vitamin c - the elixir of life? information and activities for post-16

Information and A,ctivities, related to the Health-care Industries, (on Post-I 6 Science~ourses

CHEMICALS FOR HEALTHY LIFEThis project is producing resources to support post-16 science courses.Each resource was developed initially by a teacher researching possiblestories and activities in an industrial company that makes products whichcontribute to health care. The resources provide information and guidancefor activities which relate to those undertaken by people who work in thehealth care industry. They are particularly suitable for GNVQand GSVQbut they are produced as photocopiable sheets so that they can be usedselectively to support context-related work in particular Advanced Level,Higher Grade and Certificate of Sixth Year Studies courses. They alsoprovide opportunities for students to engage in investigational project workand to produce evidence of their achievement in key skills.

The project has been sponsored by the followingorganisations:

Glaxo Wellcome plcHydro Polymers LtdSmith & Nephew Group Research CentrePfizer LtdSims Portex LtdSmithKline Becham plcAstraZenecaHoechst UKLtdRoche Products Ltd

Other published titles in the series are:

TABLET-COATING:not just a pretty colourStudents use microbiological, IR spectroscopic and titrimetric techniques tocarry out investigations which reflect the work of people who develop andmonitor the quality of coatings on medicinal tablets.(science, chemistry, biology, health studies)

EMERGENCY CARE:blood bags and tracheal tubesThis pack is concerned with the work of people who develop and test differentblends of PVC for use in medical equipment such as blood bags and tracheal tubes.(science, chemistry, physics, health studies, environmental issues)

11I1'1'11~~ll~II~~II~1N02450

CHEMICALS FOR HEALTHY LIFE

VITAMINC- the elixir of life?

Information and Activitiesfor Post-I6 Science Courses

AdvancedGNVQand GSVQ

Science

AS & A level,& Higher Grade

Biology and Chemistry

BTEC Project workNational Diploma Biology andHealth Science Chemistry

CHEMICALS FOR HEALTHY LIFE is a series of resources which usecontexts from the health-care industries to introduce post-16 students toscience concepts and to the work of people within these industries. Theresources are packaged in a form which enables teachers to select activitiesand supporting resources to suit their courses. The student task sheets inthe resources can be accessed on the internet so that they can be modifiedeasily to suit particular students.

This resource has been prepared by Gavin Cowley, Oaklands School, Yorkand John Lazonby, CIEC, and is based on research and development byBeverley Forsyth, Hull College, Bert Gilmore, Roche Products Limited, andby Bernard Betts and Andy Brown, Microbiology Research Unit, Universityof York.

AcknowledgementsThe development of this resource was funded by Roche Products Limitedand by the University of York.

The authors gratefully acknowledge the assistance and advice provided bythe staff of Roche, and in particular thank Andy Brown and Bernard Betts,Microbiology Research Unit, University of York, for their development workon the fermentation investigation and the staff and students ofNorthallerton College for trialling the activities.

We would like to thank the followingpeople who gave valuable help andadvice during the preparation of this resource:

Lesley GrattonAnn LawrenceMiranda Stephenson,

Northallerton CollegeQueen Margaret's School,EscrickChemical Industry Education Centre,University of YorkUniversity of YorkChemical Industry Education Centre,University of York

David Waddington,Alan Wingfield,

Photograph acknowledgementsCover John OlivePage 13, top left Kath James

middle left Flamingo Land Theme Park and ZooPage 15 Roche Products Limited

Design Barry Perks Graphic Design

Art work Valmai FirthClare WakeAndy Brown

© Chemical Industry Education Centre

The copyright holders waive the copyright on the material which follows tothe extent that teachers may reproduce this material for use with students,but for all other purposes, permission to reproduce this material in anyform must be obtained from the Chemical Industry Education Centre. Thematerial may not be duplicated for lending, hire or sale.

ISBN 1 85342 574 5

I

CHEMICALS FOR HEALTHY LIFEVITAMIN C - the elixir of life?

About These Resources(pink paper)

Teacher's Notes(pink paper)

Data Sheet(pink paper)

Technician's Notes(green paper)

Introducing Sheet (incorporating Tasks 1 & 2)(full-colour, 10 copies)

Task 3 Sheet(white paper)

Task 3 Support Sheet(white paper)

Task 3 Science Ideas and Techniques(white paper)







Task 6 Support Sheet(white paper)


Page1

CONTENTS OFTHE RESOURCES

Synthesis of vitamin C from glucose

Investigating the loss of vitamin Cduring cooking

Measuring vitamin C content of asolution

Measuring concentrations ofsolutions

Part 1- the ideas

5

8

9

13

17

18

19

21

Part 2- the techniques 23

Part 3- practising the calculations 27

Industrial fermentation

Industrial fermenters

Monitoring product formation duringthe development of a process

Colorimetry

Investigating an industrialfermentation process

Safe microbiological procedures

29

31

35

37

39

40

41


ABOUT THESERESOURCES

This resource pack is one of a series published by the Chemicals for Healthy Lifeproject to support post-16 science courses, particularly General Vocational Courses.

Introducing Sheet

Each pack provides the followingresources for students:

Ten copies of this full-colour, folded A3 sheet are provided.It describes the 'place of work' context and so justifies thestudent tasks. It also aims to illustrate how the particularindustry and its products are important to the students'lives and the lives of others.

Where appropriate each task is supported by the followingphotocopiable resourcesprinted on white paper:

Task Sheet

Task Support Sheet

Science Ideas andTechniques Sheets

This outlines the task and indicates which other resourcesthey may need to study. These sheets are available on theinternet, http://www.york.ac.uk/org/ciec. so they can beedited easily to cater for the needs of your students and toclarify or amend the specific evidence you wish them toproduce.

This can be used at the teacher's discretion wherestudents lack the experience to undertake the more open-ended task outlined on the Task Sheet.

These help students to research and develop a knowledgeof an experimental technique and/or an understanding ofthe underlying science. For some students these could beused as the starting point for their own research, forothers they can be used as support for class teaching ofthe topics. Some of these sheets will help studentsprepare for particular items on the unit test.

There are also resources for teachers and technicians:

Teacher's Notes(printed on pinkpaper)

Technician's Notes(printed on greenpaper)

These provide notes to help with curriculum planning, onsafety issues and on each task. Where appropriate,indications of possible outcomes of tasks and answers toproblems are given.

Additional gUidance is provided where significantpreparative work for tasks is needed.

How you could use these resourcesAdvanced General Vocational Courses

They are useful additions to the resources you have available to support yourprogramme of work for GNVQor GSVQ. The complete package can be kept in a ringbinder and you can select from the student resources those sheets which you wishthem to use and so create separate student files. The followingpages tdentify eachseparate student resource and indicate how each task contributes to the GNVQandKey Skills coverage and provides opportunities for students to produce requiredevidence.

AS level, A level, Higher Grade and Certificate of Sixth Year StudiesThe activities provide opportunities for students to relate the concepts andtechniques covered to industrial contexts. The contexts can also be used as thestarting points for investigations and project work.

Health ScienceThe Introducing Sheet can be used as the starting point for a selection of theactivities for students followingcourses such as the BTECNational Diploma.

1

http://www.york.ac.uk/org/ciec.

Student resources- each box represents a separate resource

IntroducingSheet

Tasks 1 and 2

2

Task 3 Task 3Science Ideas Science Ideas

and Techniques and Techniques

Measuring Measuringvitamin C concentrations

content / of solutions/

//

/

Task 3

Loss ofvitamin C

duringcooking

//

// Task 5 Task 5

Monitoring Science Ideasproduct - - - - and Techniques

formationColorimetry

/\

/ \

/ \

/ \

/ \

Safemicrobiological

procedures

Task 6Science Ideas

and TechniquesTask 6Task 4

InvestigatingIndustrial

fermentation

Industrialfermentation

Task 4Science Ideas

and Techniques

Industrialfermenters

GNVQcoverageIn this pack, teacher and student resources are provided for tasks related to the contextof the use of micro-organisms in the industrial production of vitamin C. The resourcessupport activities which would allow students to satisfy the performance criteria andproduce evidence indicators required for the Advanced GNVQin Science.

Performance criteria covered by each task.

Task/Perf. Criteria

Science Elements 1 2 3 4 5 61.1 Investigate health and safety practices in laboratories 3-5

1.2 Select analytical strategies 1-5 1-5

1.3 Carry out analyses and evaluate results 1-4 1-4

4.1 Investigate organisms as sources of useful products 1-5

4.2 Investigate the selection and breeding ot organisms forcommercial purposes

4.3 Investigate constraints on commercial production tromorganisms

Key Skills ElementsN 3.1 Collect and record data 1-7 1-7

U 3.2 Tackle problems 1-4,6 1-9

M 3.3 Interpret and present data 2-4 1,2 1-5 1-5

C 3.1 Take part in discussions 1-5

0 3.2 Produce written material 1-5

M 3.3 Use images 1-3

3.4 Read and respond to written materials

Science ideas and techniquesThe table below summarises the main science ideas and techniques which students are eitherassumed to know or expected to develop a knowledge and understanding of during the unit.

Assumed knowledge Knowledge developed or revised• basic microbiology, including • concentrations of solutions

the differences betweenbacteria and fungi • titration techniques

• the use of structural formulae • colorimetryto represent organic molecules

• aseptic techniques• oxidation and reduction

• microbial fermentation

• industrial fermenters

• continuous and batch processes

• scale-up of industrial processes

The activities provide opportunities for students to prepare for the test specifications ofUnit 1 (Focus 3-7). and Unit 4 (Focus 1-5).

3

GNVQScience evidence indicators arising from each task

I I I I I I v I~faiffof useful products from named I I I I I I·~ Irange of useful products from named I----+---+--+--+----+-"'~~organism '---_--& __ ....•...__ ...•...__ "--_---1.__ ....•

Elem Evidence indicators1.1 CASE STUDY safety in a student activity

NOTES on range of hazards(bio, chern & phys)

PRESENTATION on safety in the lab

1.2 PRESENTATION 2 analyses from 4 categories(environment, living, lab or industry)each analysis to include 6 points1 qualitative analysis

1 quantitative analysis

1.3 2 qualitative analyses to include 4points1 inorganic

4 lAB.REPORTS

1 organic

2 quantitative analyses to include 5points1 inorganic

1 organic - Task 3 - titrationTask 6 - colorimetry

4.1 SAMPLE organism grown by the student

NOTES reason for selecting organism andthe conditions

DESCRIPTION glucose produced by photosynthesis

process that produces a namedproduct by a plantprocess that produces a namedproduct by a micro-organism

LIST

4

TASK jOpportunities to

I I I I I I I


TEACHER'SNOTES

SafetyAlthough GNVQScience courses require students independently to plan many of theiractivities, the responsibility for safety in their practical work still lies with the teacher/tutorwho is in charge of the students. It is assumed that the following apply.

A risk assessment will exist for all practical activities. This will have been prepared bythe teacher/tutor, or by the student and checked by the teacher/tutor.

2 Plans produced by students for practical activities will be checked by the teacher/tutor.3 Practical work is conducted in a properly equipped and maintained laboratory.4 Any mains operated equipment is properly maintained. Any appliances brought in from

outside the school or college are safety checked using the employer's Portable ApplianceTesting programme before use.

5 Any fume cupboard operates at least to the standard of DES Design Note 29, FumeCupboards in Schools, (1982), HMSO.

6 Rules for student behaviour are strictly enforced.7 Eye protection is worn whenever there is a recognised risk to the eyes.8 Care is taken with normal laboratory operations such as heating substances and

handling heavy objects.9 Good laboratory practice is observed when chemicals, living organisms and materials of

living origin are handled.10 Students are taught techniques that minimise risks for such activities as pouring

chemicals, heating or smelling chemicals, and for handling micro-organisms.11 Fieldwork takes account of any guidelines produced by the employer.

Risk AssessmentsRisk assessments should be prepared using the General Risk Assessments in publisheddocuments modified as appropriate to suit local circumstances. The followingare importantsources of information:DfEE, (1996), Safety in Science Education, London: HMSOASE, (1996), Safeguards in the School Laboratory (lOth Edition), Hatfield: Association forScience EducationASE, (1988), Topics in Safety, Hatfield: Association for Science EducationCLEAPSSSchool Science Service, (1989), Hazcards, Uxbridge: Brunel UniversityCLEAPSSSchool Science Service, (1989), Laboratory Handbook. Uxbridge: Brunel UniversitySSERC, (1991), Preparing COSHH Risk Assessments for Project Work in Schools, Edinburgh:Scottish Schools Equipment Research CentreHMI. (1993), Microbiology: An HMI Guide for Schools and Further Education. London: HMSO

The DfEE (1996) publication provides guidance on safety and on the legal posttion, detailednotes on a wide range of specific hazards and an extensive bibliography. This publicationstresses the need for risk assessments to be part of the documentation in daily use ratherthan on forms which may "not be consulted regularly" and "are not likely to be modified whenan activity is changed".

GNVQstudents have to prepare risk assessments as part of the evidence indicators forElement 1.1 and this provides the opportunity to establish the practice of using riskassessments as working documents which are integral to each practical activity and whichneed to be modified as plans are modified. The student task sheets stress that they have toprepare risk assessments for their plans and have them checked by their teacher beforestarting practical work, and that if they modify their plan they should consider whether or notthe risk assessment needs modifying.

It seems likely that students will find this easier if they adopt a standard approach toassessing risk. One possibility, based on that used in the individual investigation in SaltersAdvanced Chemistry, Activities and Assessment Pack. Heinemann, is to work through thefollowing steps.

1 List all of the chemicals (approximate quantities and, when in solution, concentrations),items of equipment and operations to be used.

2 Identify and note the hazards associated with the chemicals, equipment and operations.

3 Note the precautions which they plan to take when using hazardous substances andequipment and when carrying out hazardous operations.

5

Introducing SheetThis sheet establishes the context for the activities. It emphasises the special significance ofvitamin C and the need for a daily intake. The story of vitamin C provides a case study ofresearch from the first observation of the effects of natural products through to thedevelopment of commercial methods for the production of the active ingredient, vitamin C.The debate about the Recommended Daily Allowance (RDA)for vitamin C is introduced,together with some of the wider uses of the chemical in the food industry. The commercialproduction of vitamin C provides the context for the use of a micro-organisms to produce auseful product, and so the tasks which followcan be seen as tackling problems which aretypical of those faced by people working in this industry.

It would be useful if students could read and discuss the sheet and do Task I, Lind'sinvestigation Task 2, Getting all that you need (the instructions for which are given on thesheet) in small groups. These tasks are designed to encourage students to interact with thematerial on the sheet and to provide opportunities to cover key skills.

For later tasks, students will need to be aware of the sorbitol to sorbose step in theproduction of vitamin C from glucose. A summary of the reactions in all steps is included onthe Data Sheet, page 8, at the end of these notes for your information. You may wish to makethis available to some groups of students.

Task 3 Determination of the vitamin C content lost into solution during the cooking ofpotatoesThe method of quantitative analysis for vitamin C involves titrating a solution of2,6-dichlorophenol-indophenol (DCPIP)with the solution of vitamin C. The end point isindicated by the disappearance of the blue colour of DCPIP. This method involves the use oftitration techniques. However, if students are told that 1 cm-' of a 10 mg% solution of DCPIPreacts with 0.1 mg of vitamin C then their calculation will not be based explicitly on anequation for a reaction. The Task 3 Science Ideas and Techniques Sheet - Measuring thevitamin C content of a solution provides background information on this particular technique.The answer to the example on the sheet is that the tablet contains 1010 mg of vitamin C.

If this is taken as an opportunity for students to develop or check their understanding of theprinctples and techniques of titrations then they will need to work through Task 3 ScienceIdeas and Techniques Sheet - Measuring concentrations of solutions, Part 1, 2 and 3.

Task 3 Support Sheet is available for students who require more guidance when planning thisinvestigation.

Task 4 Industrial fermentationThis task provides useful background information prior to students embarking on the maininvestigation on fermentation (Task 6). It should enable students to appreciate how thefermenter which they use is related to those used in industry. The task is supported by Task4 Science Ideas and Techniques Sheet - Industrialfermenters and it provides opportunities forstudents to cover some communication key skills.

Further information and activities concerned with industrial fermentation are available in theSalters' GCSE BiologyUnit, Industrial Microbes, available from:The Science Education Group,University of York,Heslington, York YOlO 5DD Tel 01904 432524

A computer simulation of the operation of an industrial fermenter is available from:Biochemical Engineering Education Scheme,Department of Biochemical Engineering,University College London,TOrrington Place,London WC1E 7JE Tel 020 7 419 3246

Task 5 Monitoring product formation during the development of a processThis uses the context of the development of an industrial process as a justification for usingcolorimetry to monitor the concentration of product formed. Task 5 Science Ideas andTechniques Sheet - Colorimetrij introduces the principles of the technique and the Task sheetrequires students to process some typical results. They will find that the concentration ofglucose obtained by carrying out the process under conditions A is 0.03 mol dm', whereaswith conditions B it is 0.005 mol dm-3.

,

I

1-

6

Task 5 provides essential background for their investigation of fermentation in Task 6 wherethey will monitor the production of sorbose by colorimetry. It also provides opportunities forthem to cover some key skills.

Task 6 Investigating an industrial fermentation processThis assignment requires students to use a variety of techniques, including microbiologyand colorimetry. Task 4 introduced students to fermentation and fermenters and Task 5to colorimetry. Task 6 Science Ideas and Techniques Sheets will prepare the students forthe use of safe microbiological techniques. It is advisable to demonstrate thesetechniques to the students and at the same time explain why the precautions arenecessary. The need for some background teaching is assumed.

The prior experience of Task 5 is assumed but if preferred the colorimetry could betackled either as a group or taught exercise. In either case, if a quantitative analysis is tobe made, a calibration curve of concentration of sorbose versus absorbance/transmissionis required. Dilution of the solution following treatment with Benedict's reagent will benecessary for colorimetry as the resulting solution is opaque. Qualitative results areacceptable but obviously less discriminating.

It is envisaged that the work will be challenging for students and may last for a period ofweeks if the work is carried out individually. If students were to plan independently andshare results with others the time scale could be compacted. Task 6 Support Sheet canbe made available to students who need more gutdance.It is important that plans are checked carefully so that they are safe and practical.

The agitation of the cultures is vital as the bacterial cells do settle quickly. The access tomagnetic stirrers is not essential as long as sufficient aeration is provided - this agitatesthe solution adequately. Temperature control is desirable, as it relates to real life, butagain it is not essential. Incubation at approximately 20°C will provide measurablequantities of sorbose within a 24 - 48 hour period. Difficulties with a temperature controlsystem should not deter students from undertaking the investigation. Details on thepreparation of bacterial cultures and suppliers are provided in the Technician's Notes.

CalibrationThe followingdata are included as an illustration. The data were obtained at a roomtemperature of 23°C, using a Griffin colorimeter (path length 1 cm). The red filter wasused to measure percentage transmission of the solution, with a blank made using a 1%yeast extract solution and Benedict's reagent. It is essential to make certain that theprecipitate is well suspended prior to the extraction of a sample. In this case a dilution of1 part in 50 of distilled water was necessary.

Note that with the quantities of sorbose solution and Benedict's suggested this techniqueis quantitative for the range 0 - 2% sorbose and above this concentration there wascontinued oxidation of the red Cu" to green Cu2+.

Concentration ofsorbose (%)

o0.20.40.60.81.01.21.41.61.82.0

Transmitance (%)

10092888381767469625652

The transmitancevalues representthe means of fivereadings, roundedto the nearest %.

Suggested supporting resourcesDavies, M., Austin, J. & Partridge, D. (1991), Vitamin C: its chemistry and biochemistry, Royal Society ofChemistryDenby, D. (1996), There's more to vitamin C than brussels sprouts. Chemistry Review, Vol 5, No 5,28-29Katz, J. & Sattelle, D. (1988), Biotechnology in Focus, Hobson ScientificLowrie, P. & Wells, S. (1994), Microbiology and biotechnology, Cambridge Modular ScienceTaylor, J. (1990), Micro-organisms and biotechnology, MacmillanLowrie, P. & Wells, S. (1991), Micro-organisms, biotechnology and disease, Cambridge University Press

7


SYNTHESIS OF VITAMIN C FROM GLUCOSE

DATA SHEET

Synthesis of vitamin C from glucose

CHO CH20HH OH H OH

HO H Step 1 HO H••••H OH

Aqueous solution of H OHH OH glucose is reduced to H OHI CH20H sorbitol by hydrogen CH20Hin presence of a nickel

catalyst.glucose sorbitol

Step 2

Sorbitol is convertedto sorbose bymicrobial oxidationin a series offermenters.

Step 4

The dipropanonesorbose is oxidisedby sodiumchlorate( 1)with anickel catalyst andthe product isrearranged in Step 5with acid catalystsin a mixture ofsolvents to formcrude ascorbic acid.

8

HO Step 3=0

H

In Step 4, one -CH20H isoxidised to -C02H. Butbefore that is done, theother four -OH groupsare protected fromoxidation by reactingthem with propanone inpresence of sulphuricacid. The product isdipropanone sorbose.

dipropanone sorbosesorbose

oStep 5

HO OH

dipropanone- keto-gulonic acid ascorbic acid


TECHNICIAN'S NOTES

Advance planningSome of the items required for the construction of the coffee jar fermentersneeded for Task 6 will not be part of usual stock. It is intended that thefermenters will be reusable so the initial outlay for equipment should not beseen as prohibitive as the cost per fermenter, at the time of writing, is actuallyvery little (less than £5.00 per unit). Possible sources of the equipment arelis ted below.

200 g coffee jars (Nescafe and Maxwell House are the most suitable)micro centrifuge tubes (Eppendorf, 1.5 em" volume - BDH)Suba-seals -(Sigma-Aldridge, number 20, 4.3 mm internal diameter, I.D., cat. Z12,470-2)Nalgene autoclavable tubing (LABjFDAjUSP VI Grade 0.25" I.D.) - Philip Harris)Nalgene autoclavable tubing (LABjFDAjUSP VI Grade 0.375" I.D.) - Philip Harris)Gilson pipetteman tips (5 cm-' - Philip Harris)sterile disposable syringe filter units (0.2 micron pore - Sartorius)19G hypodermic syringe needles (Philip Harris)sterile plastic syringes (Philip Harris)Araldite Rapid epoxy (widely available)Gluconobacter oxydans 8036 - available from either the National Culture of Industrialand Marine Bacteria Ltd., 23 St. Machar Drive, Aberdeen, AB24 3RY or Dr BernardBetts, Microbiology Research Unit, Department of Biology, University of York, York,YOlO 5DD.

Equipment and materials that may need to be acquired in advance:

Task 3 Investigating the loss of vitamin C during cooking

Sample preparationfresh raw potatoes100 cm-' of 10 mg% DCPIP solutionburettes1 dm-' of 1M ethanoic acid25 and 100 cm-' measuring cylinders250 cm-' conical flasksmortars and pestles or preferably a blenderdistilled water

Task 6 Investigating an industrial fermentation process

Construction of a coffee jar fermenterEquipment (per fermenter)

coffee jar (at least 200 g) plus lidAraldite Rapid epoxy (or similar)2 microcentrifuge tubes (Eppendorf) of 1.5 cm3 volume2 Suba -seals2' Nalgene autoclavable tubing (0.25" I.D.)2' Nalgene autoclavable tubing (0.375" I.D.)2 Gilson pipetteman tips (5 cm3 volume)non-absorbent cotton wool

9

I

I

1. __

Diagram of assembly

heat source

10

air out air in

'" V19G

Suba-sealseptum

aitoclavable PVCtubing

inoculation/additions port

sampling port

growth mediumcoffeejar

magnetic stirrer bar

magnetic stirrer

Instructions for assembly

Wash, dry and remove labels from coffee jar.

2 Make 4 circular holes in the lid (2 ~ 1 cm in diameter, 2 ~ 1.5 ern indiameter) using either scissors or a drill. The edges need to be filedsmooth. A third method, requiring the use of a fume cupboard, is to heat acork borer, one size down from that required, in a Bunsen flame and use itto melt a hole through the lid. If done carefully this method is best andremoves the need for filing. The Gilson pipetteman tips should fit snuglyinto the holes.

3 Remove the lids from the microcentrifuge tubes and remove a small part ofthe bottom of each tube and the Gilson tips. Slot each tube into itsrespective hole. The lip will stop the tube passing through the lid.

4 Insert a Suba-seal into the top of each microcentrifuge tube and fix inplace with Araldite.

5 Slide a 15 - 20 em length of 0.25" I.D. Nalgene tubing over the bottom ofone microcenrifuge tube. This will be the SAMPLING PORT. The other willbe the INOCULATION PORT.

6. Slide a 20 - 25 em length of 0.25" I.D. Nalgene tubing over the bottom ofone pipette tip (this tubing may need heating to fit). Snip the lowerportion of the Nalgene several times with scissors and fix a disc shapedpiece of Nalgene over the end with Araldite to block it. This port will bethe AIR IN PORT and the perforations in the tubing will make aeration moreeffective. The other pipette will be the AIR OUT PORT.

7. Fill both the AIR IN and AIR OUT PORTS with non absorbent cotton woolbut not too tightly!

8. Attach a length of 0.375" J.D. Nalgene to the top of each pipette tip. Theseshould be long enough to enable connection to a pump/vacuum tap foraeration purposes.

10. Add the medium (and a magnetic stirring bar if this is to be used) prior toautoclaving. Place a piece of cotton wool in each of the AIR IN/OUT PORTSfor autoclaving and cover the top half of the fermenter with foil.

9. Seal all joints with Araldite and allow to dry.

11. Autoclave immediately for 15 minutes at 121°C and 17 Ib/in2 .THE LID MUST BE LOOSENED FOR AUTOCLAVING.

12. Seal around the joint of the jar and lid with sticky tape.

The lids of Nescafe and Maxwell House seem to autoclave better than others.

11

Preparation of the micro-organism prior to experimentation

Gluconobacter oxydans 8036 is suggested for use in this experiment and is anADCP Class One organism. An alternative organism is Acetobacter pasteurianus11664.

Grow as streak plates on yeast sorbitol agar or yeast glucose agar by incubationat 30°C for 48 hours. Supply for students should be in a yeast sorbitol brothmedium, grown, with shaking, or aeration, at 30°C for 24-48 hours.

RecipesYeast sorbitol agar Yeast glucose agar

D-sorbitol (BDH)yeast extractagardistilled water

20.0 g10.0 g15.0 g1.0 drn-'

D-glucose (BDH)yeast extractagardistilled water

20.0 g10.0 g15.0 g1.0 dm-'

Yeast sorbitol medium

D-sorbitol (BDH)yeas t extractdistilled water

20.0 g10.0 g1.0 drn-'

Sample preparation for fermentation of sorbitol practical

Equipment and materials

coffeejar fermenters100 cm-' of bacterial culture (in yeast sorbitol medium)19G hypodermic needles1 cm-' syringes5 cm' syringescolorimeter·cuvettessyringe filtersD--sorbitolyeast extract medium (lg /100 cm3)balanceBenedict's reagentdistilled water250 cm-' volumetric flasks

All equipment should be sterile and sterile technique must be used inpreparation of media.

The first part of the practical may involve students making up solutions ofsorbitol for use in the fermenters, alternatively, stock solutions of differentconcentrations could be provided. The medium needs to be sterilised byautoclaving inside the fermenters.Make sure that the lids are loosened for this.

Following sterilisation the fermenters can be inoculated with the bacterialculture and then attached to vacuum pumps or a vacuum tap. It is likely thatthe connection of a number of fermenters in series will be necessary andtechnical assistance at this stage would be advisable.

For safety reasons the fermenters should be surrounded with a mesh jacket toprevent the shattering of glass should it break.

AT THE END OF THE EXPERIMENT THE CONTAMINATED JAR MUST BEAUTOCLAVED OR SUITABLY DISINFECTED. PARTS THAT HAVEDETERIORATED DURING THE AUTOCLAVING CAN EASILY BE REPLACEDAFTER STERILISATION.

12

CHE.MICALS FOR HE.ALTHY LIFE.

Nutrition InformationTypical Values % RDA Per tabletVitamin C 100 60mg





TASK SHEET

Task 3INVESTIGATING THE LOSS OF VITAMIN C DURING COOKING

An old fashioned treatment for acne was to make teenagers drink thewater from boiled vegetables! Often we find that these old remedies havesome basis in science. This particular cure may also fall into thiscategory. It is a common misconception that vitamin C is broken downby cooking. This is not true.

It is true that fruit and vegetables that are left for a long time before beingeaten suffer vitamin depletion but cooking simply extracts the vitamin C.The reason for this is that vitamin C is water soluble and boiling softensthe cells of the vegetable and causes the vitamin C to be released into thewater. So the vegetable water is high in vitamin C content and its use intreating acne may have links with a deficiency of vitamin C causingscurvy and its associated facial sores.

Your taskA manufacturer of baby food is concerned that one of its products whichcontains potatoes is losing a large amount of its vitamin C content in thecooking stage. They are keen to replenish any lost vitamins in order toaddress customer preferences. They have asked for advice from yourlaboratory on the quantity of vitamin C that needs to be added to therecipe, after the cooking stage, to restore the original vitamin content.

They have provided you with the following information:

• cooking time is 12 minutesimmersion in unsalted, boilingwater,

• 10 g of potatoes are includedin each jar of baby food.

• Design a method to investigate how much of the original vitamin Ccontent is lost in the cooking process. You will find the Task 3Science Ideas and Techniques Sheet helpful in your planning.

• You must produce a risk assessment for your plan. Do not startpractical work until you have had your plan and the riskassessment checked by your teacher. Each time you modifyyourplan, check to see if the modifications affect your risk assessment.

• Write a report on which recommendations for the addition ofvitamin C to the recipe can be based. Include a brief description ofyour test method and a justification of it in your report.

17


SUPPORT SHEET

Task 3SUGGESTED METHOD OF ESTIMATING VITAMIN CLOSS

You will find it useful to have read Task 3 Science Ideas and TechniquesSheet, Measuring the vitamin C content of a solution before carrying out thefollowingprocedure.

Cut the potatoes immediately before the experiment.

Method1. Weigh out 10 g of raw, peeled potato. Put it in a mortar with two

spatulas of washed sand and 25 cm-' of 1Methanoic acid solution.Grind the mixture for S minutes.

Alternatively, put 100 g of raw, peeled potato in a blender with250 cm' of 1Methanoic acid and blend the mixture thoroughly.

2. Filter the mixture through mineral wool, into either a 100 em"measuring cylinder or a volumetric flask.

3. Make up to 100 em" with distilled water, if necessary.

4. Fill a burette with the potato solution.

S. Put l cm' of DCPIP(10 mga/o)into a conical flask and place this on awhite tile beneath the burette.

6. Carry out a titration, followingthe instructions in the Science Ideasand Techniques Sheet, and record the volumes of potato solution usedto reduce the DCPIP.

7. Repeat this process using the water from boiling 10 g of potato. Use asuitable volume for the boiling and again make it up to 100 cm-'withdistilled water. Wait for the solution to cool before titration as thismay affect results.

8. Use your results to calculate the mass of vitamin C in the volume ofboiled water used in the titration and then in the total volume ofwater from cooking the potatoes. Report your conclusions.

18

L_-------- -,-

CHEMICALS FOR A HEALTHY LIFEVITAMIN C - the elixir of life?

SCIENCE IDEAS ANDTECHNIQUES

Task 3MEASURING THE VITAMIN C CONTENT OF A SOLUTION

Vitamin C (ascorbic acid) is easily oxidised to dehydroascorbic acid:

o

HO-C-H

o

HO OH o o

This reaction occurs naturally in air, catalysed by an enzyme within thecells of plants. The process is also accelerated by cooking in copper pans,due to the presence of traces of copper ions.

The reaction can be used as the basis of a method for quantitativelyanalysing the vitamin C content of a solution. To do this the solutionmust be reacted with an oxidising agent which reacts rapidly with thevitamin C and, through a colour change, provides a method of detectingwhen the reaction is complete. The blue dye, 2,6-dichlorophenol-indophenol (DCPIP for short), is a suitable oxidising agent.

The blue dye is reduced to a colourless compound by vitamin C and sothe point at which the complete reduction of a known quantity of DCPIPoccurs will be revealed by the disappearance of its blue colour.

A convenient way to do this is to use DCPIP solution with aconcentration of 10 mg per 100 cm-' distilled water. 1 em" of this solutionwill react with 0.1 mg of vitamin C.

A known volume of the DCPIP solution is placed in a conical flask andthen titrated with the vitamin C solution until the point at which theblue colour disappears. If you need to check your knowledge andunderstanding of the procedure for carrying out titrations, work throughthe Science Ideas and Techniques Sheet, Measwing the concentrations ofsolutions.

You could check your understanding of how to process the results of thistype of titration by working through the example on the following page.

19

Vitamin C tablets - quality controlA manufacturer of vitamin C tablets carriedout periodic checks of tablets to make surethat they did contain the stated minimumamount of vitamin C.

A vitamin C tablet which was labelled as a1000 mg tablet was crushed and dissolvedin distilled water and the solution furtherdiluted by distilled water to make 1000 em"of solution. This was further diluted bymaking 25 em" of the solution up to250 cm-' with distilled water.

This solution was titrated into 25 em" ofDCPIP solution (10 mg per 100 cm-'] in aconical flask until the blue colourdisappeared. One rough and three accuratetitrations were carried out. The results aregiven in the table:

Titration resultsBurette reading at 26.45 26.25 25.20 25.80finish /cm3

Burette reading at 1.05 1.00 0.00 0.50start /cm3

Volume added 25.40 25.25 25.20 25.30(titre) / ern"

Rough 1 2Average of accurate titrations

325.25cm3

Calculate the mass of vitamin C in the tablet.

20

Task 3MEASURING CONCENTRATIONS OF SOLTIONS (Part 1 - the ideas)The chemical reactions which occur in our bodies and help to keep usalive all occur in aqueous solution and so it is not surprising that theactive ingredients of most medicines are soluble in water. This meansthat in the pharmaceutical industries there is frequently a need tomeasure how much of a particular substance is dissolved in a certainvolume of solution (the concentration of a solution).

Units of concentrationConcentrations can be given as themass of dissolved substance per unitvolume of solution, for example, in theunits grams per litre (g 1-1).

CHEMICALS FOR HEALTHY LIFEVITAMIN C - the elixier of life?

(1 litre is 1000 cm-' or 1 cubicdecimetre and so g 1-1 is usuallyexpressed as g dm-').

Different conventions are used underdifferent circumstances so it isimportant that the units being usedare given.


On some medicine bottlesconcentrations of ingredients aregiven in mg per 5 ml spoonful.

It is frequently more useful for chemists to measureconcentration in moles per dm-'. where a mole is oneformula mass (in grams) of the substance. The followingexample, shows the difference between these two types ofunits.

If 80 g of sodium hydroxide are dissolved in water,and the solution made up to 1 dm-' with morewater, then the concentration of the solution is80 g dm".

graduation mark

The formula of sodium hydroxide is NaOH.The relative atomic masses of the elements presentare:

NaoH

Therefore the formula mass of NaOHTherefore the mass of 1 mole of NaOH

3Idm

23161

23 + 16 + 1 = 4040g

If a solution of sodium hydroxide contains 80 g dm",then it contains 2 mol dm".

The number of moles per cubic decimetre of a solution is sometimes calledthe molarity of the solution.

This solution of sodium hydroxide is a 2 molar solution which isabbreviated to a 2M solution.

Check that you are able to calculate the concentrations of solutionsusing both systems of units by completing questions 1-5 in Part 3 of thissheet.

21

Using molarities to measure theconcentrations of solutions

If you want to find the concentration of a particularsolution of sodium hydroxide,

and you have a solution of sulphuric acid of knownconcentration available,

you can start by finding out how much of the sulphuricacid solution reacts with a known volume of the sodiumhydroxide solution.

This is done by measuring out a particular volume of thesodium hydroxide solution, say 25 em". Then slowly addthe sulphuric acid solution and record how much of ityou have added when it has reacted with all of thesodium hydroxide. NaOH

You can tell when this point has been reached (called the endpoint) by having a few drops of an indicator present which changes colourwhen the reaction is complete. This process is called a titration.

If you found that you needed exactly 20 em" of the 0.1M sulphurtc acid to reactwith all of the sodium hydroxide, then you can use these data to calculate theconcentration of the sodium hydroxide solution. To do this you need to knowthe balanced chemical equation for the reaction, which is:

2NaOH(aq) + H2S04(aq) -7 Na2S04(aq) + 2H20(l)

From this you can see that 2 moles of NaOH react with 1 mole of H2S04,You then need to calculate the number of moles of H2S04 used.1000 cm-' of O.lM H2S04 contains 0.1 moles of H2S04

20 ern- of O.lM H2S04 contains 0.1 x 201000

0.002 moles of H2S04

From the balanced equation you knowthat this would react with 0.004 moles of NaOH

This number of moles of NaOH must have been present in the 25 cm-' of solution used.Therefore 1 dm-' of this solution would contain 0.004 x 1000

250.16 moles of NaOH

The number of moles in 1 dm ' is the molarity of the solution, so this is a 0.16M solution.1 mole of NaOH is 40 g and so 0.16 moles 40 x 0.16

6.4 g

The concentration of the solution of sodium hydroxide is 6.4 g dm-'.

Questions 6 and 7 in Part 3 of this sheet provide other examples of titration results.You can use them to practise using these type of data.

When should you use a titration?You can select titration as a suitable quantitative analytical technique tomeasure the concentration of a substance dissolved in water if the followingconditions are met:

• there is another soluble substance that it will react with• the reaction between the substances is rapid• there is some means of detecting when the reaction is complete• you know the balanced chemical equation which represents the reaction

22



Task 3MEASURING CONCENTRATIONS OF SOLUTIONS (Part 2 - thetechniques)Sometimes it will be necessary for you to make a solution of knownconcentration to use in a titration to find the concentration of anothersolution. A solution whose concentration is known accurately is called astandard solution. Sometimes you will be provided with a standardsolution which has been purchased from a supplier or made up for you.Ifyou make your own you have to followa set procedure so that you do itas accurately as possible.

A standard flask is used to allow the volume of thesolution to be measured accurately. Standard flasks aresometimes called volumetric flasks. When filled correctlyto the graduation mark on the neck of the flask, theycontain the stated volume when the solution is at thetemperature written on the flask.

graduation mark

Making a standard solutionThe followingstages are involved in making up astandard solution: 250 em3

20°Cclass B

• decide on the volume of solution you need andselect a suitable volumetric flask,

• work out the mass of solid you will have to dissolve in this volume togive a solution of the required concentration,

• weigh the solid accurately,

• dissolve the solid in a small volume of the solvent (usually distilledwater, but it can be an aqueous solution such as dilute sulphuricacid),

• transfer the solution to the volumetric flask and then add furthersolvent to make the solution up to the final volume.

Weighing out the solidAccurate weighing is carried out with the solid in a weighing bottle (noton a piece of paper). Weigh the bottle empty and then containing thesolid. Usually it is not necessary to weigh out the exact mass you havecalculated provided the mass is close and you record it accurately so thatyou can calculate the final concentration of the solution.

Making up the solution1 Take a clean beaker and rinse it with distilled water. Tip the solid

from the weighing bottle into the beaker. Rinse out the weighingbottle two or three times with distilled water, transferring thewashings to the beaker each time. It is important that all the solidgoes into the beaker.

2 Add enough distilled water to the beaker to dissolve the solidcompletely. Do not fill the beaker more than half full. The dissolvingmay take some time so be patient. It helps to stir but take care not tolose any of the solution by splashing. Remember to wash thesolution off the stirring rod into the beaker.

23

3 Wash out the volumetric flask a couple of times with distilled water.Pour the solution from the beaker into the flask through a glassfunnel. Wash down the walls of the beaker with a small quantity ofwater and pour the washings into the flask. Repeat this several timesand then rinse the funnel into the flask. Remove the funnel.

graduation mark

250 cm20°Cclass B

4 Using a measuring cylinder and then a wash bottle, carefully adddistilled water until the solution is 1 em below the graduation mark.Nowadd water slowly from a clean dropping pipette until the bottomof the meniscus is just touching the graduation mark. When you aredoing this you must have your eye level with the graduation mark.

meniscus

5 Stopper the flask and invert it several times to mix the contentsthoroughly. Immediately label the flask with the name andconcentration of the solution.

24

Carrying out a titration

What is a titration?The process of adding one solution to a fixed volume of another solutionuntil the reaction is just complete is known as a titration. The volumeof solution added is measured accurately.

As explained in Part 1 if the concentration of one solution is knownaccurately, you can use the balanced equation for the reaction to workout the concentration of the other solution.

• burette and stand• small funnel for filling the burette• white tile• pipette• pipette filler• conical flasks (usually 250 cm-')• wash bottle and distilled water• small beaker for waste solution

Titration apparatusYou must work carefully and measure volumes accurately to obtainreproducible and precise results in a titration. All glassware must beclean and you must use distilled water to make up the solutions. Belowis a list of the basic titration apparatus.

A pipette is designed todeliver a fixed volume ofIiquld.

A burette is designed todeliver a measurable butvariable volume of liquid.

Preparing the burette1 Fill the burette with distilled water and check carefully for leaks. If the

burette leaks, check that the tap is clean and pushed well into thesocket. If the tap is the type which needs greasing, clean it and regrease.Discard the distilled water.

2 Using a small funnel, add 5-10 cm-' of the prepared solution to theburette. Remove the funnel. Take the burette from the stand and tip androtate it to wash the inside surface with the solution. Run out thesolution into the waste beaker.

3 Clamp the burette vertically and, using the funnel, fill the burette withthe prepared solution so that the meniscus is above the zero mark. Placethe waste beaker under the burette and open the tap until the solutionfills the jet, making sure there are no air bubbles. Remove the funnel.

4 Carefully run out the solution until the bottom of the meniscus is onzero (or just below). The burette is now ready for use.

25

Pipetting out solution into the conical flasks5 Take a pipette of appropriate size (25 ern" is commonly used) and rinse it

with distilled water. Using a pipette filler, draw up a few ern" of theprepared solution. Remove the filler. Tip and rotate the pipette to washthe inside surface with the solution. Then discard the solution into thewaste beaker.

7 Replace the filler and draw up the solution into the pipette until themeniscus is 1-2 cm above the graduation mark. Now very carefully, withyour eye on level with the graduation mark and keeping the pipettevertical, allow the liquld level to fall slowly until the bottom of the meniscusis on the line.

8 Run the liquid into a clean conical flask keeping the pipette vertical. (Theflask need not be dry; it may be wet with distilled water.) When themovement of the meniscus stops, there will be a small quantity ofsolution left in the tip of the pipette. Dip the tip of the pipette below thesurface of the Iiquid in the flask for 3 sec. Do not tap or blowout theremaining liquid.

9 Repeat the process to transfer equal portions of solution into three moreconical flasks. If an indicator is being used, add the required quantity.

Performing a rough titration10 Write down the burette reading to the nearest 0.05 ern", To do this

accurately your eye must be level with the meniscus. Place the conicalflask containing the solution on a white tile under the burette and run inthe solution from the burette 1 ern" at a time until, on shaking, thecolour changes permanently. Your left hand should operate the tap whileyour right hand shakes the flask. At the end point, note the new burettereading. Subtract your initial reading from this value to work out thevolume of solution added. This is known as the titre. This first valuewill only be approximate, so do not include it when you work out theaverage value.

Performing an accurate titration11 Refill the burette if necessary. Repeat the titration with a fresh portion of

solution in the second conical flask. As the rough end point isapproached, add a drop at a time, shaking the flask after each addition,until one drop causes the colour to change. (Near the end point rinsedown any splashes on the sides of the flask with distilled water.)

12 Repeat the the titration two or three more times until you have two titreswithin 0.1 ern" of each other as shown in the sample results.

Use of a standard solution of sodium carbonate to determine theconcentration of a solution of hydrochloric acid

Titrationresullts

Rough 2 3

Pipette solution: 25 em" of0.0502 mol drrr ' Na2C03Indicator: methyl orange(3 drops)Burette solution: hydrochloricacid of unkown concentration

Burette reading at 23.40 22.95 45.80 23.10finish fcm3

Burette reading at 0.10 0.00 22.95 0.20start /cm''Volume added 23.30 22.95 22.85 22.90(titre) f cm '

Average volume added from burette = 22.9 ern" (average of titrcs 1, 2 and 3)

Use this average volume and the balanced equation for the reaction,

Na2C03(aq) + 2HCI(aq) ~ 2NaCI(aq) + CO2(g) + H20(l)

to calculate the concentration of the hydrochloric acid in mol dm>. Use themethod given in Part 1 of this sheet.

26

I



Task 3MEASURING THE CONCENTRATONS OF SOLUTIONS (Part 3 - practisingthe calculations)

1. How much sodium carbonate. Na2C03. would you need to weigh out tomake 1 dm-' of O.lM sodium carbonate solution?

2. How much sodium carbonate, Na2C03, would you need to weigh out tomake 250 em" of a solution with a concentration of 0.2 mol dm-'?

3. If you had prepared a standard solution of sodium carbonate whichcontained 2.12 g of Na2C03 in 250 cm-' of solution, what would themolarity of this solution be?

4 Ifyou transferred 25 ern" of iron(II) sulphate solution, with aconcentration of 0.12 mol dm", to a conical flask, how many moles ofiron(H) sulphate would the flask contain?

5. If you found that 25 ern" of a solution of sodium chloride contained0.002 moles. what would the concentration of this solution be in mol drn"?

6 A sample of sodium carbonate was known to be contaminated withsodium chloride. You are given the task of analysing the mixture tofind the percentage purity of the sample of sodium carbonate. Sodiumcarbonate solution can be titrated with hydrochloric acid, whereas thesodium chloride solution does not react with hydrochloric acid. Theequation for the reaction between sodium carbonate and hydrochloricacid is:

Na2C03(aq) + 2HCI(aq) ~ 2NaCl(aq) + H20(1) + CO2(g)

You weighed out 1.25 g of the mixture and dissolved it in distilledwater to make 250 cm-' of solution.

You then titrated 25 ern" portions of this solution with 0.105Mhydrochloric acid, using methyl orange as indicator. The average valueof your accurate titrations was 21.2 ern". Calculate the mass ofsodium carbonate in 1.25 g of the mixture and hence the percentagepurity of the sample.

7. Dilute hydrogen peroxide solution can be used as an antiseptic,however, solutions of it gradually deteriorate as it decomposes to waterand oxygen. The concentration of a hydrogen peroxide, H202, solutioncan be determined by titrating it with potassium manganate(VII).KMn04' solution. The hydrogen peroxide solution is acidtfled, prior totitration, by adding dilute sulphuric acid. This is called a redoxtitration because the potassium manganate(VII) is reduced and thehydrogen peroxide is oxidised. The ionic equation for the reaction is:

2Mn04 "[aq] + 5H202(aq)+ 6H+(aq) ~ 2Mn2+(aq)+ 8H20(I)+ 502(g)

It is a complicated equation, but the only information you need from itis that 5 moles of hydrogen peroxide react with 2 moles of potassiummanganate(VII) .

You are given the task of finding the concentration of a particularhydrogen peroxide solution which you suspect has deteriorated.25 crn-' of hydrogen peroxide solution, after acidifying with dilutesulplruric acid, reacted completely with 22.5 cm-' of 0.02M potassiummanganate(VII) solution. Calculate the concentration of the hydrogenperoxide solution in mol drrr-' and then in g drrr".

27

28

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TASK SHEET

Task 4INDUSTRIAL FERMENTATION

Scientists working on the research and development of biochemicalprocesses first demonstrate that a product, which is potentially ofcommercial value, can be obtained on a laboratory scale by a particularsynthetic route.

Usually the next stage involves production of the substance on a pilotscale which is larger than laboratory scale but still much smaller thanthe production scale. This would typically be between 40 and 2000litres. The first step is to develop a theoretical model of the productionscale process and the scale it down to pilot scale for testing. The pilotplant should model as closely as possible what the final production plantis expected to be like.

You are part of the development team that is working on the design of aproduction plant for the conversion of sorbitol to a sugar called sorboseby a fermentation process. The conversion of sorbitol to sorbose is animportant step in the production of ascorbic acid (vitamin C). Thedevelopment of an efficient fermenter would make a significantcontribution to the profitability of a process for the manufacture ofvitamin C.

In this activity you will learn about the process of fermentation and thedesign of industrial fermenters. You will find the Task 4 Science Ideasand Techniques Sheet a useful starting point for your research.

Your TaskMuch of the development work for fermentation processes involvesmicrobiologists. These are speclalist scientists who study microbes, theirbiochemistry and the conditions which favour their growth. The resultsof their research have to be passed on to other people, who consider theirsignificance and make decisions about investment in new plant.Therefore, the microbiologists are required to be good communicators aswell as good scientists. In some instances this will mean they have toexplain their work to non-scientists.

You have been asked to explain how a fermenter works to a managementteam (ofnon-scientists).

• Find out about industrial fermenters and produce a poster thatcould be used to explain how a fermenter for the conversion ofsorbitol to sorbose would work.

• Include a list of factors that affect the rate at which the bacteriagrow and other factors which will contribute to the profitability ofthe process and to the production of a safe product.

• Indicate on your poster how the design of the fermenter will enableeach of the factors to be controlled.

29

30

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----------- --- - - -~----


Task 4INDUSTRIAL FERMENTERSWhat is fermentation?Most people think of the production ofbeer or wine when they hear the wordfennentation. During the production ofwines and beers, sugars are convertedinto alcohol (ethanol) and carbondioxide. Yeast, a cellular fungus,metabolises (uses in chemicalprocesses) sugars and, underanaerobic conditions (in the absenceof oxygen) converts them into ethanoland carbon dioxide. For example,

glucose ethanol

Originally the term fermentation wasused solely for this process.

Nowadays the term is used for anyprocess involving the production ofsubstances by micro-organisms,whether in the presence or absence ofoxygen.


In home Wine-making, some yeast ismixed with a solution oj grape juice in asterilised container and kept in a warmplace. The container is fitted. with abubble trap which allows carbon dioxideto escape but does not allow oxygen orunwanted micro-organisms to enter thecontainer.

Industrial fermentation processesBacteria and yeasts are used in many, commercially important, industrialprocesses to make products such as the enzymes used in washingpowders, antibiotics and the hormone insulin as well as ethanol.As outlined in the Iniroduciriq Sheet, an example of such a process is themanufacture of vitamin C. One step in the multistage process ofconverting glucose into vitamin C is the oxidation of sorbitol to the sugar,sorbose. This is a fermentation process which uses a particular type ofbacteria. It is important example of where the conversion of one chemicalto another can be brought about more effectivelyby a living micro-organism than by conventional chemical reactions.

HO-C-H

IH-C-OH

IH-C-OH

ICH20H

sorbitol

fermentation

CH20H

IC= 0

IHO-C-H

IH-C-OH

IHO-C-H

ICH20H

sorbose

31

The Jennenters Jor brewing beer used to be opentanks and the progress oj the fermentation wasmonitored by taking out samples oj the liquidand measuring its specific gravity whichchanges as the alcohol content increases.

The design of fermentersThe home wine-making kit shown on the previouspage is called a fermenter or bioreactor.

In a modern brewery theJermenters are largestainless steel cylinders.These bioreactors aredosed containers and theconditions under which thefermentation is carried outare monitored andadjusted electronically. Inthe fermenter in thediagram the controlinstruments and the inputand outputs are all locatedinside the building. Thebulk of the bioreactorsstructure protrudes abovethe rooJ oj the building.

Fermentation in industry is done on a huge scale. One fennenter canhold up to 100 000 litres of medium. Even the pilot plants, which areused in development work, hold around 50 litres. The model fennenterthat you may use in Task 6 is simply a scaled down version of the realthing and it works on exactly the same principles.

The industrial fermentation process for converting sorbitol to sorbose,unlike home wine-making, is a continuous rather than a batch process.In a continuous process, starting materials are fed continually into thereactor and the product mixture is removed continually. When decidingwhether to use a continuous or batch process the factors which need tobe considered include the following.

Continuous process• the reaction must occur at a reasonable rate• suitable for high tonnage production• may be uneconomical if run below full capacity• the initial cost of building the plant will be high• the process is more easily automated and controlled• using the equipment for a single process reduces risk of contamination• labour costs are low

Batch process• can be used for slow reactions• suitable for small tonnage production• equipment costs are low and it can be used for different processes• no product is being produced during the emptying, cleaning and refilling stages• contamination is more likely• labour costs are relatively high

From what you have learned so far about the sorbitol-sorbose reaction,what factors are likely to have influenced the decision to use acontinuous process for this stage in the manufacture of vitamin C?Find out whether continuous or batch processes are used in beerproduction and what factors have influenced the choice of process.

32

I-

I

Common features of fermentersAll fermentation processes, whether done on production scale, pilot scaleor laboratory scale require facilities to

• add and remove material from the fermenter,

• monitor and adjust the conditions in the fermenter.

It must be possible to carry out these operations without introducing any microbialcontamination from the equipment, the material being added, the atmosphere or thepeople operating the equipment. The necessary operations include:

• sterilisation ofequipment

• input of the medium

• inoculation

• sterile aeration

• temperature control

• pH control

• agitation

• removal of product

The fennenter must be made from a material which iseasy to sterilise, so most industrial fermenters are madefrom stainless steel.

The medium is the sterile solution of the reactant, inthis case sorbitol, and any other nutrients (food)required by the micro-organism. In a continuousindustrial process this must be added continually at acontrollable rate.

A small quantity of the culture (the mixture containingthe micro-organism) is added through a port (a self-sealing entrance).

For aerobic fermentations, such as the sorbitol tosorbose process, a continuous flowof sterile air oroxygen is passed into the mixture in the fermenter.

The rate of growth of micro-organisms is temperaturedependent and the optimum rate will occur over a fairlynarrow temperature range. If the temperature is too lowthe rate will be too low and if it is too high the micro-organisms will be destroyed. It must be possible tomonitor the temperature and to adjust it by heating orcooling the mixture. In industry these processes aredone automatically.

The rate of growth of many micro-organisms isinfluenced by pH and so it is often necessary to monitorand adjust pH during the process. In industry this isdone automatically.

Where the process uses oxygen one of the problems issupplying enough to the micro-organism. The efficiencyof the absorption of oxygen is improved if the mixture iscontinually mixed. This is usually done by stirring withpaddles mounted on a rotating central shaft. Over-vigorous stirring can damage the micro-organism. Theamount of gas being absorbed by the micro-organismcan be measured by monitoring the flowof gas into andout of the bioreactor.

In the industrial process there has to be a mechanismfor the continual removal of product. In both batch andcontinuous processes there may be a need to have aport through which samples can be removed for testing.

When you use the model fennenter in Task 6, check which of the aboveoperations can be carried out with your fermenter.

33

Scaling up afermentation processIn Task 6 you have to scale down the fermentation process so that youcan carry it out in your laboratory. In industry, when a new fermentationprocess is being developed, chemical engineers have to scale up theprocess which has been developed first on a laboratory scale and then ona pilot scale by microbiologists. This is a very important stage in thedevelopment, as building the new fermentation plant for large scaleproduction will require a large financial investment and yet its predictedperformance has to be based on projections from the much smaller pilotscale project.

Factors influencing scale upThe starting materials used in the laboratory process are likely to havebeen very pure chemicals, purchased from suppliers in small quantities.For the production-scale process it must be possible to purchase largequantities of starting materials of appropriate purity,on a regular basis and at an appropriate cost.

The chosen route must be thoroughly researched so that yields andreaction times are known for a wide range of reaction conditions. Theaim is to select a robust process which will perform adequately even whenthe conditions vary slightly.

The equipment used in the production scale process is likely to be to becylindrical in shape rather than spherical as used in the laboratory. Itwill also be made of stainless steel or steel lined with glass. Thesedifferences will influence the addition of reactants and the rate oftransfer of energy when heating or cooling reactants.

For exarnple, if the temperature is controlled by a jacket around thecylinder through which coolant passes, then the critical factor is the ratioof surface area of the cylinder in contact with the jacket to the volume ofreactants. As the reactors become larger, this ratio becomes smaller therate of cooling decreases.

The yield of a chemical reaction isthe percentage of the reactantwhich is converted into theproduct. On a laboratory scaleyou would normally aim for this tobe as high as possible. On aproduction scale, the benefits ofan increased yield have to bebalanced against the increase intime to obtain this yield.

C/

-Yield

\\\

\

//

I./1 - + Cost

./

./

Time~

The maximum yield is obtained at time C, but theoptimum yield from a cost point of view is at timeB.

Suggest explanations for the changes in costbetween times A and B, and between times Band C.

The most cost effectivemethod of converting sorbitol to sorbose on aproduction scale is to operate a series of four continuous fermenters.Sorbose crystals are then extracted by means of a series of crystalltsattonsof the sorbose broth from the fourth fermenter.

34


TASK SHEET

Task 5MONITORING PRODUCT FORMATION DURING THEDEVELOPMENT OF A PROCESS

As you will have realised in completing Task 4, the control of theconditions for fermentation processes are aimed at maximising theprofitability of the process. Any increase in yield needs to be consideredagainst any increase in costs arising from changing the conditions. Forexample, changing conditions may change the energy costs. In order tomeasure the effect of a change the product needs to be monitored in someway which can provide quantitative data. One such technique is calledcolorimetry. You will find it useful to read Task 5 Science Ideas andTechniques Sheet before continuing with this task.

One industrial process that uses fermentation and can be monitored bycolorimetry is the production of glucose from potato starch. Enzymes arefirst extracted from the bacteria and then added to the starch mixture toconvert it to glucose. The conditions for this are critical and changes inconditions will affect the yield. It is important to be able to monitor theproduct formation. Glucose is a colourless compound but when reactedwith a reagent called Benedict's reagent, and heated, it forms an orangesubstance which can be quantitatively monitored by colorimetry.

The colorimeter was used to determine the percentage transmission forsolutions of glucose of known concentrations after reacting withBenedict's reagent. These results are given in the table below.

Concentration of % transmissionglucose (mol dm -3)

1.0 90.1 290.01 380.001 610.0001 75

Product mixture % transmissionfrom

conditions A 35conditions B 47

The enzyme reaction on potato starch was then carried out under twodifferent pH conditions (A and B) and the same amount of Benedict'sreagent added to the product in each case. The percentagetransmissions for the product mixture for conditions A and B are given inthe next table.

Your taskPresent the standard data on a suitable graph and use this calibrationcurve to determine the concentrations of product in the solutions fromconditions A and B. Recommend which conditions, A or B, wouldtherefore be the better to use in this process.

35

36

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Task 5COLORIMETRY

Colorimetry is a very sensitive technique which can be used to determinethe concentrations of solutions by comparison with standards of knownconcentration. It can be used for any coloured solution or for turbid(cloudy)suspensions such as a suspension of yeast cells.

The scientific principle on which it is based is a simple one. It is that theintensity of the colour of a solution is directly related to theconcentration of the solution. So the more concentrated the solutionthe darker in colour it becomes. If light is passed through a colouredsolution, some of it willbe blocked (absorbed) and more light is absorbedwhen the solution is more concentrated.

The amount of light emerging from the solution (transmission) or theamount absorbed (absorbance) can be measured. Solutions of the samecompound, under the same conditions and of the same concentrationwill absorb light to precisely the same extent.

absorbance ex: concentration of coloured solution

Why are substances coloured?Sun light, the visible part of the electromagnetic spectrum, is a mixture ofall of the colours of the rainbow and each of these colours corresponds toparticular wavelengths. When sunlight strikes an opaque object, if all ofthe different wavelengths of light which make up the light are reflected,the object will appear white. When an object appears coloured it meansthat wavelengths of light corresponding to certain colours are absorbedby the object, and the light which is reflected by the object and entersyour eye willbe made up of the remaining colours.

For example, if the substance which is the product of the reaction you areinterested in appears orange when observed in white light, what colour oflight should you shine through the substance to monitor how itsconcentration is changing? It appears orange because the blue part ofwhite light has been absorbed by the substance and so the light reflectedinto your eye is made up of the remaining components of white light andappears orange. This means blue light willbe most sensitive to changesin concentration of an orange substance. So if you were analysing anorange coloured solution you would use a beam of blue light. The easiestway to obtain blue light is to use a blue filter, that is a piece oftransparent material which, as light passes through it, absorbs all thecolours except blue.

Blue is the complementary colour of orangebecause if blue is absorbed the objectappears orange and if orange is absorbed theobject appears blue. The complementarycolours are often shown in the form of acolour wheel. Complementary colours areopposite each other. This wheel helps you topredict what would be the most efficientcolour of light to use to monitor theconcentration of a coloured substance.

37

A colorimeterThe key components of a colorimeter are shown in the simplified diagram.

An Internal lightsource: produces abeam which is shonethrough the sample tobe analysed.

A coloured filter:chosen so that thewavelength of lightreaching the sample isthat which is mostsensitive to changes inconcentration of thecoloured substance.

It is contained within alight-proof box: tomake sure that noextemallight is enteringthe apparatus. Thiswould interfere withreadings.

A meter whichdisplays either thefraction of lighttransmitted orabsorbed.

A photocell whichconverts thetransmitted light intoan electric current.

A cell holder: holds thetube which contains thesample, usually a 1 emwide glass or plastic tube.

Light which has passedthrough the solution(transmitted light).

The current generated in the photocell will depend on the amount oflight transmitted through the solution. This in turn will depend onthe concentration of the coloured substance in the solution. Thecurrent will be greatest when the concentration of the colouredsubstance is lowest. Some meters show this as either the fraction ofthe light transmitted or absorbed, although strictly it is theabsorbance which is proportional to the concentration of thecoloured substance.

Using a colorimeterAfter selecting an appropriate filter, it isnecessary to obtain a calibration curve foryour colorimeter. A series of solutions ofknown concentration are prepared andthe fraction of the light either absorbed ortransmitted is measured for each solution.A graph can then be plotted of eitherpercentage absorbed or transmitted(y axis) against concentration of thecoloured substance (x axis).

100

transmission (%)

concentration

If the % absorbed or transmitted by asolution of unknown concentration ismeasured then it is possible to use thegraph to find its concentration.

• Why is it important that all thesolutions are placed in the same oridentical tubes before measuring theirabsorbance?

38


TASK SHEET

Task 6INVESTIGATING AN INDUSTRIAL FERMENTATION PROCESS

The microbiology team has the responsibility for establishing the mostfavourable conditions for the maximum production of sorbose fromsorbitol, one of the steps in the production of vitamin C (see theIntroducing Sheet). Your team has found that 30°C is the optimumtemperature for the bacterium to grow. The growth medium that youhave developed is a simple one, consisting of a 1% yeast extract solutionplus sorbitol. It is desirable to make the process as cheap as possible.

Your task• Devise a series of experiments which, using a laboratory scale

fermenter, will allow you to investigate how the concentration of theraw material (sorbitol) used influences the concentration of theproduct (sorbose) formed. You have been asked by the productionteam to find out if there is a concentration of sorbitol above whichthere is a reduction in the yield of sorbose.

• You must prepare a risk assessment for your plan. Do not startpractical work until this has been checked by your teacher.If you need to modify your plan, check if the modifications affectyour risk assessment.

• Prepare a report whichidentifies how the operations you are able to carry out with yourfermenter compare to those used in industrial fermentation (seeTask 4 Science Ideas and Tehcniques Sheet, Industrialfermenters),outlines your recommendations for the best concentration ofsorbitol to use and describes how you analysed your results andcame to your conclusions.

Steps in the fermentation process:• Sterilisation of equipment by steam or chemical means.

• Production of medium (1% yeast extract solution plus up to 5%sorbitol).

• Inoculation of the sterile medium with bacterial culture (1 cm ' per200 em" of medium).

• Incubation of culture (optimum temperature 30°C) for up to 48hours.

• Extraction and analysis of product from the fermenter.

To investigate the effect of different concentrations of sorbitol on theproduction of sorbose you will need to use the standard microbiologicaltechniques outlined on the Task 6 Science Ideas Sheet, Safemicrobiological procedures.

In addition you will need to have completed Task 5 and have a clearunderstanding of the use of colorimetry as an analytical technique whichis appropriate for monitoring the concentrations of a sorbose solutionafter it has reacted with Benedict's reagent.

Before starting this task, check the evidence that you need to produceand make sure that you keep this in mind as you plan and carry out thetask, and when preparing your report.

39

CHEMICALS FOR A HEALTHY LIFEVITAMIN C - the elixir of life?

Task 6INVESTIGATING AN INDUSTRIAL FERMENTATION PROCESS

SUPPORT SHEET

Using a micro-organism to convert sorbitol to sorbose

Equipment and materials needed:

a coffeejar fermenteryeast extract brothbacterial culture in yeast sorbitol brothsterile syringes and 19G needlessterile syringe filtersbench lamp (60W)D-sorbitolBenedict's reagentaccess to a vacuum tap and a colorimeter

Each one oj the experiments in theJollowing series will take 2 separateperiods oj practical work, 24 hours apart, to complete.

Use aseptic technique throughout.

1. Make up the yeast extract broth as instructed by your teacher(250 em:' will be enough for one experiment).

2. Choose concentrations of sorbitol between 0 - 5% to investigateand work out how much you need to add to your 250 cm' ofbroth. Make up your solution by dissolving the sorbitol in thebroth.

3. Add your broth to your fermenter and ask your teacher toautoclave it. The lid must be loosened for autoclaving.Autodaving may have to be done ovemight and so be sure to buildthis into your plan .

4. When your nutrient is sterile you can inoculate it with thebacterial culture. You need to aseptically remove 1 cm-' of theculture and inject it into your broth using a sterile needle.

5 Provide a flow of air through the broth by connecting yourfermenter to a vacuum tap or pump. Place a bench lamp25-30 cm away from your culture. Incubate for 24 hours.

6. After incubation, remove a sample of your culture with a sterilesyringe and needle, connect a syringe filter to the syringe andfilter to remove the bacterial cells.

7. Take a 2 cm' sample of the filtrate and test for reducing sugarsas in Task 5. Use colorimetry to determine the concentration ofsorbose in the sample. Repeat with additional samples if required.

8. Record your results from the series of experiments and describehow changes in the concentration of sorbitol influences theproduction of sorbose by fermentation.

40



Task 6SAFE MICROBIOLOGICAL PROCEDURES

What is involved in microbiological investigations?Many investigations involve culturing (growing under controlledconditions) micro-organisms. This usually involves the followingbasicsteps:

1 Preparing a sterile (freefrom micro-organisms)substance (medium) forthe micro-organism togrow on. The mediummust provide thenutrients needed by themicro-organism.

~~1 ....···· . ······.1

2 Transferring the micro-organism to the mediumand putting a lid on thedish.

3 Leaving the dish at theappropriate temperature(incubating) so that themicro-organism will grow.

4 Observing and recordingwhat growth hasoccurred.

41

Why take precautions?Microbiologicalaerosols are the mainsource of risk. These are tiny droplets ofwater with micro-organisms, such asbacteria, in them. The droplets are releasedinto the air while you perform normallaboratory operations. Even if the water inthe droplets evaporates, the suspendedmaterial in them is so small that it willremain airborne for some time.

Pathogenic micro-organisms are harmfulmicro-organisms. Although micro-organisms that you use in your experimentsare considered safe for school use, youcannot be sure that pathogenic strains willnot develop or that there will not be anypathogenic contamination. It is safer toregard all micro-organisms as potentiallypathogenic and take necessary precautionsto protect yourself and your colleagues.

Micro-organisms are present all around us - in the air, on our hands andon the surface of objects. If you are doing an experiment involvingparticular micro-organisms, then your experiment will become invalid ifthere is contamination by other micro-organisms. You have to usetechniques which avoid such contamination. These are called aseptictechniques. Aseptic means free from germs.

You have to take precautions to protect;

yourself

42

and your experiment.

Protecting yourself and your colleaguesWhen culturing micro-organisms it is usually safer to do this on a solidmedium (agar) rather than in a liquid (broth). This avoids spills andaerosol formation, but whichever type of medium is used the followingprecautions should be taken.

1 Wear clean protective clothing and eye protection.

2 Wash your hands before and after practical work.

3 Make sure cuts and scratches are covered with plasters.

4 Wipe the top of your bench with disinfectant before and after eachpractical session.

5 Never put anything in your mouth. This means you should not licklabels, chew pens, pencils or fingers and always use teat-pipettes fortransferring material. Never pipette by mouth.

6 Report any spillages of microbiological material to your supervisor whowill know how to deal with it.

7 Make sure any instruments used for transferring micro-organisms aresterilised before and after use - see the next section on aseptictechniques.

8 When incubating cultures thelids of Petri dishes should besecured with self-adhesive tape,but not completely sealed. Theyshould not be opened forexamination.

9 All used cultures and glasswareshould be moved to anappropriate place after use priorto autoclaving by yourtechnician or teacher. Anautoclave is like a large pressurecooker into which the equipmentcan be placed and heated to ahigh temperature so that all themicro-organisms are destroyed.

10 Micro-organisms should never be isolated from potentially dangeroussources such as human fluids, body surfaces, drains or sinks.

43

Protecting your experiment by using aseptic techniquesThe purpose of these techniques is to avoid contamination by micro-organisms fromthe air or by putting sterile equipment in contact with non-sterile surfaces such asyour hands, clothes or the bench.

(If you have not used a technique before, your teacher will demonstrate it to you)

1 The medium must be autoclaved after preparation. This will usuallybe done for you so that you will be provided with sterile medium.

2 Containers need to be openedwhen pouring the nutrientmedium from a bottle and whensamples of micro-organisms aretransferred from a container tothe medium.

Containers should be opened forthe minimum time and in thecase of Petri dishes by tilting thelid away from the body ratherthan by lifting it off.

The screw-cap of a containershould be unscrewed as shownin the diagram and kept in thehand - not put on the bench.

The neck of the container shouldthen be sterilised by flaming(turning briefly in the flame of aBunsen burner) this kills anymicro-organisms living aroundthe neck.

The sample can then be removed.The neck should then be flamedagain and the cap replaced,taking care not to touch the hotneck of the container.

3 Instruments used for transferring micro-organisms should be sterilised before andafter use.

If it is a metal loop (inoculator) itshould be held in a Bunsen flame.

If it is a glass teat-pipette itshould be placed in disinfectantor autoclaved.

Other sources of informationHMI, (1993), MicrobiologyAn HMIguidefor schools andfurther education,London: HMSODfEE, (1996), Safety in Science Education, London: HMSOASE, (1996), Safeguards in the School Science Laboratory, Hatfield:Association for Science Education

44

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