CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF SIWES

The student industrial work experience scheme (SIWES) is an appreciable skill training

programme which form part of the minimum academic standard in Nigerian universities. The

scheme is a participatory programme involving universities, polytechnics, and technical colleges

students of various institution in Nigeria.

SIWES was established by ITF in 1973 to solve the problem of lack of adequate practical

skill by Nigerian graduates of tertiary institutions.

The Scheme exposes students to industry based skills necessary for a smooth transition

from the classroom to the world of work. It affords students of tertiary institutions the

opportunity of being familiarized and exposed to the needed experience in handling machinery

and equipment which are usually not available in the educational institutions.

One of the primary goals of the SIWES is to help students integrate leadership

development into the experiential learning process. Students are expected to learn and develop

basic non-profit leadership skills through a mentoring relationship with innovative non-profit

leaders.

By integrating leadership development activities into the Industrial Training experience,

we hope to encourage students to actively engage in non-profit management as a professional

career objective. However, the effectiveness of the SIWES experience will have varying

outcomes based upon the individual student, the work assignment, and the supervisor/mentor

requirements. It is vital that each internship position description includes specific, written

learning objectives to ensure leadership skill development is incorporated.

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 Participation in SIWES has become a necessary pre-condition for the award of Diploma

and Degree certificates in specific disciplines in most institutions of higher learning in the

country, in accordance with the education policy of government.

Operators - The ITF, the coordinating agencies (NUC, NCCE, NBTE), employers of labour and

the institutions. Funding - The Federal Government of Nigeria Beneficiaries -Undergraduate

students of the following: Agriculture, Engineering, Technology, Environmental, Science,

Education, Medical Science and Pure and Applied Sciences.

Duration - Four months for Polytechnics and Colleges of Education, and Six months for the

Universities.

1.2 OBJECTIVE OF SIWES

Specifically, the objectives of the student industrial work Experience scheme are to:

1. To provide an avenue for students in institutions of higher learning to acquire industrial

skills and experience in their various course of study.

2. To provide students with an opportunity to apply their knowledge in real work and actual

practice.

3. To make the transition from school to the world of work easier.

4. To enhance students contacts for later job placement.

5. To ascertain if the students who participated were really attached to organizations related

to their field of study.

6. To identify the specific organizations that the students under study were mostly attached.

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7. To establish the extent, the knowledge acquired during the SIWES programme was

relevant to their course of study.

8. To identify the limitations to the actualization of the objectives of SIWES programme.

9. To enlist and strengthen employer’s involvement in the entire educational process of

preparing university graduates for employment in the industry.

10. To bridge the gap between the theory and practice underwent by students in various

course of study.

11. To expose students to work methods and techniques in handling equipment and

machinery that may not be available in their various institutions.

12. To prepare students for the work situation they may meet after graduation.

13. To enable students, access their interests and sustainability for chosen professions.

14. To strengthen employers involvement in the whole education process of preparing

university graduates for employments in the industries.

15. Provide students the opportunity to see the real world of their discipline, apply their

theoretical knowledge and consequently bridge the gap between the classroom and the

real work situation

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 CHAPTER TWO

FEDERAL INSTUTUTE OF INDUSTRIAL RESEARCH OSHODI (FIIRO)

DESCRIPTION OF THE ESTABLISHMENT OF ATTACHMENT

2.1 LOCATION AND BRIEF HISTORY OF THE ESTABLISHMENT

The Federal Institute of Industrial Research Oshodi (FIIRO) is a parastatal under the

agency of the Federal Ministry of Science and Technology. FIIRO was the idea of an economic

mission sent to Nigeria in 1953 by the World Bank. The mission’s observation was that

industrial research activities in Nigeria were diffused and uncoordinated with no definite

direction. Consequently, a decision was reached to set the Institute in 1956.

2.2 VISION STATEMENT

To be the foremost Centre of Science and Technology based research and development

for the industrialization and socio-economic advancement of the nation.

2.3 MISSION STATEMENT

To conduct and promote market-driven research and development for the

industrialization and socio-economic development of the country.

2.4 REVIEWED MANDATE OF FIIRO

 Research and development of food and agro – allied processing technologies.

 Research and development into pulp and paper processing.

 Research and development into packaging and product design.

 Design and fabrication of equipment prototype.

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2.4.1 OBJECTIVES / MANDATES OF ESTABLISHMENT

 To engage in research and development up to pilot plant stage.

 To conserve Nigeria’s foreign exchange earnings by reducing dependence on foreign

goods through the developments of local substitutes from locally available raw materials.

 To improve the nutritional qualities of Nigerian foods and their suitability for industrial

processing.

 To improve the indigenous, traditional techniques of food production which are labour-

intensive, time consuming and energy snapping.

 To engage in the design and fabrication of machinery and equipment, development of

foundry processes and materials, casting of irons and aluminum production and

machining of spare parts.

 Processing of ceramic materials and other solid based minerals for industrial use,

including development of ceramic glass and mineral technology.

 To engage in technology, transfer to the public through licensing training courses and

capital good acquisition. To also offer consultancy services to individuals, public and

private institutes in Nigeria and overseas.

 To undertake the preparation, publication and dissemination of useful technical

information to industries and researchers.

2.4.2 LOCATION

The institute is located at FIIRO road, off Agege Motor road, Cappa Bus Stop, Oshodi

Lagos Nigeria.

5
2.5 VARIOUS DEPARTMENTS/UNITS IN THE ESTABLISHMENT AND
THEIR FUNCTIONS

Fig. 1: (FIIRO) Organization Flowchart

The Institute operates through seven departments and each department is made up of

divisions, sections and units. The departments are:

 Administration, Finance and Environmental Technology

 Biotechnology

 Chemical, Fibre and Environmental Technology

 Planning, Technology Transfer, and Information Management

 Project Development and Design

 Food technology

 Production, Analytical and Laboratory Management

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 The functions of the Research Departments are as follows:

1. Biotechnology Department

Specializes in the use of microorganisms or part of microorganisms to improve and produce

foods, beverages and chemicals such as enzymes, wines, alcohol, organic acids and glucose

syrup. Attention is paid to the utilization of industrial and domestic wastes to produce goods and

services. The divisions under biotechnology includes

 Enzyme Technology

 Waste Management and Fermentation

 Molecular biology and Genetics

 Food and Quality Control Service

Here I was posted and assisted in the production of different enzymes and cultivation of

edible mushroom at the following Divisions:

2. Chemical, Fiber and Environmental Department

Research is conducted in chemical and fibre science with the following objectives:

 to provide chemical raw materials for industries

 to develop new technology for environmental pollution abatement

 to investigate and develop industrial processing of locally sourced plant fibres

 to provide technical assistance to local industries

3. Project Development and Design

This division engages in the design and fabrication of machinery and equipment. It also

engages in the development of foundry processes and materials, precise analysis of Metallurgical

materials, casting of iron and aluminium, and machining of spare parts.

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4. Food and Analytical Services Department
The Food Technological Division conducts research into preservation and processing of

indigenous agricultural produce up to the pilot plant stages with the aim of reducing post-harvest

losses and upgrading traditional technological for food processing and preservation. The division

is also involved in the development of intermediate products for utilization of food and allied

industries.

5. The Analytical Service Division

This Division handles chemical, microbiological and toxicological analysis of raw

materials, semi-finished products as well as finished products.

6. Planning, Technology Transfer, and Information Management

This department comprises of Planning and Monitoring; Technology Marketing and

Extension; and Library, Documentation, and computer Services.

 The Library and Documentation Sections offers complete and tailored information and

services to industries, entrepreneurs, researchers in their fields of interest and area of

endeavours.

 The Technological Marketing and Extension Division brings FIIRO’s achievements to

the public through training courses, exhibitions, trade fairs and visits to small-scale

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 CHAPTER THREE

DESCRIPTION OF ACTUAL WORK DONE IN BIOTECHNOLOGY DEPARTMENT

3.1 MICROBIAL PRODUCTION OF ENZYME

Enzyme are biological molecules made up of proteins that act as catalyst and help

complex reactions occur everywhere in life. Virtually all reactions in the body are mediated by

enzymes. Enzymes are biochemical catalysts produced by all living things. The most

distinguishing property of an enzyme in its catalytic nature is its specificity and selectivity.

Enzymes catalyze only a specific reaction involving a specific substrate hence their great value

for industrial purpose. Enzyme molecules contain a special pocket or cleft called the active site.

The active site contains amino acid side chains that participate in substrate binding and catalysis.

The substrate binds the enzyme, forming an enzyme–substrate (ES) complex. Another major

characteristic of enzymes is their sensitivity to the conditions in which they operate; they are

functional only within a specific range of temperature, pH and presence of co-factors, inhibitors

etc. A very useful property of enzymes as catalysts is that they are generally required in very

small or minimal quantities.

Different enzymes are being produced by various microorganisms including fungi,

bacteria, yeast and they can be regarded as important product obtained for human needs through

their microbial source. The major advantage of using microorganisms for the production of

enzymes is that microorganisms grows at a very rapid rate after they have been cultured under

specific conditions (a nutrient medium, regulated temperature) and this will in turn speed up the

production of the enzyme, that bulk production is economical and microorganisms are easy to

manipulate to obtain enzyme with desired characteristics. They can be subjected to strain

improvement, mutations and other such changes by which the production of enzymes can be

9
optimized. Industrial applications of enzymes have increased and has helped a lot of industrial

sectors such as textile industries, detergent industries, food and paper industries. The different

uses enzymes include saccharification of starch, production of beverages like beer, treatment of

digestive disorders and production of cheese from milk and removal of stains.

Steps involved in the microbial production of enzyme

 Sub culturing of the microorganism to be used

 Sterilization of equipment and materials

 Preparation of Mineral Medium

 Preparation of substrate

 Introduction of inoculums into the substrate

 Fermentation process

 Crude enzyme extraction

 Crude enzyme assay

 Crude enzyme purification

3.2 CELLULASE ENZYME

Cellulase is one of the most useful enzymes in industries. Cellulase is usually produced

by fungi, bacteria, actinomycetes, but fungi are widely reported to be the most common

producers. This could be as a result of the central roles of fungi in biogeochemical cycles with

emphasis in carbon cycle. Bacteria, which has high growth rate as compared to fungi has good

potential to be used in cellulase production. The application of bacteria in producing cellulase is

not as commonly used as the use of fungi in cellulase production. The greatest potential

importance is the ease with which bacteria can be genetically engineered. This is needed

especially in order to enhance cellulase production. The story for fungal cellulase is bit different,

10
with a lot of advantages to bacterial cellulase bothering around less technical know-how in

production, generation of large amount of cellulase, and use of cost effective machineries in

production of cellulase from fungi.

Celluloses are regarded as the most important renewable resource for bioconversion, and

many cellulosic substances have been successfully converted to simple sugars for production of

single Cell Protein, sweeteners etc. This enzyme family is grouped into endolytic (endo-1, 4-b-

glucanase, EC 3.2.1.4) and exolytic (cellobiohydrolase, EC 3.2.1.91) enzymes. Complete

depolymerization of cellulose to glucose requires another sort of cello-oligosaccharide-degrading

enzyme, 1, 4-b-glucosidase.

Microbial cellulases play important roles in fermentation processes to produce alcoholic

beverages including beers and wines. These cellulases have the ability to improve both quality

and yields of the fermented products. The production of fruit and vegetable juices requires

improved methods for extraction, clarification, and stabilization. Cellulases also have an

important application as a part of macerating enzyme complex (cellulases, xylanases, and

pectinases) used for extraction and clarification of fruit and vegetable juices to increase the yield

of juices. The cellulase in this case acts as a macerating agent/enzyme.

Pre-treatment of livestock feed with cellulose enzyme also eliminates anti-nutritional

factors present in the feed base. Cellulase can effectively hydrolyse the anti-nutritional factor,

cellulose, in the feed materials, thereby nutrients more available to the animal.

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3.2.1 METHOD USED IN MICROBIAL PRODUCTION OF CELLULASE

1. PRE-TREATMENT OF BREWER’S SPENT GRAIN (BSG)

The Brewer’s Spent Grain that was used was obtained from Nigeria Brewery as a waste

product. The BSG served as a substrate for the micro- organism in cellulase enzyme production.

a) Hydrolysis of BSG Using 35% of 1N HCL

The substrate (50g) BSG was pretreated with 15ml of 35% 1N HCl and autoclaved for

30minutes at 121°C. The pretreated sample was washed with sodium acetate buffer at pH 4.6,

Oven dried at 40°C for 72 hours and reweighed. The experiment was carried out under sterile

condition and in duplicate with control.

b) Pretreatment of Brewer’s Spent Grain Using 10% of 1N NaOH

The BSG was meshed and 50g was weighed into two different 500ml of beaker. The

samples were mixed thoroughly with 10% of 1N NaOH and transferred into muslin bags. The

samples were autoclaved for 30 minutes at 121°C and washed with sodium acetate buffer at pH

4.6.The pretreated BSG’s were oven dried at 40°C for 72 hours and reweighed.

c) Preparation of Mineral salt for Cellulase Production

Mineral salt medium was compounded to give the micro- organism the required nutrient

for their growth.

KH2PO4 3g

MgSO4.7H2O 1g

CaCl2.H2O 0.5g

ZnSO4.7H2O 1.6g

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FeSO4.7H2O 0.05g

CoCl2.H2O 0.5g

Distilled water 1L

10% of Cellulose was introduced into the pretreated BSG as well as mineral salt.

3.2.2 ISOLATION AND SCREENING FOR MICRO-ORGANISMS

THE POTENTIAL FOR PRODUCING CELLULASE

 Soil samples were collected from two different locations which are rich in cellulose

waste, which was the paper mill.

 1g of soil was transferred to 10ml of distilled water inside a test tube.

 Serial dilution was carried out to 10-6 and 0.1ml of the soil suspension was spread on the

already sterilized Potato dextrose agar(PDA) for fungal isolates, while Nutrient agar was

used for bacterial isolates(1% CMC was added to the medium before sterilizing).

 The cultured medium was incubated at 28°C for 7 days

 The cultures grown was sub-cultured to get a pure culture

 Pure culture was transferred on to the potato dextrose agar and nutrient agar slants for

fungal and bacterial isolates respectively

a) Screening of microorganisms

 Microbial isolates were individually inoculated on CMC-agar plates and incubated for 2

days.

 The plates were flooded with0.1% Congo red for 20 min and washed with 1 M NaCl for

15min.

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 The clear zone formed by the isolates indicates that the microorganism is viable to

produce cellulase enzyme.

b) Extraction of Cellulase

 In the extraction of enzymes there are two types of fermentation processes which are;

solid state fermentation and sub-merged fermentation.

 For this extraction process solid state fermentation was employed.

 The fermentation process took place for five (5) days under normal room temperature (i.e

26oC).

 3000ml citrate buffer (containing citric acid and sodium acetate) was prepared and was

adjusted to pH 6.5

 When it was time for the extraction (i.e the fifth day), the already prepared buffer

solution was pour inside a bowl the substrate was brought out and was added into it.

 The mixture was left for 30minutes so as to homogenise.

 A clean muslin cloth was placed on top of a container and the mixture was poured

through it so as to get the liquid crude enzyme.

 The enzyme was stored inside a freezer.

c) Cellulase Assay

Steps involved in cellulase assay;

 0.01M of citrate buffer was prepared using trisodium citrate and citric acid. The pH was

adjusted to 5.

 1% cellulose was prepared

 1ml of substrate (Cellulose) was added into 3 different test tubes.

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 One of the test tubes was labelled blank.

 Inside the same test tubes 1ml of the enzyme (Cellulase) was pipetted inside it.

 For the blank 1ml of distilled water was added into it.

 Afterwards they were incubated at 50oC for 30minutes.

 0.5ml of Dintrosalicyclic acid (DNS) was added to the test tubes including the blank.

 The solution were boiled at 100oC for 10minutes using water bath.

 The absorbance reading was taken using spectrophotometer.

d) Protein determination of cellulase

 6 different test tubes were washed and dried using incubator.

 The test tubes were divided into two, i.e. 3 for the enzyme and the other 3 for blank.

 0.2ml of the enzyme was added into the test tubes.

 0.8ml of distilled water was then added into the test tubes.

 0.5ml of alkaline solution was added also into the test tubes.

 It was allowed to stand for 10minutes.

 After 10minutes 0.5ml of folin C solution

 After which the solution was placed in a dark room for 30minutes.

 The absorbance reading was taken at 600nm.

3.3 TANNASE ENZYME

Tannase enzyme catalyses the hydrolysis of gallic acid esters and hydrolysable tannins.

Tannase enzyme is produced by plants and microorganism and it is industrially used as catalysts

in the manufacturing of gallic acid. It belongs to the family of hydrolases, specifically those

acting on carboxylic ester bonds, with E.C number of EC 3.1.1.20. Its systematic name is Tannin

15
acylhydrolase. Tannase is a key enzyme in the degradation of gallotannins, a type of

hydrolysable tannase which is present in a diverse group of microorganisms including rumen

bacteria.

The main commercial application of tannase at present are in the preparation of

instantaneous tea, action on tea polyphenols, used in the production of gallic acid, beer chill

proofing and wine making, Tannase can also be used to reduce tannin levels wherever desirable

such as fruit juice.

3.3.1 MICROBIAL PRODUCTION OF TANNASE USING SOLID STATE

FERMENTATION

Mainly when using solid state fermentation process a fungi is involved in the

fermentation process. The fungi used in this case is Aspergillus niger.

 Sub-culturing of Aspergillus niger

Sub-culturing of the micro-organism was done from already existing slant which ws used

to preserve the specie of the organism in other to get pure strains of the organism and a 24hr old

organisms.

 The work bench was sterilized using 70% ethanol and cotton wool

 10g of potato dextrose agar was weighed and poured into a clean beaker

 150ml of distilled water was added to it and then homogenized using a magnetic stirrer.

The mixture was then sterilized using the autoclave at 1210c for 15mins

 The solution was the poured into Petridis and allowed to solidify, which was then oven

dried at 400c

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  An inoculating loop was flamed until it turned hot red and was allowed to cool before

using it to pick some colony from the slant and placed on the newly prepared media

 The media was then incubated at 300c for 48hrs.

 Sterilization of equipment and material

Muslin clothes were washed, and dried with hot air oven for 2hours.The fermentating

tray was washed and dried and then placed in a hot air oven with the surface covered with foil

paper at 1000C for 2hrs.

 Screening of microorganism
A fungal strain was isolated using tannase screening agar medium containing;

 1% (w/v) Tannic acid 3g

 NaNO3 0.9g

 KH2 PO4 0.3g

 MgSO4 0.15g

 KCl 0.15g

 FeSO4 0.003g

 Agar Agar 9g

The reagents were all mixed together inside 300ml distilled water and the spores was

cultured inside the medium for 24hrs. A clear zone on the plate indicates that the microorganism

is viable to produce tannase.

 Preparation of Mineral water

The mineral medium was prepared and added together to give the microorganism the

required nutrient.

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  CaCl2 0.3g
 (NH4)2SO4 0.9g
 KH2PO4 0.3g
 MgSO4 1.5g

After which 300ml of distilled water was added and mixed together.

 Preparation of Substrate (Sorghum Pormace)

 300g of sorghum pormace was weighed and was sterilized at 121oC for 1hr

 The fermenting tray was sterilized at 200oC for 2hrs.

 The substrate was then poured inside the fermenting tray

 300ml of the mineral salt was added to the substrate and mixed using the hand covered

with latex gloves. Then 3g 1% tannic acid was added into it.

 Two drops of tween 80 as added inside a sterile water.

 The sterile water was then used to scrap the spore of the organism and then poured inside

the substrate and was mixed together using hand covered with latex glove. And it was

covered with foil paper immediately to avoid contamination.

 The substrate was left for 5days at room temperature to ferment.

 Extraction of Tannase enzyme

 3000ml of citrate buffer was prepared and its pH was adjusted to 5.0

 The buffer solution was poured inside the already fermented substrate and mixed together

to homogenize

 The mixture was filtered using muslin cloth.

 The filtrate was discarded and the solution was then labeled as crude tannase enzyme.

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 Tannase Assay

BLANK TEST CONTROL

1. 0.6ml of citrate buffer was 0.5ml of substrate solution 0.5ml of the substrate solution
poured inside a test tube (sorghum pomace) was added was added into a test tube and
into the test tube and 0.1ml of 0.1ml of tannic acid was
tannic acid was also added added into the test tube.
into the same test tube after
which 0.1ml of the enzyme
was added into the solution.

2. Incubate at 300C for 5minutes.

3. After incubation 0.6ml of Rhodanine was poured into the test tube.

4. Incubate at 300C for 5minutes.

5. 0.4ml of KOH was added into the test tube.

6. Incubate at 300C for 5minutes.

7. Dilution was carried out on the solutions, 8ml of distilled water was poured inside all the test
tubes so as not the make the solution sticky.

8. After then Absorbance reading was taken and recorded.

3.4 MOISTURE CONTENT DETERMINATION IN FOOD SAMPLES

This is used to determine the moisture content of two unknown samples

 Procedure

 Petri dishes were washed and cleaned with D.H2O and dried in an oven
 It was then allowed to cool to room temperature using the desiccator
 The petri dish was weighed and labeled as W1
 10g of the sample (Yellow) was placed on the petri dish and labeled as W2

19
  The sample was then placed in an oven to dry for 1hour at 1050C
 10g of another unknown sample(white) was put inside a petri dish
 The samples was weighed before and after drying.
 The results we recorded.

Foil(white) (g) Glass(yellow) (g)

Weight of sample + Dish 4.50 38.60

Weight of dish alone 2.50 36.60

Weight of dish + dry sample 4.50 38.20

Weight of sample 2.00 2.00

Percentage moisture content was determined using

% Moisture = W2 W3 x 100
W1

3.4.1 DETERMINATION OF TITRABLE ACIDITY

The aim of this experiment is to determine the amount of titrable acid that is present in a

drink sample. The drink that was used for this experiment was FIIRO’s pineapples fruit juice.

 Materials used

 Pipette
 Measuring cylinder
 Retort stand
 Burette
 Beaker

 Reagents used

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  Phenolphthalein

 0.1M NaOH

 Pineapple juice.

 Procedure

 5ml of the fruit juice was pipette into the beaker and 2 drops of phenolphthalein was

added.

 NaOH solution was put in the burette for titration then the mixture in the beaker was

titrated until a pink colouration was obtained.

 These procedure was repeated four times and the results were recorded.

 Result

Rough 1st titre 2nd titre 3rd titre

Final volumes of 42.50 43.30 44.00 44.70

acid used

Initial volume of 42.60 42.50 43.30 44.00

acid used

Total volume 2.50 0.80 0.70 0.70

used

Average titre = 0.80+0.70+0.70 = 0.75

3
= 1.175 x 0.75 = 0.88

3.5 PRODUCTION OF COCONUT OIL

This experiment was carried out by extraction of oil from coconut.

21
 Material used

 Coconut water

 Knife

 Bowl

 Muslin cloth

 Waving blender

 Procedure

 The coconut was cracked with a hard object and then dehusked

 It was then cut into smaller pieces using small knife.

 It was washed and place in a waving blender and warm water was added for it to be

blended into a paste.

 The milled coconut was poured into a wide bowl with the addition of water. It was mixed

and allowed to settle for about 5minutes.

 Then the mixture was poured into another bowl using a muslin cloth which was squeezed

to get milk out.

 It was then allowed to sediment, then the coconut cream was obtained and placed in a

clean pot and was heated up and stirred continuously until it started to bubble at the top

and a clear oil began to show.

 The oil was gently scooped into a sterilized bottle.

3.6 WATER ANALYSIS USING WET DIGESTION METHOD

This experiment is used to determine the amount of trace elements present in water

sample using Aqua regia

 Materials used

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  Beaker
 Conical flask
 Filter paper
 Pet bottles
 Pipette
 Standard flask
 Reagents used

 Hydrochloric acid(HCl)

 Nitric acid(HNO3)

3.6.1 PREPARATION OF AQUA REGIA

 30ml of Hydrochloric acid was added into a beaker.

 10ml of nitric acid was added into the 30ml HCL inside the beaker making the solution

40ml (Aqua Regia).

 Procedure

 20ml of the water sample was measured into a clean beaker

 10ml of Aqua regia solution was poured inside the beaker containing the water sample

and was mixed together.

 The beaker was placed on a hot plate to allow the water to evaporate.

 For Blank

 10ml of Aqua regia was poured into a beaker and was allowed to evaporate on a hot

plate

3.6.2 PROCEDURE FOR DIGESTION

 The evaporated beakers were rinsed with deionized water (do not discard).

 The water inside beaker was emptied inside a 100ml standard flask

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  Continue rinsing the beaker pouring inside the standard flask until it reaches 100ml

 Filter paper was used to filter the water into a sample bottle.

 The sample bottle was taken for AAS analysis(Atomic Absorption Spectrometer)

3.7 WORK DONE & EXPERIENCE GAINED IN THE MUSHROOM DIVISION;

MUSHROOM CULTIVATION (OYSTER MUSHROOM)

3.7.1 EDIBLE MUSROOM CULTIVATION (OYSTER MUSHROOM) USING SAW

DUST AS SUBTRATE
Mushrooms are neither plants or animals but belong to a separate

group of organisms called fungi. They are fleshy, spore-bearing fruiting bodies of fungi

typically produced above ground, on soil or on their food sources. They lack the usual

green matter present in plants, therefore cannot use solar energy as their source of food.

They also grow on dead and decaying organic materials. The mushroom fruiting body may be

umbrella-like or of various other shapes, size and colour. Commonly, it consist of a cap

or pileus and or stipe. But others have additional structures like veil or annulus, a cup

or volva. Mushrooms are known for their nutritive and medicinal values. Mushrooms were

known to the ancients as “ The mushroom of immortality” because it had the reputation

of promoting health and longevity, boosting the immune system and reducing the risk of

life-shortening conditions such as cardiovascular disease and cancer. As a fruit body of an

edible white rot fungus, oyster mushroom belongs to Pleurotus, Pleurotaceae, Agaricales,

Basidiomycota. In nature, oyster mushroom appear in cluster on dead trees from late fall to

spring and are distributed almost all around the world.

3.7.2 TECHNIQUE USED FOR THE CULTIVATION OF OYSTER MUSHROOM

DESCRIPTION OF ACTUAL WORK DONE
During my stay as an industrial trainee at the waste utilization laboratory of

biotechnology department, the major type of mushroom cultivated was the oyster mushroom

24
(Pleurotus pulmonarius) using different substrate such as sawdust, cotton waste, banana leaves

Achen Stover. It involves some technological elements which are in consonance with those

exhibited by our common agricultural crop plants. For example, there is a vegetative growth

phase when the mycelia grows profusely and a reproductive (fruiting) growth phase when

the umbrella-like body that we call mushroom develops. After the vegetative (mycelia)

phase has reached maturity, the next stage is the induction of fruiting. This is the time

the mycelia growth tips should be retarded by regulating the factors such as switching

on the light, providing fresh air and lowering temperature can trigger fruiting. Oyster

mushroom (Pleurotus spp) cultivation undergoes the following major phases which are:

 Tissue culturing

 Spawning

 Substrate preparation

 Fruiting and harvesting.

 Packaging

3.7.3 TISSUE CULTURING

The first stage in any mushroom cultivation process is to obtain a pure mycelia

culture of the specific mushroom specie. After this decision has been made, it is

important to determine if that specie possess organoleptic qualities acceptable to the

indigenous population or to the international market. If suitable substrates for cultivation

are plentiful and if environmental requirements for growth and fruiting can be met

without excessively costly systems of mechanical control.

Procedures for tissue culturing:

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 Preparation Of Agar Media: Mushroom culture usually require two standard

formulas which are: Potato Dextrose Agar (PDA), Malt Extract Agar (MEA).

 For Malt Extract Agar 50g of MEA was dissolved in 1L of distilled water. While for

Potato Dextrose Agar, the manufacturer’s prescription should be followed as to the

required preparations.

 1g of Yeast extract was added to the mixture as a nutritional supplement.

 0.02g gentamycin sulphate was added as an antibiotic and covered with an aluminum

foil.

 The solution was homogenized, and was sterilized using the autoclave at 1210c for

15mins

 The medium was then allowed to cool and poured in to petri dish, then allowed to

solidify and dry

3.7.4 FRUITING CULTURE

 The mushroom which was to be used was selected and sterilized using alcohol in which

a cotton swab soaked in 70% alcohol is first used to wipe the surface of the

mushroom in order to remove any dirt or external damaged tissue.

 After alcohol sterilization, mushroom was divided into two halves with a scalpel

and bits from the collar region (i.e junction of cap and stalk) then transferred to

pre-sterilized PDA/MEA culture medium.

 This was then incubated at 250C for 7days.

 Mycelia growing from edges is carefully transferred to MEA/PDA slants and again

incubated for 2-3 weeks to obtain pure cultures.

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3.7.5 DEVELOPMENT OF SPAWN GRAINS
Spawn is the seed required for growing mushroom. It is the vegetative mycelium

from a selected mushroom cultured on a convenient medium like wheat, pearl millet,

sorghum grains etc.

Spawn production is a fermentation process in which the mushroom mycelium

will be increased by growing through a solid organic matrix under controlled

environmental conditions. The purpose of the grain spawn is to multiply the mycelium to

a state of vigour such that it will rapidly colonize the selected bulk growing substrate.

The grain is an important nutrient support as well as a vehicle for the eventual even

distribution into the growing medium of the mushroom inoculants. Individual grain

becomes coated with mycelium and becomes a mycelia capsule.

 Procedure

 Bottles were washed with soap and water, then sterilant (sodium metabisuphide) also

used in rinsing the bottles.

 The bottles were then air-dried for some minutes and then carefully arranged in an oven

for 1hour for drying (this was done for proper sterilization.)

 10kg of sorghum grains was poured into an aluminium pot and boiled

 5g of calcium carbonate was then added to the grain

 5g of calcium phosphate was also added to the grains and stirred

 The grain was per boiled for about 30mins and poured into a sieve in order to drain and

dry up.

 After drying the grain was poured into the already sterilized bottles and after which they

were corked

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  The bottles were then autoclaved at1210c for 45mins

3.7.5.1 INOCULATION OF SPAWN

 The inoculation room was sterilized using ethanol

 The spawn bottles were taken into the room and opened

 A sterilized spatula was used to pick colonies from the mother spawn and placed in the

new spawn bottles and corked back

 The bottles were then incubated at room temperature for about 3 to 8 days for complete

ramification

3.8 SUBSTRATE PREPARATION

Oyster mushroom can utilize various kinds of substrate than any other

mushrooms. Considering the conclusion of the worldwide survey that about 200 different

wastes are available as oyster mushroom substrate.

The main nutritional sources for oyster mushroom are cellulose, hemicellulose and

lignin. Oyster mushroom requires much carbon and less nitrogen source than button

mushrooms (Agaricus bisporus) but most of the main substrate materials are: barley straw,

bean pods, cinnamon leaves, citrus fruit peels, coconut fibre pith and coir, coconut husk,

coffee sawdust, wood logs, corn cobs, corn fibre, corn stalks, cottonseed hulls, cotton wastes,

lemon grass leaves, maize straw, oat straw, paper waste, rice straw etc.

For the cause of this production Saw dust was used as the substrate. Based on sawdust

substrate, it is important to select true species that are both favorable for the growth of

mushrooms and easily available.

 Procedures
 About 100kg of Sawdust, 20kg of rice bran was measured and mixed with water

depending on the quantity of mushroom to be produced.

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  10g of calcium carbonate was added with continuous mixing; even distribution was

ensured throughout the entire substrate. The substrate moisture is usually 65%.

 A palm test method was done to check whether the mixture has the proper water content

or not.

3.8.1 FILLING AND COMPRESSING

 The prepared substrate mixture was filled into bags before each treatment, this was done

in order minimize contamination risk.

 The filled bags are then properly compressed for fast mycelia colonization.

3.8.2 OIL DRUM PASTEURIZATION

In oil drum pasteurization, the first grates were fixed into a notch in the drum, water is

then filled to about 15cm below the grates. The prepared bags were stacked in a layer and then

on the grates. The lid is then placed and the lid rim is sealed, water is boiled and the heating is

maintained for 4hours from the time when the vapor is set to rise. The pasteurization time

depend in the bag size and substrate material.

3.8.3 INOCULATION OF SUBSTRATE

 The inoculation room and gloves were cleaned and disinfected with 70% alcohol

solution.

 Also, the work bench and hands were swabbed to avoid contamination.

 A spoonful of spawn was placed in the substrate bags and corked as quickly as possible

for secured sterile operation.

3.8.4 INCUBATION OF THE SUBSTRATE

 The inoculated bags were moved to the incubation room were higher temperature and

higher humidity are required for the proper growth of mycelia.

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  Bags were incubated at an optimum temperature of 20-25°C in darkness since spawn run

does not require light.

 Full ramification took about15-25days depending on the bag size, substrate material and

condition.

3.8.5 FRUITING

Mushrooms are quiet sensitive to growth condition unlike other crops. The correct

temperature enables them to grow well in growing house. The light and ventilation influence the

colour, size and texture of the mushroom.

Already ramified substrate bags were brought out from the incubating and kept in the

fruiting room where the growth conditions such as temperature, humidity, light and ventilation

was properly monitored. Substrate bags were punctured for the mushroom to shoot out of the

bags

3.8.6 WATERING AND HARVESTING

The substrate bags were watered 3 to 4 times daily to maintain a humid environment for

proper growth of mushroom. This was done until the mushroom shoot is ready for harvesting

(Harvesting is done by direct removal of the mushroom from the substrate)

3.8.7 PACKAGING

The freshly harvested mushroom is packed in tetra packs and sealed up with transparent

polyethene bags.

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 CHAPTER FOUR

LIST OF INSTRUMENTS & THEIR FUNCTIONS

3.4 INSTRUMENTS USED AND THEIR FUNCTIONS

1. INCUBATOR: This equipment is used for the cultivation\growing of microorganisms

2. WEIGHING BALANCE: Weighing balance is used in the measurement of solid and powded

samples.

3. MAGNETIC STIRRER: It is a laboratory equipment which is used to homogenize a

mixture of solid and liquid.

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4. KHJELDAR APPARATUS: This is an automated laboratory equipment used in

determination of percentage protein in food samples.

5. DISTILLER: This equipment is used in the distillation of water so as to get a pure water.

6. SHAKER INCUBATOR: A shaker incubator is a device which is used to provide optimal

conditions for cell growth, it is used in the mixing of liquid samples. The shaker incubator

can be used for growth of just about any kind of cell including bacterial cultures, tissue

cultures and yeast.

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7. AUTOCLAVE: A high pressure device which is used to sterilize laboratory wares and

samples.

8. DESSICATOR: This device is used to cool already sterilized sample to prevent the sample

from gathering moisture as it cools.

9. UV SPECTROPHOTOMETER: This is used to measure the wavelength of light over a

wide range of electromagnetic spectrum.

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10. MICROSCOPE: It is used in the viewing of microorganism.

11. FERMENTING TRAY: It is used in the carrying the solid substrate during solid state

fermentation of enzyme.

12. MOISTURE ANALYSER: A moisture analyzer provides accurate and precise

determination of the moisture content of samples. It works in a way that water interacts with

infrared light.

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13. WATER BATH: It is laboratory equipment used to boil liquid samples.

14. FREEZE DRYER: Used to convert liquid samples to powdered or solid form.

15. LAMINAR AIRFLOW CHAMBER: This is a carefully enclosed bench designed to

prevent contamination of biological samples, or any particle sensitive materials.

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 CHAPTER FIVE
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

5.1 SUMMARY

In the last 6 months Student Industrial Work Experience Scheme at Federal Institute of

Industrial Research Oshodi (FIIRO). I learnt a great deal of biochemical processes ranging from

the microbial productions of enzymes, cultivation of edible mushroom, determination of ash

contents in food samples. Majority of the processes were not new theoretically but were new

practically. The SIWES program helped me to bridge the gap but my theoretical and practical

knowledge in relation practical life experience, the experience has also helped me, during my

interaction with students from other departments, to appreciate how Biochemistry is related to

other disciplines such as microbiology, chemistry, biotechnology, food science and technology,

among others and how interaction with these disciplines will yield results in building new

technologies.

5.2 CHALLENGES ENCOUNTERED DURING THE PROGRAM

 Limited number of apparatus which rendered work to be tedious.

 Inconsistent power supply which made errors occur during practicals

 Lack of stipends from both government and the establishment during and after the course

of the industrial training program.

 Over working of the Industrial Trainees as cheap labour which made the training stressful
rather than being interesting.

5.3 CONCLUSIONS
Upon completion of my SIWES programs at Federal Institute of Industrials Research

Oshodi, I have learnt to appreciate the initiative of learning outside ones comfort zone and also

36
to letting students know would be faced in the outside world. I will also like to conclude that the

SIWES program has been a huge success and has so far achieved its aim of producing

competent, skilled and adequately trained scientists, engineers and Technologists. I have gained

a lot at the Federal Institute of Industrial Research, Oshodi (FIIRO).

I have also been able to improve my communication and presentation skills and thereby

developed good relationship with my fellow colleagues at work. I have also been able to

appreciate the connection between my course of study and other disciplines in producing a

successful result.

5.4 RECOMMENDATIONS OR SOLUTIONS TO THE PROBLEMS

 The various institutions involved in directing the affairs of the student’s Industrial Work

Experience Scheme should work hand-in-hand to look into the 6 months duration being

assigned for the industrial training.

 Over working of students on industrial training as cheap labour should be discouraged

 The organization and the federal government should endeavour to give out stipends to

encourage trainees during the course of the work experience scheme.

 Views and ideas from Industrial Trainee on research issues should be seen as a welcome

one, as one of the ethics of a good researcher is to keep an open-mind, be unbiased in

thinking and to build a strong personal relationship with people involved with the

research.

 The school should help a good number of their student secure appropriate organizations

for placement as this is the major problem of the scheme and is being complained about

by the students almost every year. It should also allow time for proper supervision of

students. There should be better liaison between the Companies, University and ITF.

 The SIWES program should be taken serious by prospective students.

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