i

OPTIMIZATION OF SOLID STATE FERMENTATION PROCESS ON

SAWDUST USING Aspergillus niger

BY

OGUCHE MATTHEW DANJUMA

10AC000136

A REPORT SUBMITTED TO THE DEPARTMENT OF ANIMAL

SCIENCE, COLLEGE OF AGRICULTURAL SCIENCES,

LANDMARK UNIVERSITY, OMU-ARAN, KWARA STATE, NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR OF AGRICULTURE (HONOURS) IN

ANIMAL SCIENCE.

JUNE, 2015

ii

OPTIMIZATION OF SOLID STATE FERMENTATION PROCESS ON

SAWDUST USING Aspergillus niger

BY

OGUCHE MATTHEW DANJUMA

10AC000136

A REPORT SUBMITTED TO THE DEPARTMENT OF ANIMAL

SCIENCE, COLLEGE OF AGRICULTURAL SCIENCES,

LANDMARK UNIVERSITY, OMU-ARAN, KWARA STATE, NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR OF AGRICULTURE (HONOURS) IN

ANIMAL SCIENCE.

JUNE, 2015

iii

CERTIFICATION

I hereby declare that this research was carried out by me

OGUCHE MATTHEW DANJUMA and this report contains exactly my own account of the

research. I also declare that this report is original and authentic and has not been previously

submitted elsewhere. All citations and sources of information are duly acknowledged by

means of reference.

Certified by:

DR. R.A. ANIMASHAHUN

(SUPERVISOR) __________________

Signature/ Date

DR. B.T. ADESINA ___________________

(Head of Department) Signature/ Date

iv

DEDICATION

This project is dedicated to my Heavenly Father the creator of Heaven and earth for His

unending grace and faithfulness in my life and for seeing me through this research

successfully, to God alone be all the glory.

v

ACKNOWLEDGEMENT

To God alone be all the glory for His faithfulness, support and grace in my life, He has

blessed me beyond measures. He personally saw me through this project and I cannot

adequately thank Him for His endless grace and love that provided me with the strength to

finish this project.

I am forever grateful to Dr David O. Oyedepo (Chancellor, Landmark University), for the

opportunity given me to be part of this Divine agrarian revolution.

I wish to express my profound and inexhaustible gratitude to my supervisor

Dr R.A. Animashahun whose efforts contributed immensely to the success of this project. I

appreciate his fatherly consideration, careful review and for creating adequate time to run

through this project to make appropriate corrections despite his busy schedule.

I am also very grateful to Dr O.B. Akpor of the Department of Biological sciences for his

supervision and constructive criticisms throughout the project, Sir! Your contribution and

moral support to this research is priceless, may God richly bless you.

I appreciate the Head of Department of Animal science Dr B.T. Adesina and all my lecturers

in the Department of Animal science especially Dr. P.A. Aye, Dr (Mrs) Alabi, Dr. B.A.

Ajayi, Mr Adejumo, Dr Afolayan and Dr Soyombo.

To my parents, Mr & Mrs Oguche, who have given me nothing but the best, I could wish for

nothing more than the care you have offered and am forever indebted for your spiritual,

financial and moral support.

To my siblings, Bob-Joseph, Emmanuel, Mariam, Gabriel, Abraham and baby David. I am

indeed very grateful for your love, encouragement, prayers and assistance all through this

period.

My heartfelt gratitude goes to my Atinuke Akinradewo Idowu for her unlimited patience and

care especially during the laboratory work of this project. You are special, God bless you

immensely.

vi

My sincere gratitude also goes to my project partner Ilesanmi Ifeoluwa for her kind

accommodation, trust, efforts and immense contribution throughout the period of this project.

I acknowledge my project group members Ali Victor Nda, Oluwashina Boluwatife,

Oyeladun Oluwafemi, Musa Olamide, Adeniji Elizabeth and Fatiregun Tolulope for their

cooperation and dedication to work.

Sincere appreciation is expressed to the Technicians of both the Animal science and

Biological science laboratory who offered assistance to me during the laboratory research.

Furthermore, I acknowledge my friends and course mates Lovesternwill Durrell

(Victorybouy) Isaac whyte, George, Bunmi, Tayo, Alads, Dele, Dare, Richard, Moyo,

Muyiwa, Japheth, Peter, Etieno, Kenny, Ebun(mummy), Tomi, and Yettymaron for their

efforts in ensuring that this work was completed successfully.

The support and contribution of everybody toward the realization of this project is prove that

God truly does work through people. God bless you all.

vii

ABSTRACT

This study aimed to determine the optimum condition for the nutrient improvement of

sawdust under solid state fermentation while varying the initial moisture level of the

substrate using a filamentous fungus Aspergillus niger. The substrate (sawdust) was

subjected to three experimental treatments and control based on its initial substrate : moisture

ratio, these treatments includes A (1:1), B (1:2) control, C (1:3), D (1:4) and the fermentation

period lasted for fourteen days. 8 conical flasks were allotted to each moisture level and each

conical flask contain 20g of sawdust, and 20mls, 40mls, 60mls of moisture was added to the

treatments respectively; samples were withdrawn every 2 days from each treatment for

analysis. The result revealed that the best pH range best for the activities of Aspergillus niger

was recorded to be between 6.20 to 4.60 with the optimum pH for crude protein production

recorded at 4.60 on Day 14. Crude protein content of all the treatments increased during the

fermentation process. Highest increase of 42.35 % was recorded in Treatment D (1:4) after

14 days of fermentation while Treatment B (1:2) recorded a 23.86% decrease in crude

protein on Day 14. The optimum dry matter content of the fermented substrates was also

recorded in Treatment D (1:4) with 25.00% increase on Day 8. There was a build-up in the

ash content also, and the optimum build up was recorded in Treatment D (1:4) on Day 14

yielding 48.5% .General increment in all the parameters analysed were recorded in in

Treatment D (1:4) on day 14.The results of this study indicated that Aspergillus niger

performs best at a 1:4 substrate-moisture ratio for the improvement of the nutrient value of

agro industrial by products. The study reveals that optimal increment of the crude protein

content of sawdust can be achieved when the substrate (sawdust) is fermented, using

Aspergillus niger in a solid state fermentation for 14 days.

Keywords: Sawdust, nutrient improvement, Optimization, Aspergillus niger, solid state

fermentation.

viii

Table of contents

Contents page

Certification....................................................................................................... iii

Dedication…………………...…………………………………………….………… iv

Acknowledgement............................................................................................. v

Abstract…………………………………………………………………………….... vii

Table of contents.......................................................................................... ……... viii

List of tables.............................................................................. ……………… xii

List of figures………………………………………………………….................... xiii

List of plates………………………………………....................................... …… xiv

CHAPTER ONE

1.0 Introduction.......................................................................................... 1

1.1 Background of the study......................................................................... 1

1.2 Statement of the problem........................................................................ 3

1.3 Justification of the project..................................................................... 4

1.4 Objective…………………..................................................................... 5

CHAPTER TWO

2.0 Literature review.………………………………...…………………………. 6

2.1 Agro-industrial by-products (AIBPs)…………………….……….…........... 6

2.1.1 Agro-industrial by-products in Nigeria…………………………………….... 7

2.1.3 Economic importance of agro industrial by-products ……………………… 9

ix

2.1.4 Limitations of agro-industrial by-products………………………...………... 10

2.1.5 Improvement of agro industrial by products in Nigeria……………..……… 12

2.2 Saw dust………………….………………………………………………….. 12

2.2.1 Sawdust as feed…………………………….………………………………... 14

2.2.2 Some other uses of saw dust…………………………………….………… 15

2.3 Fermentation……………………………………………………………….... 16

2.3.1 Fermentation process for improvement of food…………………………... 16

2.3.2 Sub-merged fermentation (SmF) or liquid fermentation (lf)…………..… 17

2.3.3 Solid state fermentation (SSF)……………………………………………. 18

2.3.4 Differences between SSF and SmF……………………...……………….. 20

2.3.5 Factors that influence SSF………..……………………………………….. 22

2.3.5.1 Biological factors ……………………………………………………….. 22

2.3.5.1.1 Type of microorganism ………………………………………………... 22

2.3.5.1.2 Inoculum ……………………..……………………………………….... 23

2.3.5.1.3 Substrates ………………………….…………………………………… 24

2.3.5.1.3.1 Starchy substrates……………………..……………………………… 24

2.3.5.1.3.2 Protein containing substrates…...………………….…………………. 25

2.3.5.1.3.3 Cellulosic or ligno-cellulosic substrates………...…...……………….. 25

2.3.5.1.3.4 Substrates with soluble sugars…………………………...……...…….. 26

2.3.5.2 Physico-chemical factors ………………………………………………….. 26

2.3.5.2.1 Moisture content …………..……………………………………………... 26

2.3.5.2.2 pH ………………………………………………………………………... 27

2.3.5.2.2 Temperature ……………………………………………………………… 27

2.3.5.2.3 Gaseous environment …………………………………………………….. 28

2.3.5.2.5 Particle size………………………………………………………………... 29

x

2.3.5.3 Mechanical factors …………………………………………………………... 30

2.3.5.3.1 Agitation/mixing …………………………………………………………... 30

2.3.6 Perceived advantages of SSF …………………………………………………... 32

2.5 Aspergillus spp as a biocatalysts………………………………………………… 33

2.7 In vitro digestibility studies……………………………………………….…… 34

CHAPTER THREE

3.1 Location of the experiment……………………………………………………… 35

3.2 Microorganism and substrate …………………………………………………… 35

3.3 Experimental setup…………………………………………………………….. 36

3.4 Determinations of results………………………………………………………. 37

3.4.1.1 pH determination………………………………………………………….. 37

3.4.1.2 Moisture determination……………………………………………………. 38

3.4.1.3 Dry matter determination…………………………………………………. 38

3.4.1.4 Ash determination…………………………………………………………. 38

3.4.2.1 Crude protein determination………………………………………………. 39

3.6 Data analysis………………………………………………………………….. 40

CHAPTER FOUR

4.0 Result……………………………………………………………………….. 41

4.1 Effect of initial moisture level on the pH of the substrate……………………… 41

4.2 Effect of initial moisture level on dry matter content of the substrate………… 43

4.3 Effect of initial moisture level on the moisture content of the substrate………. 45

xi

4.4 Effect of initial moisture level of the substrate on ash………………………… 47

4.5 Effect of initial moisture level on crude protein …………………………………….. 49

CHAPTER FIVE

5.0 Discussions and conclusion……………………..……………………….………………51

5.1 Discussions…………………………..…………………………………………………..51

5.2 Conclusions……………………….………………………...……………………………54

5.3 Recommendations…………………………………………………………..……………55

xii

LIST OF TABLE

Table 2.1 Classification of agro-industrial by-products (AIBPs) readily available in Nigeria

and their examples…………………………………………………………………………. 7

Table 2.3 Differences between SSF and SmF…………………………………………….. 20

Table 2.4 The main advantages of SSF listed and summarized within four categories...……31

Table 3.1 Composition of each experimental treatment……………………………………..36

Table 4.1 Changes in pH of the fermented substrate at different levels of initial moisture

treatment during the 14days period of fermentation………………………………………...41

Table 4.2 Changes in dry matter of the fermented substrate at different levels of initial

moisture treatment during the 14days period of

fermentation……………………………...…………………………………………………43

Table 4.3 Changes in moisture of the fermented substrate at different levels of initial

moisture treatment during the 14days period of

fermentation…………………………………………………………………..……..………45

Table 4.4 Changes Ash of the fermented substrate at different levels of initial moisture

treatment during the 14days period of

fermentation…………………………………………...............................................……..47

Table 4.5 Changes Crude protein of the fermented substrate at different levels of initial

moisture treatment during the 14days period of

fermentation…………………………………………………………………………..……49

xiii

LIST OF FIGURES

Fig 4.1: Variation in pH of sawdust at different levels of initial moisture treatment during the

14 days period of fermentation…………………………………………………………..… 41

Fig 4.2: Variation in dry matter content of sawdust at different levels of initial moisture

treatment during the 14 days period of fermentation……………………………………... 43

Fig 4.3: Variation in moisture content of sawdust at different levels of initial moisture

treatment during the 14 days period of fermentation………………………………………. 45

Fig 4.4: Variation in Ash content of sawdust at different levels of initial moisture treatment

during the 14 days period of fermentation…………………………………………..……. 47

Fig 4.5: Variation in Crude protein content of sawdust at different levels of initial moisture

treatment during the 14 days period of fermentation………………………………………. 49

xiv

LIST OF PLATES

Plate 2.1: Inappropriate disposal of saw dust leading to environmental pollution………..…12

Plate 2.2 Cattle at Iowa eating 70% sawdust ration……………………………...................14

1

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Nutrition has been established as one of the major constraints to survival and satisfactory

productivity of livestock in Nigeria (Oso et al., 2014), and report currently shows that feeds

and feeding presently constitute about 70-80 % of total production cost in intensive livestock

production especially in poultry and pig production (Bentil, 2012).

The competition between livestock feed industries and other sectors for conventional feed

ingredients often push the price of finished feed upwards thereby increasing the total cost of

livestock production (Iyayi and Aderolu, 2004).

The skill of manipulating feed ingredient to maximize productivity is paramount, if a stable and

cost efficient production in livestock enterprise is to be achieved (Oso et al., 2014); because the

availability of good quality, good quantity and cheap compounded feeds is important for the

success and rapid expansion of the livestock industry in Nigeria. This is particularly true of the

intensive livestock enterprises – poultry, pigs and rabbits, whose performance depends mainly on

the use of concentrate and balanced compounded feeds. Therefore adequate feeding and proper

nutrition is the single most important constraint facing the livestock industry in Nigeria today.

Several problems relating to the inadequate supply, high cost and poor quality of feeds have

seriously threatened the poultry industry in recent times (Oso et al., 2014).

The use of conventional feedstuffs such as grains for domestic consumption and production of

industrial products such as ethanol and biofuel have caused the increasing cost of this

conventional feed ingredient (Lawrence et al., 2010).

2

Agro-industrial by-products and crop residues makes up a large portion of animal feed resource,

which at this point is still largely unexploited. With ongoing and past research carried out on the

potential of these by-products and crop residues only little practical application has been

achieved (FAO, 2012). There is need to improve scientific knowledge for the utilization of low

cost locally available agro-industrial by-products which is essential in intensive monogastric feed

production in order to reduce the feed cost (Swain and Barbuddhe, 2008).

FAO (2012) also reported that Production of livestock would be achieved at a lower cost,

resulting in improved net profit and foreign exchange savings if appropriate agro-industrial by-

products are used. The physical and chemical property of by-products determines the efficient

use as alternative feed ingredient, which influences production system outputs (Umesh et al.,

2014).

Inexpensive biotechnology like fungal biotechnology using solid state fermentation has been

used as a tool for the effective conversion of these wastes into useful products; its application can

increase the protein and soluble sugars and reduce the complex carbohydrates of these wastes

there by enhancing its efficiency for usage (Iyayi and Aderolu, 2004).

Sawdust is readily available as waste product throughout Nigeria because of the constant

conversion of timber by saw mills into domestic furniture and other wooden products (Pat-

Mbano and Nkwocha, 2012). United Nations Development Program (UNDP) have supported

projects through implemented programs such as the Global Environmental Facility-Small Grants

Program (GEF-SGP) for the conversion of sawdust to briquettes as a cheap but efficient

alternative to fuel-wood used by bakeries and other similar businesses, it‟s also an efficient

3

means of disposal, sawdust can also be exploited for the production of decorative items like

flower vases, arts and craft products, and particle boards. (UNPD Nigeria 2012).

Since sawdust is available throughout the year in many developing countries, Nigeria inclusive,

possible utilization of sawdust in livestock feed will reduce the cost of production livestock

production. Also biological and chemical treatment of these lignocellulose wastes, could

improve their digestibility (Oke and Oke, 2007).

Ogah (2009) reported that the estimated volume of sawdust waste generated by sawmills is about

3.89 million cubic meters per year and increases with industrial growth. This figure shows

tremendous potential for the adequate availability of saw dust for use as an alternative feed stuff

if the proper biotechnological application is used to enhance its nutritive value.

1.2 STATEMENT OF PROBLEM

The livestock industry in Nigeria has been ranked second to crop production as a result of

underutilization of local feed resources and agro-industrial by-products which could be used in

the formulation of good quality livestock feed, and thereby bring down the cost of production of

livestock. The rapid success and growth of the livestock industry in Nigeria thus depends on the

availability of good quality, quantity and cheap compounded feeds (Oso et al., 2014).

With increase urbanization the furniture industry in Nigeria is constantly faced with the problem

of waste sawdust disposal (Pat-Mbano and Nkwocha, 2012; Omonigbo and Olaniyan 2013). A

study observed that the greatest causes of air pollution problem in the Nigerian environment is

atmospheric dust arising from many industrial processes of which sawmill industries is included

(Pat-Mbano and Nkwocha, 2012).

4

1.3 HYPOTHESIS OF THE STUDY

Null hypothesis: that bio degradation through solid state fermentation using Aspergillus

niger doesn‟t have any effect on the crude protein value of sawdust.

Alternative hypothesis: that bio degradation through solid state fermentation using Aspergillus

niger has effect on the crude protein value of sawdust.

1.4 JUSTIFICATION OF THE PROJECT

Sawdust is abundantly available, due to the fast growth recorded in the building construction

sector there have been high increase in the establishment of sawmills in different parts of the

country to satisfy the growing demand of wood (Pat-Mbano and Nkwocha, 2012).

By-products like sawdust which are mostly lignocellulosic with a complex make up of

polysaccharides (lignin, cellulose, and hemicellulose) can serves as substrates for both bacteria

and fungi, which are able to break them down into simple sugars (Iyayi, 2004).The potential of

saw dust has not being fully exploited in Nigeria (Oke and Oke, 2007), as its disposal is mainly

by dumping or burning (UNDP 2012).

The completion of this project will help to address the following:

1. Reduction in the level of pollution in the environment.

2. Reduction in the cost of production of feed for livestock farmers.

3. Increased campaign for the benefits associated with sawdust as a feed ingredient in

livestock farming.

5

1.1 OBJECTIVES OF THE STUDY

1. Optimization of solid state fermentation process by determining the best

physiochemical condition for the process.

2. Optimization of substrate level in the solid state fermentation of saw dust using

Aspergillus niger.

3. To determine the changes in crude protein values of sawdust through degradation

by Aspergillus niger in solid state.

6

CHAPTER TWO

LITERATURE REVIEW

2.1 AGRO-INDUSTRIAL BY-PRODUCTS (AIBPs)

Agro industry with emphasis on food production is defined as the post-harvest activities

involved in the transformation, preservation and preparation of agricultural production for

intermediary or final consumption (Wilkinson and Rocha, 2008).

A by-product is a secondary product derived from a manufacturing process or chemical

reaction. It is not the primary product or service being produced (WTO, 2014). Therefore

agro-industrial by-products (AIBPs) refer to the by-products derived from agricultural-based

industries as a result of processing of the main products. The processing of Agricultural crops

and animal products has led to the generation of vast quantities of AIBPs which can

alternatively be used to feed animals to salvage a threatening problem to the environment

when improperly disposed (Bentil, 2012).

By products from a wide range of plants, animals and other industrial processes have been

studied and found to possess certain nutrients composition which could be exploited as dietary

ingredients for livestock (Falaye, 1993). The possibility of using these by-products for

livestock feeding has been explored but Processing remains a major hindrance to their

maximum utilization (Iyayi and Aderolu, 2004).

7

2.1.1 AGRO-INDUSTRIAL BY-PRODUCTS IN NIGERIA

Over the past few years In Nigeria, because of the competition between livestock and humans

for conventional feedstuffs, there have been need to examine closely the potentials and

advantages of locally available agro industrial by-products as possible substitutes for the

conventional feedstuffs which are dwindling in supply and escalating in their cost (Falaye,

1993).

Iyayi and Aderolu (2004) reported that agro-industrial by-product are abundant in Nigeria;

among the common ones are brewers dried grain, rice bran, palm kernel meal, corn bran,

cassava peels and sawdust.

8

Table 2.1 Classification of agro-industrial by-products (AIBPs) readily available in

Nigeria and their examples.

AGRO INDUSTRIAL BY-PRODUCTS (AIBPs)

SOURCE

Examples

1 Flour milling.

Wheat Bran

Wheat Offal

Wheat Middling

2 Brewing.

Brewers Dried grain (BDG)

Brewers grain Press water

Brewers dried

yeast

3 Rice processing. Rice Bran

Rice Hulls

Rice Polishing

4 Sugar processing. Cane Molasses

Bagasse

5 Root crop.

Cassava peel

Yam peel

Potatoes

6 Cocoa beverages. Cocoa pod Meal

Cocoa Bean Shell

7 Oil Milling.

Groundnut Cake (GNC)

Coco Cake (CNC)

Palm Kernel Cake (PKC)

Soybean cake (SBC)

8 Abattoir.

Blood Meal

Horn and Hooves meal

9 Intensive Livestock Dried poultry Waste(DPW)

Production.

Cattle Manure

Maggots Meal (MGM)

source: (Aye 2014)

9

2.1.3 ECONOMIC IMPORTANCE OF AGRO INDUSTRIAL BY-PRODUCTS

Before now Nigeria depended almost exclusively on imported feed ingredients for the

formulation and production of compounded feeds, however with the economic recession and

the ban imposed on the importation of the major constituents of livestock feeds, especially

grains, many entrepreneurs and farmers in the livestock industry that are unable to withstand

the tough competition have fallen by the wayside and are out of business. Under this

condition wide variations exist in feed supply and hence high prices which have resulted in

the present low level of productivity of the animals regardless of the system of management

(Egbunike and Ikpi, 2000).

The importance of the used of AIBPs in Nigerian cannot be overemphasized since it has been

establish that there is incessant scaring cost as well as irregular supply of notable

conventional ingredient, especially those with high protein concentration like fish meal

(Falaye, 1993). Cheaper protein sources from AIBPs therefore becomes of great significance

in achieving cheaper feed production (Falaye, 1993).

Egbunike and Ikpi (2000) reported that there is a supply of at least 738,271.6 tonnes of agro

industrial by-products nationally, and 52 million tonnes of crop residues in the cereal belts of

Nigeria. Judicious use of these in conjunction with the grass and pasture carryover from the

rainy seasons in form of hay or silage will minimize the dry season weight loss in our

animals, especially ruminants and encourage acceptable weight gains while reducing calf and

herd mortalities. In the case of non-ruminants, the ban placed on the importation of feed

resources has been partially contained by the use of these non-conventional feed resources.

10

In a study carried out to provide information on the profitability on the use of some selected

by-products after fermentation; diets in which the biodegraded by-products replaced maize

produced eggs at a lower cost than the standard commercial diet. Among the by-products use

in the test diet, brewer‟s dried grain (BDG) produced the lowest cost of egg production as it

reduced the cost of egg production by 28.30% compared with 11.32% reduction for

rice bran (RB) and 24.53% for palm kernel meal (PKM). The study therefore concluded that

Utilization of these fermented by-products can replace up to 50% of maize in conventional

layers diets (Iyayi and Aderolu, 2004).

2.1.4 LIMITATIONS OF AGRO-INDUSTRIAL BY-PRODUCTS.

According to Egbunike and Ikpi (2000) the limiting factors on the use of AIBPs can be

categorized into two, and these includes: constraints in the use of by-products and crop

residues and constraints in the use of research results.

The first category include: bulkiness, location in areas with lower population density, poor

nutritive value and unsuitability for direct animal use. The latter includes the following:

i. Lack of appropriate terms.

The use of different local names in different localities and by Researchers constitutes a

problem; there is need for the adoption of standard system for describing crop residues and

by-products. An example is the use of “cassava peels” to describe a mixture of the peel, flesh

and some discarded tubers most times.

11

ii. Lack of biological screening

It is known that some of these products contain some toxic materials that may be harmful to

animals when used for long time; because most of the experiments have been on short-term

basis, it has not been easy to adopt some of the recommendations arising from research

results blindly.

Theobromine and hydrocyanic acid contents in cocoa husks and cassava peels/leaves,

respectively, tacitly caution against long-term utilization of these by-products especially for

breeders. For examples, report has shown that the long-term feeding of cassava peels to

breeding nanny goats causes abnormal embryogenesis resulting in the birth of stunted

neonates that have very little chance of surviving.

iii. Contrasting responses of animals of different species, physiological state and ages.

Recommendations drawn from some results tend to ignore the fact that different species or

classes of animals e.g. ruminant and non-ruminants respond differently to agricultural

by-products and crop residues. Also animals of different physiological status (pregnant or

not) and ages would obviously respond differently to these material. Often the ages and live

weight of experimental animals are not indicated while sometimes pregnant or sick animals

are used thus making adoption of results there from difficult.

iv. Conflict between the goals of the researcher and the farmer

In many instances the Researchers work in complete isolation of the farmers who are the end

user of the result, under such circumstances these results are clear wastage of time and funds

as they are not adopted by the farmers; in few cases their adoption has been a failure.

12

This may partially explain the almost regular non-repetition of the research findings by the

farmers and the lack of development of proper packages for the transfer of research findings

from the researcher to the farmer by extension expert.

2.1.5 IMPROVEMENT OF AGRO INDUSTRIAL BY PRODUCTS IN NIGERIA

In Nigeria today, development of good road network and the opening up of the rural areas for

development have solved the issue of the bulkiness and location of easily accessible

by-products. Research results have shown that supplementation with molasses, non-protein

nitrogen (urea and poultry manure) and chemical (NaOH) and physical (grinding pelleting

and extrusion) treatments improve the nutritive value and intake of by-products (Aye, 2014).

However physical treatment using Inexpensive biotechnology like fungal biotechnology in

solid state fermentation has been used as a tool for the effective conversion of these wastes

into useful products; its application can increase the protein and soluble sugars, and reduce

the complex carbohydrates of these wastes (Iyayi and Aderolu, 2003).

2.2 Saw Dust

Sawdust or wood-dust is a by-product of cutting, grinding, drilling, sanding, or

pulverizing wood with a saw or other tool; it is composed of fine particles of wood; it is also

the by-product of certain animals, birds and insects which live in wood, such as the

woodpecker and carpenter ant. It can present a hazard in manufacturing industries, especially

in terms of its inflammability. Sawdust is also the main component of particleboard (Anon,

2015).

13

It is estimated that the volume of sawdust waste generated by sawmills in Nigeria is about

3.89 million cubic meters per year and this waste generation increases with industrial growth

(Ogah, 2009).

In Germany there have been efficient utilization of small sawmill residues such as sawings

and sawdust, more than half of this material is being used for the production of wood based

panels, mainly particle board. A growing percentage becomes pressed to pellets or briquettes

for energy use, and so the resources are becoming scarce in that country ( Kürsten and Militz,

2005). Clean sawdust has become a high price market since the summer of 2008 in the

United States averaging $50 or more and ranging from $600 to $1,200 per truckload of

material (Dan-Buren, 2012).

However Sawdust in many countries is still regarded as a troublesome by-product of

sawmilling operation and often disposed of as landfill or incinerated, thus causing

environmental problems (Wood report, 2012)

Plate 2.1: Inappropriate disposal of saw dust leading to environmental pollution

Source: (wood report, 2012).

14

2.2.1 Sawdust as feed

Sawdust is a lignocellulolytic material with varying biomass composition, the major

component is cellulose (35-50%), followed by hemicelluloses (20-30%) and lignin

(10-25%), in addition to minor components such as protein, oil and ash that make up the

remaining fraction of lignocellulosic biomass (Hong et al 2011). Lignocellulolytic materials

are not easily digested by non-ruminants, however in the nineteenth century Scientists were

able to justify the addition of sawdust to ordinary bread by claiming not only its nutritional

value but its digestibility. The subject of „sawdust bread‟ got quite a bit of journal space at

the time on account of the possibility of it assisting the feeding of the poor at little cost to the

rich during the times when wheat prices were high (Foodie, 2011).

Report shows that cattle at Iowa consume a 70% sawdust ration without any detrimental

effect to their health (Orlan, 2014). Belewu and Popoola (2007) reported that Feeding of

Rhizopus treated sawdust to WAD goat improved the feed intake, feed efficiency and body

weight gain of the experimental animals, hence fungal treatment of sawdust could be a tool

for increasing the performance characteristics of WAD goat as well as solving the problem of

environmental pollution.

Although scares information exists on the utilization of sawdust by-product by chickens

Oke and Oke (2007) reported that the daily live weight gain increased as the level of sawdust

in the experimental diets increased up to 80 g/kg and declined at 100 g/kg inclusion rate, the

study concluded that sawdust up to 80g/kg level of inclusion in broiler diets, did not have any

detrimental effect on weight gain.

15

Oyster mushroom (Pleurotus ostreatus) is preferred because it‟s excellent flavor and taste

(Bhattacharjya et al 2014). Carbon, nitrogen and inorganic compounds sources are required

as nutritional sources for mushroom to grow, organic substrate like rice and wheat straw,

cottonseed hulls, corncob, sugarcane bagasse, sawdust, waste paper, leaves containing

cellulose, hemicellulose and lignin can be used as mushroom substrate to supports the

growth, development and fruiting of mushroom (Chang and Miles 2004).However

Bhattacharjya et al (2014) reported that sawdust substrate is a better mushroom substrate

compared to the previous Scientists research work.

Plate 2.2 Cattle at Iowa eating 70% sawdust ration.

Source: The gazette

2.2.2 SOME OTHER USES OF SAW DUST.

Apart from serving as an alternative in both livestock and human diets, sawdust is being

utilized for other industrial and aesthetic purposes; in both livestock husbandry and pet care,

sawdust offers definite advantages as bedding material, they reduce the strong ammonia odor

in the animal houses, in addition it provides warmth for the animals.

16

Researchers in Iowa (U.S.A.) have found that lining underground drainage tiles with wood

chips can filter out about 70 percent of the nitrates stemming from dead plants, human waste

and crop fertilizers. As the wood decomposes, bacteria transform the nitrates into nitrogen

gas (Wood report, 2012). Laboratory tests have shown that all nitrates could be effectively

removed if the water is held within the system long enough (Kürsten and Militz, 2005). The

Forestry Research Institute of Nigeria (FRIN) has developed ceiling boards and floor tiles

from saw dust which were presented at the World Exposition EXPO 2000 in Hanover

Germany (Wood report, 2012).

2.3 FERMENTATION

Fermentation is the chemical transformation of organic substances into simpler compounds

by the action of enzymes (complex organic catalysts) which are produced by microorganisms

such as molds, yeasts, or bacteria (Shurtleff and Aoyagi, 2004). Fermentation in food

processing is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids

using yeasts, bacteria, or a combination thereof. The science of fermentation is also known

as zymology or zymurgy (Anon, 2015).

2.3.1 FERMENTATION PROCESS FOR IMPROVEMENT OF FOOD.

Enzymes act by hydrolysis; a process of breaking down or predigesting complex organic

molecules to form smaller compounds and nutrients. For example, the enzyme protease

breaks down complex protein molecules first into polypeptides, dipeptide and peptides, then

into numerous amino acids, which are readily assimilated by the animal body. The enzyme

amylase works on carbohydrates, reducing starches and complex sugars to simple sugars; and

the enzyme lipase hydrolyzes complex fat molecules into simpler free fatty acids.

17

The word "fermentation" is derived from the Latin meaning "to boil," since the bubbling and

foaming of early fermenting beverages seemed closely akin to boiling (Shurtleff and Aoyagi,

2004).

Fermented foods often have numerous advantages over the raw materials from which they

are made; fermentation not only makes the end product more digestible, it can also create

improved flavor, texture, appearance and aroma; synthesize vitamins, destroy or mask

undesirable or beany flavors, reduce or eliminate carbohydrates believed to cause flatulence,

decreases the required cooking time, increases storage life, transforms what might otherwise

be agricultural wastes into tasty and nutritious foods and replenishes intestinal micro flora

(Shurtleff and Aoyagi, 2004).

In livestock production silage making is an important method of conserving green fodder.

silage is the fermented product of green forages where the acids produced by anaerobic

fermentation of the sugars present in these forages are responsible for preserving them

(Alemawor et al., 2009). Fermentation occurs either by encouraging fermentation by bacteria

present in the herbage to produce lactic acid or by direct addition of weak acid solution. Even

though silage was not in common use before 1900, at present it is been accepted in all major

dairy countries (Borucki-Castro et al., 2007).

2.3.2 SUB-MERGED FERMENTATION (SmF) or LIQUID FERMENTATION (lf)

SmF is a fermentation process that utilizes free flowing liquid substrates such as molasses

and broths; the bioactive compounds are secreted into the fermentation broth. The substrates

are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients.

18

This fermentation technique is best suited for microorganisms such as bacteria that require

high moisture (Subramaniyam and Vimala, 2012).

2.3.3 Solid state fermentation (SSF)

Solid state fermentation (SSF) has been defined in many ways: many Researchers in the field

have introduced their own ways to define SSF. Viniegra-Gonzalez (1997) defined SSF as a

microbial process occurring mostly on the surface of solid materials that have the property to

absorb or contain water, with or without soluble nutrients. Pandey et al. (2000) defined SSF

as the cultivation of microorganisms on moist solid supports, either on inert carriers or on

insoluble substrates that can also be used as carbon and energy sources. SSF is described as

any process in which substrates in a solid particulate state are utilized (Mitchell et al 2000).

SSF is the growth of microorganisms on moistened solid substrate, in which enough moisture

is present to maintain microbial growth and metabolism, but where there is no free-moving

water and air is in continuous phase (Rahardjo et al 2006); Rosales et al. (2007) gave a

simple definition of SSF as where the growth of microorganisms is on solid or semisolid

substrates or support; Mitchell et al. (2011) redefined SSF as a process that involves the

growth of microorganisms on moist particles of solid materials in beds in which the spaces

between the particles are filled with a continuous gas phase. Whatever the definition, we can

understand that SSF is referring to the microbial fermentation, which takes place in the

absence or near absence of free water, thus being close to the natural environment to which

the selected microorganisms, especially fungi, are naturally adapted (Musaalbakri, 2014).

19

In recent years, SSF has received fresh attention from Researchers and industries all over the

world; this is due to several major advantages that it offers over SmF, particularly in the area

of solid waste treatment apart from the production of food and feed. SSF shows a

tremendous potential in applications to produce high value-low volume products such as

enzymes, biologically active secondary metabolites and chemicals (Musaalbakri, 2014).

Interestingly, fungi, yeasts and bacteria that were recently tested in SSF exhibited different

metabolic strategies under the two fermentation conditions, and a direct comparison of

various parameters such as growth rate, productivity and volume activity favored SSF in the

majority of cases; in addition, in most cases the cost-factor for the production of “bulk-ware”

enzymes favors SSF over SmF (Musaalbakri, 2014).

Many research works have so far focused on the general applicability of SSF for the

production of enzymes and metabolites. Food and agro-industry provide much different solid

wastes as valuable solid substrates, which have been combined with many different

microorganisms and resulted in a wide range of fermentation processes. For example,

enzymes production by SSF is a growing field due to the simplicity of the processes, high

productivity, and generation of concentrated products (Castilho et al., 2000). Another

important factor that influences the development of SSF is that both food and agro industrial

waste are rich in carbohydrates and other nutrients so that they can serve as a substrate for

the production of enzymes (Cauto and Sanroman, 2006). With the advances of biotechnology

and bioprocess nowadays, for example in the area of enzyme and fermentation technology,

many new avenues have opened for their utilization in SSF.

20

2.3.4 DIFFERENCES BETWEEN SSF and SmF

In contrast to SSF, SmF is typical 100% liquid with, possibly, some suspended solids. The

moisture content of SSF, on the other hand, is usually maintained within the range of

12 - 70%, and typically around 60% (Chen, 2013); Industrially SmF is by far the most

common operation employed in the fermentation (Hata et al., 1997).

Most researches in SmF are aimed at determining the production economics of the process

including productivity and product yields (Castilho et al., 2000) and maximizing these

parameters.

The use of filamentous fungi for the production of commercially important products has

increased rapidly over the past half-century and the production of enzymes in SmF has long

been established (Papagianni et al., 1999). SmF currently produces commercial enzymes and

several of the potential applications have been commercially exploited, primarily due to

shortage and high cost of enzymes (Viniegra-Gonzalez et al., 2003). Even though modern

SmF offers many advantages, it suffers from some major disadvantages Barros Soares et al.

(2003) reported that SmF for transglutaminase production has constraints such as a long

fermentation process, excessive foam production that prevents oxygen mass transfer and use

of expensive culture media.

21

Table 2.3 Differences between SSF and SmF.

SSF SmF

There is no free water, and the water content of substrate is in the range 12 - 70% Water is the main component of the culture

Microorganisms absorb nutrients from the wet solid Microorganisms absorb nutrients from the liquid substrates; a nutrient concentration gradient exists culture; there is no nutrient concentration gradient The culture system consists of three phases (gas, liquid and The culture system mainly consists of liquid; the liquid solid) and gas is the continuous phase is the continuous phase

Inoculation size is large, more than 10% Inoculation size is small, less than 10%

The required oxygen is from the gas phase; the process needs The required oxygen is from dissolved oxygen; there is low energy consumption a larger amount of dissolved oxygen

Microorganisms attach and penetrate into the solid substrate Microorganisms uniformly distribute in the culture system

At the end of fermentation, the medium is a wet state At the end of the fermentation, the medium is liquid substrate, and the concentrations of products are high and the concentrations of products are low

High production rate and high product yield Low production rate and low product yield

Mixing is difficult or impossible, some microorganisms are sensitive to mixing or agitation and the growth of Mixing is easy, and the growth of microorganisms is microorganisms is restricted by nutrient diffusion not restricted by nutrient diffusion

Heterogeneity Homogeneity Extraction process is simple and controllable; little Extraction process is usually complex; there is a large water amount of waste water

Low water activity High water activity

Simple fermentation bioreactor High-tech design fermentation bioreactor

Natural enrichment or artificial breeding systems Pure strains

Low raw material cost High raw material cost

Source: (Musaalbakri, 2014).

22

2.3.5 Factors that influence SSF

Its simplicity and its closeness to the natural habitat of many microorganisms confer great

advantage on SSF. Through modern biotechnology, there are new initiatives to improve and

enhance the productivity of SSF. Each microorganisms, solid substrate, and bioreactor

system plays a major role in the success of SSF. Musaalbakri (2014) classified the factors

influencing the performance of SSF into three major categories, namely:

i. Biological factors

ii. Physico-chemical factors

iii. Mechanical factors

2.3.5.1 BIOLOGICAL FACTORS

2.3.5.1.1 TYPE OF MICROORGANISM

The most important criterion in SSF is the selection of a suitable microorganism, which has

the ability to degrade the solid substrate. SSF processes are due mainly to the fermentation

activity of either fungi alone, bacteria alone, a mixture of fungi and yeasts or fungi followed

by a mixture of bacteria and yeast. The selection of microorganism is usually dependent on

the type of solid substrate, growth requirements and targeted final product (Krishna, 2005).

These general criteria will affect the fermentation design and downstream processing.

Filamentous fungi continue to dominate as an important microorganism in SSF due to their

mycelia mode of growth as well as their neutral physiological capabilities (Mitchell et al.,

2011). The use of a single microorganism, especially in industrial SSF processes, has the

advantage of improved rate of substrate utilization and controlled product formation (Nigam

and Pandey, 2009).

23

Ensiling and composting are among the processes involving several microorganisms that

exhibit symbiotic behavior; in other words, mutual growth of microbial communities and

thus mixed culture processes, as these exist in most natural habitats (Nigam and Pandey,

2009).

2.3.5.1.2 INOCULUM

Inoculum can be described as a preparation containing high numbers of viable

microorganisms, which may be added to bring about desirable changes in the solid substrate

(Wolzapfel, 1997). The age of the inoculum, the medium used for its cultivation, and

therefore its physiological state are of the utmost importance in many fermentation processes.

According to Sheperd and Carels (1983), if the inoculum used for the production of

secondary metabolites is not in the correct physiological state, a considerable decrease in

production will occur. This is because the early hours of fermentation determine the future

direction of the culture. Sekiguchi and Gaucher (1977) observed that with Penicillium urticae

the type of inoculum used greatly influences the level of secondary metabolites produced;

Smith and Calam (1980) reported that different yields were obtained in penicillin and

griseofulvin fermentation using different types of inoculum. From their study, it was shown

that biochemical factors, such as the level of enzyme activity and efficiency, were at least as

important as morphology in determining yield, being carried forward from the inoculum to

the production stage. For example, most fungi produce spores, spores inoculum are easy to

prepare and can be stored for longer periods than vegetative cells. The chances for

contamination are higher if low levels of inoculum density were used.

24

According to Nigam and Singh (1994), by increasing the inoculum quantity, the time

required for substrate utilization can be shortened and this can also aid the inoculated fungus

to displace any other microbes that may be present. This makes processes involving fungi

more flexible since the synchronization of inoculum production with the rest of the process is

not that crucial. Sporulation is generally not desirable during the fermentation itself (Mitchell

et al., 2002).

2.3.5.1.3 SUBSTRATES

Carbon sources supplied in the medium are of great importance to fungi since they provide

the carbon source needed for the biosynthesis of cellular constituents. This includes

carbohydrates, proteins, lipids, nucleic acids, and their oxidation provides energy for the cell

(Gadd, 1988). The solid substrate is a major element in SSF. In addition to providing

nutrients such as carbon and nitrogen, the solid substrate also performs the role of the

physical structure that supports the growth of microorganisms (Cauto, 2008).

2.3.5.1.3.1 Starchy substrates

Starchy substrates that have been used in SSF include rice, barley, oats, cassava, wheat bran,

cassava meal, corn meal, okra, sweet potato residues, and banana peel. Starchy substrates,

being rich in carbohydrates (important carbon source in many microbial fermentation

processes), are hydrolyzed to produce simple sugars that can be consumed by

microorganisms (Musaalbakri, 2014).

25

2.3.5.1.3.2 Protein Containing Substrates

Food and agro-industry by-products such as oil cakes are an ideal source of protein nutrients.

Their use as a solid substrate is highly favored in SSF. Pumpkin oil cake (63.52% protein )

(Pericin et al.,2008), soybean oil cake (51.8% protein) (Borucki Castro et al., 2007), sesame

oil cake (48.2% protein) (Yamauci et al.,2006), groundnut oil cake (45.6% protein) (Batal et

al., 2005), safflower oil cake (44.0% protein) (Sivaramakrishnan and Gangadharan, 2009),

rapeseed meal oil cake (42.8% protein) (Bell, 1984), cottonseed oil cake (41.0% protein)

(Ramachandran et al., 2005), mustard oil cake (38.5% protein), sesame oil cake (35.6%

protein), sunflower oil cake (34.1% protein) and canola oil cake (33.9% protein)

(Ramachandran et al., 2007protein), linseed oil cake (32 - 36% protein) (Rani and Ghosh,

2011), coconut oil cake (25.2% protein) (Ghosh et al., 2013), copra oil cake (23.11% protein)

and palm kernel oil cake (20.4% protein) (Dairo and Fasuyi, 2008) and olive oil cake (4.77%

protein) (Vlysside et al., 2004) are the most abundant agriculture by-products oil cakes, rich

in proteins (important nitrogen source in many microbial fermentation) and supported by

other nutrients such as carbohydrates and minerals, these offer a wide range of alternative

substrates in SSF for the production of various enzymes, a wide spectrum of secondary

metabolites, biomass, organic acids and biofertilizer among other uses.

2.3.5.1.3.3 Cellulosic or ligno-cellulosic substrates

Most agricultural residues contain high levels of cellulose or ligno-cellulose, which have the

potential to be used as solid substrates in SSF. These include sugarcane bagasse, soybean

hulls, wheat bran, rice hulls, rice stover, corn cob, barley husk, sugar beet pulp, wheat straw,

26

barley straw and wood. In this case, cellulolytic fungi such as Trichoderma reseei,

Trichoderma longibrachiatum, Trichoderma viride, and Aspergillus niger

2.3.5.1.3.4 Substrates with soluble sugars

Solid substrates containing significant amount of soluble sugars may be obtained from fruit

processing such as molasses, grape pomace, apple pomace, kiwi pomace, lemon peel, lemon

pulp, peach pomace, pineapple waste, sweet sorghum, fodder and sugar beets, sugar beet

pulp, carob pods, and coffee pulp,etc.

2.3.5.2 PHYSICO-CHEMICAL FACTORS

2.3.5.2.1 MOISTURE CONTENT

The water requirements of microorganisms for microbial activity can be expressed

quantitatively in the form of water activity (aw) of the environment or substrate. This gives

an indication of the amount of free water in the substrate and determines the type of

microorganisms that can grow. Saturated air is usually applied to the system as an alternative

to maintain water activity (aw) and moisture content of the fermented substrate in cases

where bioreactors are used. It is also a common practice used to avoid substrate drying. This

approach is suitable when the SSF is carried out in designated bioreactors. At high moisture

content, solid substrate particles tend to stick together and thus reduce the surface to volume

ratio of solid material. According to Mitchell et al. (2002), high moisture levels can cause

agglomeration of medium particles in SSF and lead to oxygen transfer limitations; as a result,

a great decrease is observed in the production of microbial metabolites. Hence, it is important

to provide and monitor the moisture content at an optimum level.

27

2.3.5.2.2 pH

In SSF, pH is very difficult to measure and control. This is because of the nature of solid

substrate, very low water content (lack of free water), heterogeneity in the conditions of the

systems, and due to the lack (or absence) of suitable on-line pH measurement methods

(Durand et al., 1997). There is no reliable electrode that can measure pH in the solid medium,

usually it is desirable to use microorganisms which will grow over a wide range of pH and

which have broad pH minima (Musaalbakri, 2014). Individual groups of microorganisms

react in different ways to the pH value of the fermentation environment. Bacteria generally

prefer pH values near neutrality, fungus and yeasts lightly acid pH values, and actinomycetes

above neutrality. Villegas et al. (1993) suggested using of a potentiometric electrode or a

standard pH electrode after suspending the fermented substrate in water.

2.3.5.2.2 TEMPERATURE

The problem regarding temperature arises during the SSF process due to the heat generated

from microbial activity and accumulated in the system (Nigam and Pandey, 2009).

Temperature due to heat and mass transfer effects presents difficulties in handling the SSF

process (Krishna, 2005). The heat needs to be removed from the system to avoid overheating

and thereby disturbing the growth of microorganisms and the formation of products (Pandey

et al., 2001). Therefore, in SSF, most studies on solid state bioreactor designs are focused on

maximizing heat removal (Figueroa-Montero et al., 2011; Ashley et al., 1999). The problem

becomes crucial in large-scale systems where heat evolution leads to huge moisture losses

and, under these circumstances, disturbing fungal growth (Khanahmadi et al., 2006).

28

Another problem is that heat creates condensation such that a large amount of water is

returned back to the fermented solid. This will create heterogeneity in the solid substrate.

Because of this, it is difficult to maintain the temperature at an ideal range. To overcome this,

air is usually blown into the system, to force out the heat generated via a gas outlet (Sato et

al.,1984). The flow rate of the air blown into the system needs to be taken into account to

avoid the loss of moisture content from the fermented substrate (Shojaosadati et al., 2007).

2.3.5.2.3 GASEOUS ENVIRONMENT

The gases of interest are oxygen and carbon dioxide. Oxygen must diffuse from the inter-

particle space to the biomass. Adequate supply of oxygen is required to maintain aerobic

conditions. Carbon dioxide must diffuse from the biomass to the inter-particle space .This

requirement can be achieved by aeration or mixing of the fermenting solids. Oxygen

limitation might occur at deep areas of the substrate. These can be solved by turning the

fermenting substrate through mixing processes (Lonsane et al., 1985).

2.3.5.2.4 AERATION

Microorganisms normally vary in their oxygen requirements. Oxygen or air is sparged into

the medium. Aeration plays two important roles in SSF:

(i) Meeting the oxygen demand in aerobic fermentation and

(ii) Heat and mass transport in a heterogeneous system.

Aeration provides and maintains high oxygen levels and low carbon dioxide levels in the

inter-particle solid substrates; the points to take into account with the aeration are the flow

rate and air quality. Dry air at high flow rate will have an effect on the moisture of fermented

substrate even though it has an advantage in terms of heat removal.

29

Aeration rate was shown to have a positive effect on microbial growth and product formation

(Assamoi et al., 2008; Gutarra et al., 2005; Zhang et al., 2003). Alternatively, using saturated

air is a common strategy to avoid substrate drying by maintaining moisture levels. In

addition, the rate of aeration by saturated air controls the temperature and the moisture

gradients of the solid medium (Saucedo-Castaneda et al., 1992).

2.3.5.2.5 PARTICLE SIZE

The particle size properties of solid substrates will lead to the shape, accessible area, surface

area and porosity of the solid substrates (Richard et al., 2004). Processes like chopping,

grinding and cutting create a condition for microorganisms to be active at the initial stages of

growth and increase the degradation and hydrolysis rate since the solid substrate is insoluble

(Ramana Murthy et al.,1993). The most important physical factor is the particle size that

affects the surface area to volume ratio of the solid substrate (Krishna, 2005). Smaller

particle size would provide a larger surface area per volume and allow full contact of

microorganisms with the nutrients but the diffusion of oxygen would be affected (Nigam and

Pandey, 2009); larger particle size provides small area per volume ratio and gives excellent

diffusion of oxygen but contact with nutrients is affected (Nandakumar et al., 1994). A

suitable particle size should satisfy both mycelial growth and the demand for oxygen and

nutrients (Nandakumar et al., 1996).

Particle size also affects the size of inter-particle voids and porosity (Mitchell et al., 2002).

Any change in porosity of the solid substrate bed changes the apparent density of solid

substrate and diffusion of gases into the bed, a large pore size is suitable for an adequate

oxygen supply (Pandey, 1991). If porosity is limited, the effective diffusivity of gases is less.

30

Particle size and properties may change during fermentation; these do not only affect the

growth of microorganisms, but also affect the monitoring of heat conductivity, substrate

consumption, products concentration and water content (Rahardjo et al., 2005).

2.3.5.3 MECHANICAL FACTORS

2.3.5.3.1 AGITATION/MIXING

Agitation or mixing plays the same role as aeration. In addition, agitation is a possible

alternative to solve heterogeneity problems in SSF and might improve homogeneity and

disrupt gradients (Lonsane et al., 1985; Xu and Hang 1988).

Another benefit of agitation is that air flow is more evenly distributed which improves the

conditions for microbial growth within the entire fermented bed (Suryanarayan, 2003).

however, agitation affects mycelium formation as shear forces due to agitation can destroy

the mycelium. Continuous agitation also may create problems related to cell damage

especially when filamentous fungi are used (Mitchell et al., 2011).

2.3.6 PERCEIVED ADVANTAGES OF SSF

SSF has centuries of history, but it is only in the last two decades that there has been a

concerted effort to understand bioprocessing aspects involved in SSF and to apply them to a

wide range of new products (Pandey et al., 2008). Although significant advances have been

achieved in understanding the controls of process performance, much research is still

required. Studies on SSF and some of their results have provided a substantial contribution

to the improvement of the existing and widely used technology.

31

To a certain extent, some of the research findings have widened the scope of research

activities towards a better understanding of existing SSF systems. The various advantages

that have been identified through the literature can be described based on different criteria,

namely

(1) Biological advantages;

(2) Processing advantages;

(3) Environmental advantages and

(4)Economic advantage.

32

TABLE 2.4 The main advantages of SSF listed and summarized within four categories.

Type of

advantage

Remarks

Biological

Other biological advantages:

fungi-producing spores. Spores can be used as

inoculum, can be preserved for a long time and can be used repeatedly

Processing

e loading is much higher

Other processing advantages:

simple and unrefined

-treatment and treatment of the natural resources can be very

simple

be simple since products are

concentrated

-foam chemicals

Environmental

products manufacture

Economic

- Natural unusable carbon source

which are extremely cheap, variable and abundant agro-industry and food

waste.

-friendly

proved to be economically feasible

Source: (Musaalbakri, 2014)

33

2.5 Aspergillus spp AS A BIOCATALYSTS

Aspergillus is the name used for a genus of moulds that reproduce only by asexual means,

Aspergillus species are common and widespread; they are among the most successful groups

of moulds with important roles in natural ecosystems and the human economy.

The genus Aspergillus have several species such as Aspergillus flavus, Aspergillus oryzae,

Aspergillus fumigatus, Aspergillus glaucus and Aspergillus niger.. Aspergilli are a

fascinating group of fungi exhibiting immense ecological and metabolic diversity as they

could be both pathogenic and industrially productive (Papagianni et al., 1999). These include

notorious pathogens such as Aspergillus flavus, which produces aflatoxin, one of the most

potent, naturally occurring, compounds known to man. Conversely, also included are other

fungi, such as A. oryzae, involved in the industrial production of soy sauce and sake, A. niger

used for the production of citric acid and enzymes such as glucose oxidase and lysozyme

(Masayuki and Katsuya, 2006). These fungi are also liberate enzymes that degrade lignin

present in cellulolitic materials (Abreu et al., 2007). In favourable environments

(temperature, relative humidity, luminosity) they produce ignocellulase enzymes, mainly

laccase (LAC) and Mn-peroxidase (MnP), which convert these lignocellulosic residues into

food ( Bentil, 2012).

2.6 ADVANTAGE OF USING Aspergillus niger IN SOLID STATE FERMENTATION.

Oligotrophic are organisms that can live in an environment that offers very low levels of

nutrients, they may be contrasted with copiotrophs, which prefer nutritionally rich

environments (Anon, 2015).

34

In addition to growth on carbon sources, many species

of Aspergillus demonstrate oligotrophy where they are capable of growing in nutrient

depleted environments, or environments with a complete lack of key nutrients. A. niger is a

prime example of this; it can be found growing on damp walls, as a major component

of mildew. The Uninted states Food and drug Administration (US FDA) in the united States

list Aspergillus oryzae and Aspergillus niger on its generally recognized as safe (GRAS) list

(Schuster et al., 2002).

2.7 IN VITRO DIGESTIBILITY STUDIES.

The nutritive quality of any feedstuff is evaluated by the availability of its nutrients to the

animal in question for good growth performance (Tatli and Cerci, 2006). However,

nutritional management may be influenced by several factors such as the physiological and

hormonal conditions of the animal (Cone et al., 1996). Monogastric animals such as poultry

and pigs do not have any microorganisms in the GIT unlike ruminants therefore always need

high energy diets containing readily available simple sugars (Chang et al., 1998) to

complement their energy requirements. The bioavailability of a feedstuff can therefore be

investigated outside the animal by the application of in vitro digestibility studies (Tilley and

Terry, 1963) which employs enzymatic action on feed material in a test tube to evaluate the

utilization of the feed by the animal. The only drawback with in vitro digestibility is that

these methods cannot mimic the complex and dynamic conditions in the digestive tract of

live animals where exogenous and endogenous secretions and nutrients are mixed and where

there are also interactions in concentration, inhibition, microbial, neutral and hormonal

effects though relatively simple, inexpensive, rapid and high level of precision achieved

(Tilley and Terry, 1963).

35

CHAPTER THREE

MATERIALS AND METHODS

3.1 LOCATION OF THE EXPERIMENT

This study was carried out in the Animal science Laboratory of the college of Agricultural

science Landmark University Omu-Aran, Kwara State, Nigeria.

3.2 MICROORGANISM AND SUBSTRATE

The microorganism that was used for the study is Aspergillus niger USM F4. The organism

was obtained from the Laboratory stock of the Department of Biological Science, Landmark

university, Omu-Aran, Kwara State, Nigeria.

The nutrient media used for this experiment was the Malt extract broth. which was prepared

using 40g/litre and 20g/litre of peptone added to 50mL of distilled water, and then the solution

was thoroughly mixed using a glass rod (stirrer.). The solution was heated to boil at 121 oC for

15 min and then allowed to cool to ambient temperature. After cooling, the cooled broth was

then taken to the biochemistry Laboratory where 0.05% of chloramphenicol was added to

inhibit bacteria growth.

Preparation of the Agar

Sabouraud dextrose agar was used in the preparation. 12.5g of the agar was measured into the

conical flask and 200mls of distilled water. The mixture was then cotton plugged and then

sterilized. It was placed at room temperature (25ºc) where it was solidified. Before usage for

the experiment, the organism was first cultured on sterile Sabourand dextrose agar plate, to

ascertain its purity.

36

The pure organism was then stored in Sabourand dextrose agar and stored in a refrigerator at

4oC±2

oC and withdrawn when needed. Prior to use, the fungal was cultured in Sabourand

dextrose broth and incubated at 25oC ±2

oC for 72 h. At the expiration of incubation, the

culture medium was centrifuged (TDL 5000B) at 5000 revolution per minute (rpm) for 45

min.After centrifugation, the supernatant was decanted while the cells were swashed several

times with sterile distilled water to remove the broth. After swashing the cells were then

suspended in sterile normal saline (0.85% w/v NaCl). The microbial counts in the suspended

cells was then estimated using the plate count method with cell count expressed as spore

forming units/mL, using standard microbiological techniques.

The substrate (sawdust) used for the study was obtained from a timber processing industry in

Omu-Aran, Kwara State, Nigeria. Prior to use, the sawdust was air dried for two weeks, after

which known amount of the substrate was weighed into 250 mL capacity conical flasks,

cotton-plugged and autoclaved for sterilization at 121oC for 15 min at 15 pound square

inch-gauge (psi).

3.3 EXPERIMENTAL SETUP

The study focussed on variation of moisture level which was set up with both experimental

and control test i.e. three (3) treatments and one (1) control. The weighed samples were

inoculated with Aspergillus niger aseptically in a laminar flow, separately into 32 conical

flasks with 8 conical flasks for each treatment level and the control test. Counting showed

that the parent stock contained 8.0 x106

cfu/ml (colony forming unit) at different levels for all

treatments.

37

Table 3.1 Composition of each experimental treatment.

Treatment Composition

A (1:1) 20g of substrate + 20ml of spore suspension

B(1:2) Control 20g of substrate + 20ml of distilled water

C (1:3) 20g of substrate + 20ml 0f spore suspension + 40 ml of distilled water

D (1:4) 20g of substrate + 20ml 0f spore suspension + 60 ml of distilled water

All the substrates were incubated at room temperature for 14 days; 4 samples were taken

every 2days for analysis, one sample from each treatment and one from the control.

3.4 DETERMINATIONS OF RESULTS

Physiochemical changes and some part of proximate analysis were carried out. The

fermented samples were removed from the conical flask, and physiochemical analysis was

carried out immediately. The remaining samples were air dried at room temperature before

the proximate components analysis.

3.4.1.1 pH DETERMINATION

pH gas electrode was used to check the pH of the samples; the pH scale was calibrated to pH

7 using buffer. 2ml of distilled water was added to 1g of each sample of the substrate in a

beaker and mixed thoroughly, the mixture was then left for 5mins after which the pH was

checked and the readings taken.

38

3.4.1.2 MOISTURE DETERMINATION

Empty crucibles was weighed separately at first, and then 2g of the dried samples were

weighed separately also and then later placed inside the crucible. The weight of the crucible

and the sample was then taken together. The crucible together with the sample was then

placed in an oven at 105oC for 24 hours. The samples were then removed and placed in a

desiccator and allowed to cool and weighed thereafter.

%Moisture content= wt. of sample+ crucible before drying -wt of sample+ crucible after drying × 100

Wt. of sample

3.4.1.3 DRY MATTER DETERMINATION

The dry matter was gotten after the substrate samples dried in the oven. The samples left

were the dry matter.

%dry matter= 100- %moisture

3.4.1.4 ASH DETERMINATION

Two grams (2g) of each of the dried samples was weighed into empty crucible. The crucibles

were placed in a furnace to ash at 550oc for 4hours. After ash, the crucibles were left in the

furnace for it to cool for a while before the sample were then removed and placed in a

decicator to cool. The cooled samples were then weighed.

% Ash=Weight of crucible+ ash-weight of the crucible × 100

Weight of sample

39

3.4.2.1 CRUDE PROTEIN DETERMINATION

One gram (1g) of each of the dried sample was placed into the Kjeldahl digestion flask. A

known amount of catalyst (selenium powder) was added, one gram (1g) of copper sulphate

(CuSO4) was added to the mixture, 5g of sodium sulphate (Na2SO4) was also added to the

mixture and 12ml of Sulphuric acid was finally add to each of the digestion flask and then

shaken thoroughly to ensure the samples were properly mixed with the chemical mixtures.

The determination was in 3 stages, they include; digestion, distillation and titration.

Digestion: The digester was turned on and allowed to heat until the temperature readings was

420oC, the Kjeldahl digestion flask was then moved into the digester and allowed to digest

for 1 hour, and it was then left to cool completely.

Distillation: The digested material still in the digestion flask was then moved to the

distillation unit, and a Kjeldahl nitrogen distillation apparatus (TecatorTM Kjeltec system)

was used for the distillation. The distillation lasted for 5 minutes.

Titration: The filtrate from the distiller was then moved for titration. It was titrated with

0.1N HCl acid solution.It was titrated until the colour changed from blue to pale red and the

readings recorded.

%N = (T-B) × n × VA × 100

Weight of sample (mg)

% CP = N × 6.25

Where:

N = Nitrogen

T = Titre value

40

B = Blank value

n = Normality of acid (0.1)

VA = volume of acid (nitrogen)

CP = Crude protein

3.6 DATA ANALYSIS

The results were presented using tables and graphs.

41

CHAPTER FOUR

RESULT

4.1 EFFECT OF INITIAL MOISTURE LEVEL ON THE pH OF THE SUBSTRATE

From Fig. 4.1, it is revealed that there was a progressive decrease in the pH of the substrate

throughout the period of fermentation which indicates a progressive increase in the acidity of

all the experimental treatment.

As shown in Fig. 4.1, on Day 0 all Treatments including the control maintained a slightly

acidic pH. Treatments A, C, D had a pH of 6.21, 6.22 and 6.20 respectively. The control

Treatment also had a slightly acidic pH of 6.20

On day 2( as shown in Table. 4.1), Treatment A increased slightly in acidity from pH 6.21

that was seen in Day 0 to pH 6.19, Treatment C also increased slightly in acidity from a pH

of 6.20 to 6.17. A similar trend in slight increase in acidity was also seen in treatment D as

there was a slight change in pH value from 6.20 seen on Day 0 to 6.16 noticed on Day 2.

After the entire period of fermentation of 14 days, the highest acidity (lowest pH) was seen in

treatment D with pH 4.60 on day 14 while the lowest acidity (highest pH) was notice in

Treatments B and D on Day 0 having the same pH of 6.20.

Treatment B which served a control also decreased in pH progressively from 6.20 seen on

day 0 to 5.50 seen on day 14.

42

Fig 4.1: Variation in pH of sawdust at different levels of initial moisture treatment during the

14 days period of fermentation.

Table 4.1: Changes in pH of the fermented substrate at different levels of initial moisture

treatment during the 14days period of fermentation.

TREATMENTS Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14

A 1:1 6.21 6.19 6.16 6.13 5.88 5.46 4.98 4.76

B 1:2(control) 6.20 6.17 6.16 6.07 6.07 5.93 5.61 5.50

C 1:3 6.22 6.17 6.12 5.98 5.98 5.32 4.88 4.64

D 1:4 6.20 6.16 6.03 5.98 5.98 5.20 4.82 4.60

4

5

6

7

0 2 4 6 8 10 12 14

%

Incubation period (days)

pH

A B C D

43

4.2 EFFECT OF INITIAL MOISTURE LEVEL ON DRY MATTER CONTENT OF

THE SUBSTRATE

From Fig. 4.2, it is revealed that there was a progressive decrease in the dry matter from Day

0 to Day 8 from where the dry matter began to increase again.

Treatment A maintained a dry matter of 91.50% for day 0 and day 2 then decreased

progressively from day 4 to day 8 where it was recorded to have a dry matter of 88.50% , the

dry matter started to increase again progressively from Day 8 to Day 14 where it recorded the

highest dry matter content of 93.00%.

As shown in Fig. 4.2, treatments C and D showed a very similar pattern in the change in dry

matter. Both treatment C and D had a dry matter level of 90% and this was maintained for

day 0 and day 2, both treatments also had the same dry matter of 89% on day 4, a progressive

increase in dry matter was notice from day 10 and both treatment had a dry matter of 90.5%

on day 14.

Treatment B which served as a control for the experiment had no noticeable pattern in

increase of decrease in dry matter content.

44

Fig 4.2: Variation in dry matter content of sawdust at different levels of initial moisture

treatment during the 14 days period of fermentation.

Table 4.2 Changes in dry matter of the fermented substrate at different levels of initial

moisture treatment during the 14days period of fermentation.

TREATMENTS Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14

A 1:1 91.50 91.50 91.00 89.50 88.50 90.00 91.50 93.00

B 1:2(control) 90.50 91.00 91.50 91.50 91.50 92.00 91.50 91.00

C 1:3 90.00 90.00 89.00 88.50 88.50 89.00 90.00 90.50

D 1:4 90.00 90.00 89.00 88.00 87.50 89.00 89.50 90.50

87

88

89

90

91

92

93

94

0 2 4 6 8 10 12 14

%

Incubation period

Dry matter

A B C D

45

4.3 EFFECT OF INITIAL MOISTURE LEVEL ON THE MOISTURE CONTENT OF

THE SUBSTRATE

As revealed in Fig.4.3, the initial moisture level treatment of the substrate gave varying

levels of moisture content throughout the period of fermentation. Treatment A at Day 0 and

Day 2 maintained a stable level in moisture content at 8.50%, and showed a progressive

increase from Day 4 to Day 8 where the moisture content for this treatment peaked at 10.50%

from where there was a steady decrease till the final day of fermentation where the moisture

content of the substrate for this treatment was lowest at 7.50% seen on Day 14

Treatment C showed a similar pattern as revealed in Fig.4.3, Day 8 had the highest level in

moisture content at 12.00% and Day 14 having the lowest level of moisture content at 9.50%.

Treatment D (1:4) had the highest level of moisture content of 12.50% on Day 8 and 9.50%

on day 14.

Treatment B which served as the control of the experiment showed no exact pattern of

change in the moisture content throughout the period of fermentation, and had an average of

8.62% moisture content throughout the period of fermentation.

At the end the fermentation period for Treatment A, 26.08% increase was noticed from Day

0 to Day 8 which had the highest moisture level and a 39.13% decrease was seen from day 8

to day 14, similarly for Treatment C 16.66% increases was noticed from Day 0 to day 8

while a 20.83% decrease was noticed from day 8 to 14. For Treatment D 20% percentage

increase was notice from Day 0 to Day 8.

46

Fig 4.3: Variation in moisture content of sawdust at different levels of initial moisture

treatment during the 14 days period of fermentation.

Table 4.3 Changes in moisture content of the fermented substrate at different levels of initial

moisture treatment during the 14days period of fermentation.

TREATMENTS Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14

A 1:1 8.50 8.50 9.00 10.50 11.50 10.00 8.50 10.00

B 1:2(control) 9.50 9.00 8.50 8.00 8.50 8.00 8.50 9.00

C 1:3 10.00 10.00 11.00 11.50 12.00 11.00 10.00 9.50

D 1:4 10.00 10.00 11.00 12.00 12.50 11.00 10.50 9.50

6

8

10

12

0 2 4 6 8 10 12 14

%

Incubation period (days)

Moisture

A B C D

47

4.4 EFFECT OF INITIAL MOISTURE LEVEL OF THE SUBSTRATE ON ASH

As revealed in Fig.4.4, there was a progressive increase in the ash content of the substrate for

all experimental treatments. After the 14 days period of fermentation the Ash content for;

Treatment A Changed from 2.58% to 4.58%, for Treatment C, it changed from 2.58% to

4.90% while treatment D changed from 2.60% to 5.05%.

Fig 4.4 also revealed that the control (Treatment B) had no definite change and decreased

from 2.55% seen on day 0 to 2.42% noticed on day 14 .

The observed variations translate to increases of 43.67%, 47.35%, and 48.50% for treatment

A, C, D, respectively after the total fermentation period. Therefore the Highest Ash content

of 5.05% was seen on Day 14 in treatment D.

Treatment B which served has the control had no regular pattern and there was no noticeable

increase seen in the control, since the ash content of the substrate on Day 0 is 2.55% and the

resultant ash content on day 14 is 2.41%, the ash content after the period of fermentation

could be translated to give a 5.49% decrease in the control.

.

48

Fig 4.4: Variation in Ash content of sawdust at different levels of initial moisture treatment

during the 14 days period of fermentation.

Table 4.4 Changes Ash of the fermented substrate at different levels of initial moisture

treatment during the 14days period of fermentation.

TREATMENTS Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14

A 1:1 2.58 2.66 2.89 3.20 3.31 4.10 4.27 4.57

B 1:2(control) 2.55 2.58 2.50 2.58 2.46 2.56 2.31 2.41

C 1:3 2.55 2.55 2.93 3.32 3.50 4.42 4.42 4.97

D 1:4 2.60 2.99 2.99 3.37 3.72 4.72 4.72 5.05

1

2

3

4

5

6

0 2 4 6 8 10 12 14

%

Incubation period (days)

ASH

A B C D

49

4.5 EFFECT OF INITIAL MOISTURE LEVEL ON CRUDE PROTEIN

The result shown in Fig. 4.5 indicates a progressive increase in the crude protein content of

the substrate in all treatment. In Treatment A there was a steady increase in the crude protein

content of the substrate from day 0 to day 4,however a slight decrease was noticed on day 6,

and from day 8 to day 14 there was a steady increase. The crude protein increase for

Treatment A translate into 33.67% increase when the value gotten on day 14 is compared to

that of day 0

Treatment C as shown in Fig 4.5 showed a progressive increase in the crude protein of the

substrate, with the highest crude protein level seen at day 14, the total increase translating to

39.10% increase when the value gotten on Day 14 is compared to that of day 0

Treatment D also increased progressively as the fermentation progressed. After the entire

period of fermentation the highest crude protein values was recorded in Treatment D and

gave a 42.35% increase in crude protein when the value gotten on Day 14 (4.58) is compared

with the value gotten on Day 0 (2.64), as shown in table 4.5

Treatment B which severed as the control showed no regular pattern in its crude protein

content.

50

Fig 4.5: Variation in Crude protein content of sawdust at different levels of initial moisture

treatment during the 14 days period of fermentation.

Table 4.5 Changes Crude protein of the fermented substrate at different levels of initial

moisture treatment during the 14days period of fermentation.

TREATMENTS Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14

A 1:1 2.64 2.79 2.88 2.82 3.01 3.56 3.60 3.98

B 1:2(control) 2.64 2.55 2.61 2.68 2.34 1.89 1.92 2.01

C 1:3 2.64 2.82 3.12 3.27 3.31 3.59 3.89 4.34

D 1:4 2.64 2.89 2.97 3.39 3.42 3.62 3.93 4.58

1

1.5

2

2.5

3

3.5

4

4.5

5

0 2 4 6 8 10 12 14

%

Incubation period (days)

Crude protein

A B C D

51

CHAPTER 5

DISCUSSIONS AND CONCLUSION

5.1 DISCUSSIONS

According to Soma et al., (2011), lower moisture content causes reduction in solubility of

nutrients of the substrate, low degree of swelling and a high water tension. On the other hand,

higher moisture levels can cause a reduction in enzyme yield due to steric hindrance of the

growth of the producer strain by reduction in porosity (interparticle spaces) of the solid

matrix, thus interfering oxygen transfer.

The pH of the substrate was affected by the duration of mycotic fermentation as the acidity

increased progressively for all treatment including the control. Treatment D (1:4) had the

highest level of acidity with a pH of 4.60 after the 14 days period of fermentation while the

lowest acidity (highest pH) was notice in Treatments B and D on Day 0 having the same pH

of 6.20. The progressive decrease in pH noticed is similar to (Animashahun et al., 2013) who

reported a progressive decrease in pH of cassava residual pulp from day 0 to day 21.

According to Karr-Lilienthal et al. (2005), xylan (a polysaccharide pentosan found in plant

cell walls and woody tissue) contains a low proportion (approximately 4%) of side chains of

single units of D-glucopyranosyluronic acid residues attached to the main chain by 1→2

linkage. Upon hydrolysis, xylan yields a mixture of acidic sugars that are considered to be

constituents of most agro industrial by-products. This might be one of the reasons that

explain the acidic pH value of the substrate. It can thus be stated here that the highest acidity

seen in Treatment D (1:4) as compared to other treatments can be attributed to increase in

microbial activity brought about by to increased moisture content.

52

The moisture content of a feed material gives an indication of the extent to which the

nutritive value of the feed material can be maintained, otherwise known as its shelf life. Low

moisture content is therefore required for a longer shelf life. The gradual increase in moisture

content observed in all experimental treatment from Day 0 to Day 8 could be attributed

mycelia growth of the fungi (Bentil, 2012), this observation also agrees with Bano et al

(1986), who stated that mycelia of some fungi contain some amount of moisture. The decline

of the moisture content after the eighth day could be attributed to increase in microbial

activity that led to increase in nutrients utilization as well as water for growth (Bentil, 2012).

As show in Table 4.3 and Fig 4.3, Treatment A (1:1) had the lowest moisture content after

the entire period of fermentation, this could be attributed to the low initial moisture

treatment.

The ash content of the inoculated samples had a progressive increase throughout the period of

fermentation and the highest ash was seen on day 14, this trend was noticed among all

inoculated substrate (Fig.4.4 and Table. 4.4), the control had no regular pattern. The increase in

ash content may suggest the introduction of specific mineral(s) by the inoculum that was not

investigated. The highest percentage increase in ash was noticed in Treatment D among other

treatment which peaked on day 14 with a value of 5.05% translating into 48.5% increase when

compared to the control. The general increase in the ash content could be attributed to the

enrichment of minerals by mycelia of the fungi growing on the substrate. The fermentation

process generally impacted a positive effect on the total mineral content of the substrate

compared to the control that had no regular pattern and had a 5.49% decrease in its ash content

after the period of fermentation. This result obtained actually confirms the assertion that the

observed increase in the ash content of the inoculated sawdust was as a result of the mycelia

53

growth on the sawdust substrate. The 48.5% increase noticed in treatment on day 14 makes the

sawdust a very useful feed material for animals, as minerals in diets of animals play several

roles such as development of strong bones and also needed in metabolic processes preventing

certain health-related problems associated with deficiency of minerals (Church, 1976).

The result shown in Fig.4.5 indicates an increase in the crude protein content of the sawdust

fermented with Aspergillus niger as compared to the control. The optimum level of the crude

protein was attained at day 14 of fermentation in Treatment D (1:4) with a crude protein value

of 4.58% which translated into 42.35% increase when compared to day 0. It was realized that

there was an increasing trend of the crude protein content with fermentation time. However, the

increase in the crude protein content of the inoculated sawdust compared to the control

(uninoculated) throughout the fermentation period could possibly be attributed to the secretion

of some extracellular enzymes such as cellulases, xylanases, amylases and other lignin-

degrading enzymes which are all proteins in nature in an attempt by the fungus to utilize

available cellulose as source of carbon (Oboh et al., 2002) it could also be as a result of increase

in the growth and proliferation of fungal biomass in the form of single cell protein (SCP) or

microbial protein accounting for part of the increase in the protein content after fermentation

(Oboh, 2006; Oboh et al., 2002; Oboh and Akindahunsi, 2003).

From Fig 4.5 It was also observed that protein content of the substrate increased as initial

moisture level increased. This may be as a result of the absorption of the water by the sawdust

particles leading to the swelling of the substrates for easy penetration of the fungal mycelia,

facilitation of effective absorption of nutrients from the substrates for growth and metabolic

activities as was also observed by How and Ibrahim (2004); Ibrahim et al., (2012).

54

Proteins are macromolecules that serve as energy source when metabolized and also have

structural and mechanical functions such as actin and myosin in muscle and cytoskeleton

formation (Schwarzer and Cole, 2005). Metabolism of proteins yields amino acids which

enhances the growth and well-being of animals (Zagrovic et al., 2002). Thus the increase in the

protein content following fermentation with Aspergillus niger will contribute positively to the

utilization of sawdust by animals as feed material. A. niger has been reported to have high

specific activity for cellulases and hemicellulases (Howard et al., 2003).

5.2 CONCLUSIONS

This study, which was aimed at investigating the optimum conditions under solid state

fermentation for increasing some proximate component of sawdust especially crude protein

level, by varying the initial moisture level and utilizing Aspergillus niger as the fermentation

microbe was able to reveal the following:

The Optimum initial moisture level for increasing the crude protein content of

sawdust was observed in Treatment D (1:4) with maximum increase recorded to be

4.58% after a fermentation period of 14 days.

Solid state fermentation of sawdust with an initial substrate-moisture ratio of 1:4

could give a 42.35% increase after a fermentation period of 14 days using

Aspergillus niger.

Aspergillus niger is effective for the fermentation of sawdust, increasing the crude

protein and ash content of the substrates after some period of fermentation.

Solid state fermentation is an effective type of fermentation to be utilized for the

increase of crude protein of sawdust when utilizing Aspergillus niger

55

Although this study cannot be regarded as exhaustive, as there is need for further

investigations; the provided information from this study about the optimum conditions for

increasing the crude protein content can be utilized for other research works. It has provided

information on the optimum conditions for increasing crude protein content of sawdust and

has added to the knowledge base on the topic. The knowledge of this could help in further

research works and also in effective increase of the crude protein content of rice bran which

can be utilized by monogastric farmers in feeding their animals.

These results obtained have therefore rendered the sawdust more useful as animal feed

material particularly for monogastric animals, the extensive use of sawdust subjected to

solid state fermentation could therefore reduce the environmental pollution caused by this

agro-industrial by-product.

5.3 RECOMMENDATIONS

Notwithstanding the suitability of the fungi treated sawdust as feed material for animals, it is

recommended that:

(a) An assay should be conducted to ascertain the mycotoxicity level in the fungi treated

sawdust since the strain of Aspergillus niger used in the study was not specified whether or not

it produced Aflatoxin during the fermentation process.

(b) Secondly, an in vitro digestibility studies should be conducted to be able to ascertain the

level of fibre digestibility of the sawdust in terms of available total sugars.

(c) Finally, a large-scale fermentation of sawdust and its subsequent utilization in feeding trials

of broiler or layer chicks should be investigated.

56

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