectrophotometer. The wavelength range is 340 nm to 600
nm with a nominal spectral slit width of 20 nm that is constant over the wavelength range. The
wavelength accuracy is 2.5 nm. The spectronic 20D is supplied with 1/2 inch test tubes.

SAMPLE MEASUREMENT: Absorbance

1. Remove dust cover. Turn on Power Switch clockwise. Allow the spectrophotometer to
warm up for 15 min.
2. Set the required wavelength with the Wavelength Control Knob.
3. Set the display mode to TRANSMISSION by pressing the MODE CONTROL KEY
until the LED beside TRANSMISSION is lit.
4. The sample compartment should be empty and closed. Adjust the display to 0.0%T with
the Zero Control Knob (same as power switch).
5. Fill a spec 20D 1/2 inch test tube with blank solution. The tube should be at least 1/2 full.
Wipe the test tube with tissue to ensure no liquid drops, dust or fingerprints. Place the test
tube in Sample Compartment and align the guide mark on the test tube with the guide
mark at the front of the sample compartment. Press test tube firmly into sample
compartment and close lid.
6. Press the MODE CONTROL KEY until the LED beside ABSORBANCE is lit. Adjust
the display to 0.0A with the Transmission/Absorbance Control Knob. Remove the test
tube from the sample compartment.
7. Put test tube containing sample(s) in Sample Compartment and close lid. Read
absorbance directly from display.
8. When all measurements are complete, turn off the spec 20D and replace dust cover.
Thoroughly rinse all spec 20D test tubes with distilled water, and place test tubes in spec
20D rack upside down. DO NOT PUT SPEC 20D TEST TUBES ON DISCARD
TROLLEY.

COMMENTS
1. Keep all solutions free of bubbles.
2. The display must be reset to 100%T or 0.0A every time the wavelength is changed.
 53

OPERATION OF FLOOR MODEL CENTRIFUGES

Note: If procedure varies depending on centrifuge manufacturer a step by step operation
procedure is usually located on or nearby the centrifuge or the teaching assistant will help you.

HITACHI HIGH SPEED HIMAC REFRIGERATED CENTRIFUGE

• to select or change settings the CHECK button must first be pressed (light on). The light
stays on for 16 sec. When the light is off you can no longer select, change setting or carry
out any operation, just press check button again and continue.
• When the centrifuge is turned on and the CHECK button is not pressed. The centrifuge
displays real time parameters.

OPERATION
Centrifuge tubes should be balanced by scale by adding or removing appropriate solution from
one of the tubes.

1. Turn power switch on. The indicators on the control panel are illuminated. The door lock
is released.
2. Open door. If required set the rotor gently in position and close door. Turn the rotor
lightly by hand to check that the rotor is correctly set. Remove the rotor lid and place
balanced tubes opposite each other in rotor. You cannot run the centrifuge with an odd
number of tubes. SCREW ON LID.
3. Call up memory code number or enter parameters.
Call up pre-programmed memory code number: Press CHECK button, MEMORY button,
memory code number, and CALL button. Each memory code number consists of a
specified set of operation parameter (see sheet on centrifuge cover). See below for a list of
operation parameters and how to set and store operation parameters.
OR
Real time operation (enter original parameters): see setting of operation parameters below.
4. After the parameters are set make sure the check light is still on. If not, press the CHECK
button.
5. Press the START button. The rotor starts running. The start lamp begins flashing. The
timer starts to count down.
6. The timer counts down to zero or press the STOP button. The rotor begins to decelerate.
The stop light begins flashing.
7. The rotor stops. The stop light stops flashing. A buzzer sound occurs. The door lock is
released.
8. Unscrew rotor lid and remove tubes. If required, use tweezers to help remove tubes.
Wipe out rotor if spills occur. DO NOT SCREW ON THE LID just place on top of the
rotor.
9. Close centrifuge lid and turn off power.

PARAMETERS
ROTORS NUMBER: SMALL (maximum volume 40 ml) RPR20-2 = ROTOR #7
LARGE (maximum volume 450 - 500 ml) RPR9-2 = ROTOR #13
 54

TEMPERATURE: 4 to 20 oC
SPEED: Rotor number 7 (small) - maximum speed 18,000 rpm
Rotor number 13 (large) - maximum speed 8,000 rpm
Example: for 3,520 rpm, press 3 . 5 2
TIME 0 to 99 min 59 sec, FREE
Example: for 5 min and 30 sec, press 5 . 3 0
ACCEL. Higher the value the faster the acceleration - 9 is good for basic
centrifugation.
DECEL. Higher the value the faster the deceleration - 7 is good for basic
centrifugation.
Example: for loose pellet or phase separation the deceleration number
should be decreased to 3.

SETTING OF OPERATION PARAMETERS

1. Press CHECK button, CHECK button lights up. Parameter for preceding operation are
displayed. The LED light goes off after 16 sec, it is only possible to set parameters with
the CHECK button light on. Press CHECK again if light goes out.
2. Press TEMP button. Position where setting is to be made will flash. Using the ten number
key pad, key in desired value for parameter setting. Data will be displayed in the above
setting position. Press SET button. The flashing stops and selected operation parameter is
set.
Note: if your require 4oC, set at 8oC as the centrifuge often goes below the requested
setting. Your sample may freeze if spinning for greater than 5 min at a high speed.
3. Repeat step 2 for each remaining parameter...SPEED, TIME, ROTOR NO., ACCEL., &
DECEL. Remember to press the CHECK button again if the CHECK light goes out.

STORING OF OPERATION PARAMETERS (Storing of operation parameters is optional.)

4. After setting desired parameters as above, press CHECK if light has gone out.
5. Press MEMORY, memory code number (one that is available) and the RECORD button.
There are nine available memory program code numbers. See chart on centrifuge lid for
memory programs that already exist.
 55

FINAL LAB EXAM: Industrial Microbiology 60.451

DATE: example PAGE: 1 of 2 TIME: 1.5 h
INSTRUCTOR: Dr. L. Cameron
Student Name: __________________ Student Number: _______________________

Briefly all questions on exam paper in space provided. (Spaces reduced for reference binder.)
WRITE EXAM IN PEN ONLY.

6 1. What is the function of each of the following components as used in the industrial
microbiology lab beer or wine preparation?

a) sodium bisulfite
b) gelatin
c) hydrometer
d) air-lock
e) bentonite
f) potassium metabisulphite and potassium sorbate

2 2. State function and/or principle of primary and secondary fermentation in beer making.

2 3. Wine making in your lab, unlike beer making, has an additional stage. Name stage. State
function of stage specifically relating function to components and procedure.

2 4. Outline how the liquid in your wine kit it prepared. What is the major difference between
white and red wine kit liquid preparation.

5. The following questions pertain to citric acid fermentation by Aspergillus niger performed
in your lab.
1 a) How is the Aspergillus niger spore suspension prepared? Explain the function of each
major step or solution.
1 b) The Aspergillus niger growth medium contains 30 mM sucrose. What dilutions must
be made to determine this initial sucrose concentration using a standard curve linear from
0 to 50 :g/ml sucrose. Sucrose determination assay is the same as your lab. sucrose F.W.
= 342
2 c) The following components are in the medium for citric acid fermentation by
Aspergillus niger (per liter):
2.6 g NH4NO3
2.6 g KH2PO4
0.26 g MgSO4.7H2O
8.8 g sucrose
final pH of 3.5
Outline complete protocol to prepare 100 ml of this medium for inoculation with
Aspergillus niger.
Note: you have available a microbiology lab with all necessary supplies and equipment
required.
 56

FINAL LAB EXAM: Industrial Microbiology 60.451

DATE: example PAGE: 2 of 2 TIME: 1.5 h
INSTRUCTOR: Dr. L. Cameron

2 d) When assaying for citric acid in a sample, 5 components are present in the cuvette in
addition to the citrate sample. List the 5 components and function of each with reference
to sample citrate determination.

1 e) Use the following citric acid determination data to calculate citric acid concentration.
Analyze data results.
sample Absorbance values at 340 nm of citric acid assay sample.
time
sample (zinc added to media) sample (no zinc added to media)
(hour)
sample 1 sample 2 sample 1 sample 2
initial final initial final initial final initial final
72 0.58 0.19 0.66 0.23 0.64 0.03 0.67 0.03
slope of standard curve = 40 mmole-1
sample volume analyzed = 1 ml

1 f) Explain why a sample should only be removed once from a small fermentation flask.

1 g) What environmental factors influence citrate production by Aspergillus niger?

1 h) Why is citric acid production widely used industrial product?

6. Briefly answer each of the following questions with reference to industrial microbe strain
improvement lab.

1 a) Why does the soil contain CaCO3 when preparing soil for Actinomycetes selection?

1 b) Outline procedure to quickly select antibiotic producing Actinomycetes. Do not include

experimental details

1 c) Use the following hemocytometer data to calculated the concentration (spores/ml) of

the Aspergillus suspension used to inoculate the medium.
Counts for row 1 = 24 spores, row 2 = 22 spores, row 4 = 30 spores and row 5 = 26
spores.

1 d) What are the next experiments that should be done to characterize the effective
producers of antibiotic?
__
26
-END-