PHYSICAL, CHEMICAL AND BIOLOGICAL TREATMENT OF RICE HUSK

PHYSICAL, CHEMICAL AND BIOLOGICAL TREATMENT OF

RICE HUSK TO IMPROVE ITS NUTRITIVE VALUE

RAHAT NASEER

2003-VA-196

A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE

REQUIREMENT FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

BIOCHEMISTRY

FACULTY OF BIO-SCIENCES

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES,

LAHORE

2015

To

The Controller of Examinations

University of Veterinary and Animal Sciences

Lahore

We, the Supervisory Committee, certify that the contents and form of the thesis,

submitted by Mr. Rahat Naseer, have been found satisfactory and recommend it to be processed

for evaluation by the External Examiners for the award of degree.

SUPERVISOR DR. ABU SAEED HASHMI

MEMBER DR. MUHAMMAD TAYYAB

MEMBER PROF. DR. HABIB-UR-REHMAN

In the Name of ALLAH

The Most Beneficent, The Most Merciful

i

DEDICATION

THIS ACHIEVEMENT OF LIFE IS DEDICATED TO MY MOTHER

Khalida Saleem

WHO ALWAYES PRAYED FOR ME, SUPPORTED ME AND INSPIRED ME

TO GO FOR HIGHER IDEALS

ii

ACKNOWLEDGEMENTS

In the name of almighty ALLAH, the inspirer of truth. All praise and gratitude is to

almighty “ALLAH” Who provided ease on my way, and gave me will, strength and health to

accomplish this research, who gave me the power to do, the right to observe and mind to think,

judge and analyze.

I bow my head before the HOLY PROPHET (P.B.U.H) who are a light of guidance and

role model for entire mankind.

I would like to extend my heartfelt gratitude to my respected supervisor, Dr. Abu Saeed

Hashmi, Eminent Professor, Faculty of Biosciences, UVAS whose excellent guidance,

constructive criticism, encouragement, learning me professional and applicable matters of field

and moral support that enabled me to develop an understanding of the subject.

I am grateful to the members of my Supervisory Committee Prof. Dr. Habib-ur-Rehman

Dean faculty of Biosciences and Dr. Muhammad Tayyab, Assistant Professor, Institute of

Biochemistry and Biotechnology Vice Chancellor, University of Veterinary and Animal Sciences,

Lahore for their patronage, valuable inputs, encouragement and unabated advice throughout the

study period and they gave moral support that enabled me to develop an understanding of the

subject.

I am also grateful to Dr. Saima Naveed for her technical assistance and contribution to

the fibre analysis and conducting in vivo trials and to Dr. Erum Hussain assistant professor

LCWU for her help in the characterization experiments with FT/IR. I would also like to express

my thanks to Dr. Toseef Hussain for his collaboration through the ultramicroscopic Studies.

I will always remember Faiza Masood, for our friendly PhD pep-talks and for the

technical and moral support in designing the fermentation experiments. Finally, my huge

appreciation for all the faculty members of the Biochemistry department for their cooperation,

and for accepting me as a relatively inert member of the department, especially during the final

stages of my Ph.D

iii

I am very grateful to my mentors Prof Dr. Makhdoomm Abdul Jabbar and DR.

SUALEHA RIFFAT for their unconditional support.

My loving thanks to my niece, Shanza and Samiya for adding a fresh breathe of non-

academic humor to my sometimes humdrum life.

I am indebted to my husband, Zulfiqar-ul-Hassan, for his never-ending patience, gentle

encouragement, kind technical assistance and acceptance of all sloppy standards at home and to

all my dearest Son Taha Hassan, whose company made dull moments bright and bearable.

Finally my heartfelt gratitude to all my spiritual teachers and mentors those have carved

on my heart determination and faith.

Rahat Naseer

iv

TABLE OF CONTENTS

DEDICATION--------------------------------------------------------- (i)

ACKNOWLEDGEMENT ------------------------------------------ (ii)

TABLE OF CONTENTS -------------------------------------------- (iv)

LIST OF TABLES --------------------------------------------------- (v)

LIST OF FIGURES -------------------------------------------------- (vi)

Sr. No. CHAPTERS Page No.

1 Introduction 1

2 Review of Literature 5

3 Effect of various treatment on chemical composition of rice

husk 46

4 Nutritive enhancement of Rice husk with Pleurotusflorida

through Solid state fermentation 58

5 Effect of acid and alkali on surface modification of rice husk 70

6 To investigate the feeding value of processed rice husk in

growing Lohi sheep 83

7 Summary 95

8 Annexures 97

v

LIST OF TABLES

Table No. Title Page No.

1.1 Chemical composition of untreated rice husk 53

1.2 Effect of acid on chemical composition of rice husk 53

1.3 Effect of alkali on chemical composition of rice husk 54

1.4 Effect of hydrothermal treatment on chemical composition of rice

husk

54

2.1 Composition of Basal medium for fungus growth 63

2.2 Rice husk composition after 7,14,21 & 30 day of SSF 63

3.1. Reference table showing group frequencies of respective function

groups

74

3.2. Absorbance of treated and untreated samples (SS1=untreated husk

SS2= 2%NaOH treated, SS3 =.5 N H2SO4 treated,SS4 = 4%NaOH

treated husk)

75

4. 1. Ration formulations of different experimental groups of lohi sheep 89

4.2. Nutritional Profile of different experimental Rations 89

4.3. Growth performance of Lohi sheep fed on differently treated rice

husk as a wheat straw replacer

90

4.4 Daily Feed intake of different experimental groups 92

vi

LIST OF GRAPH

Sr. No. Title Page No.

2.1 Chemical composition of untreated Basmati rice husk 64

2.2. Effect of incubation period on crude protein content of rice husk 64

2.3. Effect of incubation period on NDF content of rice husk 65

2.4. Effect of incubation period on ADF content of rice husk 66

2.5. Effect of incubation period on ADL content of rice husk 66

4.1. Weekly Weight Gain of Lohi sheep fed on different experimental rations 91

4.2. Weekly average feed intake of Lohi sheep fed on different experimental

rations

91

vii

LIST OF FIGURES

Figure No. Title Page No.

3.1 FT-IR spectra of treated and untreated rice husk 1= untreated husk,

2 =2% NaOH 3=.5%H2SO4, 4= 4% NaOH

76

3. 2 Electron micrograph of rice husk treated with 0.3 N H2SO4 76

3.3 Electron micrograph of rice husk treated 0.5 N H2SO4 77

3.4 Electron micrograph of rice husk treated with 2 %NaOH 78

3.5 Electron micrograph of rice husk treated with SEM images of 4

%NaOH

78

3.6 Electron micrograph of untreated rice husk 79

1

CHAPTER 1

INTRODUCTION

Rice grain (Oryza sativa) contains a rough and hard outer covering, called rice husk

which is inedible for human consumption and is also not being fully incorporated in livestock

feeding. Total production of rice is 6160 thousand tons in Pakistan (Pakistan economic survey

2014). As rice husk comprises 20% of the paddy rice, production of rice husk comes to 1540

thousand tons annually. Rice husk contains 35 percent cellulose, 20 percent hemi cellulose, 30

percent lignin, 18 percent pentose and 17 percent ash (Kumar, 2010). The silica embedded in the

matrix of lignin present in the outer wall of plant cell makes it a rigid structure (Park et al, 2003).

Rice husk is one of the by-products of rice threshing process. The husk is produced in the

first stage of the threshing process, when paddy rice undergoes threshing process the husk is

removed along other parts like rice polishing and bran. The percentage of rice husk in paddy rice

varies across different countries and this is influenced by various factors such as rice species,

cultivation area, soil fertility, weather, irrigation efficiency and farming practices (Bhattacharya,

et al. 1999). However, 20 percent is generally considered as a fair average for general rice husk.

Traditionally, rice husk obtained from rice thresher has been used as ingredient in

ruminant and poultry feeds but the problem of low nutrients digestibility, high silica/ash content

and abrasive characteristics are limiting factors in its utilization. According to Chun-Yang Yin

(2011) rice husk is composed of crude protein 5-6% oil, 8-12%, crude fiber 39-42% and ash

12%. Rice husk is underutilized and highly available resource. According to the International

Rice Research Institute, rice is one of the important staple food crops, daily food for more than

INTRODUCTION

2

3.5 billion people of the world. FAO (Food and Agriculture Organization of the United Nations)

has estimated world rice production of 700 million tons in 2010 (Binod et al, 2010). Since the

husk constitutes 20% of rice, vast quantities of rice husk is disposed of as waste, or burned in

open fields, polluting the environment (Kim et al, 2004).

The average composition of rice residues which includes straw and hulls is 32–47%, 19–

27% ,5–24%, cellulose hemicelluloses and lignin respectively (Binod et al, 2010), whereas in

rice hulls the percentage of cellulose is 36-40 % and hemi-cellulose is that of 12–19%

(Banerjeeet al., 2009; Saha et al., 2005; Saha and Cotta, 2007, 2008). Composition has fats

essential oils waxes, resins alkaloids, and other cellular components. The ash is 12% of total

residue mainly composed of silica (80–90%), trace amounts of Mg, Fe, Na, K2O, P2O5 (5%)

and CaO (4–1.2%) can be detected (Balconi Bevilaqua, 2010; Diel Rambo, 2009). This highly

complex nature of rice husk actually presents a potential hazard for the release of

polysaccharides.

Biochemical conversion of agricultural wastes into value added products is a subject of

great interest. To achieve this purpose it is required to modify the structure of non-digestible

carbohydrates such as cellulose and lignin to make them more vulnerable to the enzymes which

can hydrolyze into monomeric sugars. Various chemical and biological treatments can modify

the structure of agricultural residues in such an efficient manner so that ruminal enzymes can

hydrolysis embedded polysaccharides efficiently (Vadiveloo, 2000; 2003).

Several studies have been reported on utilization of rice straw as ruminant feed with or

without chemical treatment. (Shen et al., 1998, Abou-El-Enin et al., 1999) Supplementation of

rice straw with other feed stuffs or components and fermented rice husk in order to increase its

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INTRODUCTION

3

utilization were employed. Treatment regime includes various physical, chemical and biological

methods. Various methods were optimized and recommended but acceptance of these

techniques is less at farm level (Karunanandaa et al, 1992 b; Shen et al, 1999; Liu and Ørskov,

2000). Unfortunately rice husk received little attention and few studies are available. In general,

the use of rice straw and husk as an animal feed as well as its treatment is always cost

dependent for farmer (Selim et al., 2004)

Hydrothermal process proved to be an effective method of bioconversion of biomass. The

process has no environmental hazards and can be carried out even at the ambient temperatures.

(Hisaya et al. 2014)

The feeding value of the poor quality rice husk can be improved through various

physical, chemical and biotechnological methods (Selim et al, 2004).Now-a-days

biotechnological method due to their specificity and simplicity became a method of choice for

treating low quality roughages. This approach uses the appropriate microorganisms which have

ability to grow aerobically by solid state fermentation (SSF).Fungi are good candidate for this

purpose. These organisms have the capacity to grow using aerobic conditions moderately moist

substrate and optimum temperature. Solid state fermentation is an advantageous method to

degrade lignin and improve the digestibility. As fungi can secrete lignin degrading enzymes

which enhance the accessibility of hemi-cellulose and cellulose results in improved digestibility.

Fermentation of rice husk can be used as a vital source of energy for the production of cheap and

good quality biomass for poultry/livestock. At present, efforts are being made all over the world

by this method. The modern technological information regarding their culture, inoculation &

harvest has provided sufficient understanding and working tools for the mass production of

biomass protein for poultry and livestock.

INTRODUCTION

4

The aim of this study was to explore the effect of various treatments on surface and

composition of rice husk. The study was conducted using a narrow range of concentrations of

reagents so that their feeding value in small ruminants can be evaluated. Emphasis was placed on

developing a practical approach using water, acid, alkali and fungi to make it convenient for

farm use.

5

CHAPTER 2

REVIEW OF LITERATURE

Physical and Chemical Characteristics of Rice husk

Paddy rice (Oryza sativa) is grown on every continent, with the exception of Antarctica,

and the extent of paddy cultivation covers about 1 percent of the earth’s surface. Studies suggest

that more than half of the world’s population employ rice as a staple food and it is considered

second to wheat in terms of cultivation area and production. The quantum of global production

of paddy is close to 650 million tons per annum (www.maps of world.com).

Predominantly, Asia is the only continent, where rice can be cultivated during the rainy

season in the waterlogged tropical areas. Asia generates over 90 percent of world rice production

(Table 1 annexure attached). In Pakistan during 2013-14 the total production of rice was

cultivated on an area of 2789 thousand hectors with the yield of 5536 thousand tons. (Economic

survey of Pakistan 2014)

Rice husk can be defined as the outermost layer, which encases and protects a rice grain.

It is yellowish in color and has a convex shape. Typical dimensions are 4mm by 6mm.It is light

weight having a ground bulk density of 340 kg/m3 to 400 kg/m3. The exterior of rice husk is

composed of dentate rectangular elements (Mansaray and Ghaly, 2007).

The rice husk share almost same biochemical composition as that of other lingo-cellulose

wastes, that it has 40-50 % 25-30 % 15-20 % cellulose lignin and ash respectively. The moisture

content is 8- 15 percent (Prasad et al., 1998).


6

Longitudinal section of rice husk (www.fao.org/docrep/t0567e/t0567E07.htm.)


ruminant and poultry feeds but the problem of low nutrients digestibility, high silica/ash content

and abrasive characteristics are limiting factors in its utilization.

Cellulose

Anselme Payen in1839 discovered and isolated cellulose some 150 years ago. It is

abundantly available and largest polysaccharide on earth. It is the main structural component of

plants, providing support and shape to plants. It is major component of rice husk. Cellulose is

also present in bacteria, fungi, algae and even in animals. Cellulose is one of the principle

components of cell walls, which provide mechanical and chemical strength to plants. Cellulose is


7

synthesized during photosynthesis through metabolism(van Kuijk et al. 2014). Cellulose is also

main fibrous substance of plant cell wall.

Cellulose is a polymer of glucose. Glucose is present in the form of a disaccharide known

as cellobiose courtesy to β-1, 4-polyacetal linkage. The empirical formula of cellulose is

(C6H10O5) n.

Several characteristics of cellulose depends on its extent of polymerization (DP), means

how many glucose units are involved in making one polymer molecule. Generally the DP of

cellulose is between 800-10000 units; however, this number may extend to 17,000 units in some

cases (Kirk-Othmer, 2001).

The polymer is arranged in the form of long chains due to the nature of the β-

1,4glucosidic linkage that exists between the glucose molecules. The hydrogen bonds are present

on (Hussain et al. 1996)either sides of the structural monosaccharide which allow the formation

of hydrogen bonding. Consequently, the hydrogen bonds are responsible for the making a

polysaccharide composed of more than one parallel chains linked to each other (Faulon et al.,

1994).

Cellulose can exist in two forms crystalline and non-crystalline. The joining of several

polymer chains leads to the formation of micro fibrils. These microfibers are then joined to form

fibers which give cellulose a crystalline form.

Carbohydrates are the primary energy source for ruminants; due to its utilization as

ruminant feed, cellulose is an important and abundant carbohydrate. Cellulose comprises 20-40%

of dry matter of higher plants. The nutritional utilization of cellulose varies from totally


8

indigestible to completely digestible, depending largely upon lignifications. However, in addition

to lignifications, there are other inhibitors and limiting factors and intrinsic properties which alter

its digestibility. Because of these limiting factors and its intrinsic properties, digestibility of

cellulose in the digestive system is still not clear.

Hemi-cellulose

Hemi-cellulose is one of the major components of rice husk. It is a complex

polysaccharide that is composed of different sugars like xylose, glucose, mannose, arabinose and

sugar acids. The major portion of hemi-cellulose extracted from hardwood is xylan and from soft

wood is glucomannan respectively. Cropresidues composition is similar to hard wood.

Agricultural plants like grasses and straw also has xylan as a major component (Fengel and

Wegener, 1984 and Saha, 2003). In addition C6 and C5 sugars copolymer with hemi-cellulose

were also found in the plant cell wall.

Cellulose has higher molecular weight than hemi-cellulose. Hemicellulose has branches

with short side chains that consist of hydrolysable sugars (Fengel and Wegener, 1984).

Hemicellulose serves as a connection between the lignin and the cellulose fibers, making a

cellulose–lignin–hemicellulose network stronger and rigid (Laureano-Perez et al, 2005).

Temperature has a significant effect on solubility of the various hemicellulose

compounds. Solubility tends to increase with rising temperature. Galactose is the least soluble

whereas mannose is the most. Xylose glucose and arabinose occupy the middle tier. However,

the solubility of very higher molecular polysaccharide could not be estimated due to the

unknown melting points. The complete dissolution of hemicellulose in aqueous conditions starts

from 180 °C if conditions are neutral as described by(Bledzki et al. 2010). However some

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9

hemicellulose solubilizesat 150 °C is also reported (Garrote et al. 1999). The breakdown of

lignocellulose material depends on temperature, water activity and pH (Fengel and Wegener,

1984).

Acidic or Alkaline environment can facilitate the extraction of xylan from hemicellulose

whereas strong alkaline medium is required for the extraction of glucomannan (Fengel and

Wegener, 1984). Comparatively Xylan can be extracted easily.

Hemicelluloses if subjected to thermal-chemical treatment exhibit sensitivity(Isa et al.

2011)During the treatment the lateral groups of hemicellulose react first leading to the

breakdown of hemicellulose backbone

Lignin

Lignin after cellulose and hemicellulose is the abundant carbohydrate in nature and also

the part of rice husk. Lignin is a hetero polymer amorphous compound consisting of three

phenylpropane units namely p-coumaryl, coniferyl and sinapyl alcohol. All these units are linked

through different linkages mainly ß-O-4 and α-O-4ether linkage. Other linkages include 4-O-5

ether and carbon-carbon bond. The main function of lignin is to provide impermeability

structural support, oxidative stress and resistance against microbial attack to plants. The lignin

iswater insoluble and devoid of optical activity, unable to rotate the plane polarized light. All

these complex linkages make lignin a hard and resistant material for ruminal degradation (Fengel

and Wegener, 1984).

Lignin starts to dissolve into water at 180 C if conditions are neutral ((Isa et al. 2011).

The solubility of the lignin in different reagents like acid, alkaline or even in neutral conditions









10

rests upon its precursor from which they are formed which could be either p-coumaryl or

coniferyl, or sinapyl alcohol or all of them ((Bazargan et al. 2014).

Lignin is a polymer of aromatic ring containing compounds which can be formed through

a metabolic pathway and functions as a protective layer for the plant cell walls. In nature across

the year these substances grow and decay. It is estimated that around 7.5x1010 of cellulose are

consumed and then regenerated every year (Kirk-Othmer, 2001), thereby, making it the most

usable organic compound in the world.

Apart from the three basic chemical compounds, cellulose hemicellulose and lignin,

water is also part of rice husk. In addition to these small amounts of proteins, minerals and other

components can be found in the rice husk composition.

Fiber is a nutritional entity which is defined as much by its biological properties as its

chemical composition (Van Soest et al. 1993). With regard to forages, it has been traditionally

defined as the complex of dietary nutrients that are relatively resistant to digestion and are slowly

and only partially degraded by herbivores (Van Soest 1982). By this definition, fiber is

composed of structural polysaccharides, wall proteins, and lignin. The main anti quality role of

lignin in forages is in limiting digestion of the structural polysaccharides cellulose and

hemicellulose (Moore and Hatfield 1994).

Lignification controls the amount of fiber that can be digested and, therefore, has a linear

and mostly significant effect on the digestible energy (DE) and importance of the agricultural

residue (Jung and Allen 1995). Lignification also influences the amount of dry matter that can be

consumed by an animal (Mertens, 1994). The undigested portion of the forage passes slowly

through the digestive system and contributes to the fill effect of the diet. The greater the


11

concentration of degradable fiber in the diet the less dry matter an animal can consume.

Therefore, lignifications impacts forage nutritive value by both decreasing DE concentration and

limiting dry matter intake (Moore et al. 1993).

Lignin is a distinct chemical entity of plant cell walls and has been recognized for over

100 years (Sjostrom 1981) however research is still in progress regarding its structure,

biosynthesis, and measurement. Anabolism of lignin is a complex process which is manifested in

the resultant complexity of lignin molecules. While generalized structures for lignin have been

drawn, it is not yet possible to definitely determine the complete structure of any isolated lignin

molecule, let alone the structure of lignin in the plant cell wall. Because there is no well-defined

and standard reference lignin structure is available measurement of lignin concentration is

relative and very much depends on methodology. All of these ambiguities have made it very

difficult to clarify the roles of lignin in plant growth and development, and the mechanism by

which lignin limits cell wall digestibility.

Lignin can be characterized as a polymer formed in phenyl-propanoid pathway derived

from mono-lignols in vascular plants. Some reviews provide excellent coverage of the process

and biochemistry of lignification (Baucher et al. 1991). Lignin is deposited in the plant cell walls

as part of the cell maturation process after cell elongation has ceased. Based on difficulty of

lignin extraction from the cell wall, it has been concluded that lignin is chemically bonded to

carbohydrates and probably to proteins in the cell wall to form complex macromolecules. The

only cross-linking structure of lignin to other cell-wall components that has been definitively

characterized is the bonding of lignin to arabinoxylans in grasses via ferulic acid molecules

(Ralph et al. 1995), even though chemical extraction information does indicate ∝-ether bond of


12

lignin directly to polysaccharides (Watanabe 1989). Undoubtedly there are other lignin cross-

linked structures that involve polysaccharides and proteins both.

Lignin found in gymnosperms is 95 % Guaiacyl-type lignin whereas angiosperms

generally deposit significant amounts of both guaiacyl- and syringyl-type lignins. Small amounts

of p-hydroxyphenol lignin are present in most plants (Lapierre et al. 1988). As more lignin

mutants and transgenic plants involving the lignin pathway have been characterized, it has

become apparent that this pathway is actually more of a web than linear (Sewall et al. 1997b).

Because of this web structure and the ability of plants to incorporate non-typical

phenylpropanoid precursor molecules, the results of biotechnological manipulation of the

pathway have yielded unexpected results and unique lignin structures (Orlandi et al. 2014).

Apparently lignin plays such an important role in plant development that alternative

routes and precursors can be used to provide the amount of lignin necessary for normal

development. When lignin concentration has been significantly reduced through biotechnology,

nonviable plants result. Deposition of lignin in the cell wall of grasses appears to involve ferulate

esters of arabinoxylans as nucleation sites. In annual ryegrass (Loliummultiflorum Lam.), lignin

cross-links to ferulates consisted only of structures that would form if monolignols reacted with

the ferulates directly rather than polymeric lignin reacting with ferulate esters (Orlandi et al.

2014).

Unlike ferulic acid, p-coumarate (the other major cell wall hydroxycinnamic acid

ingrasses is esterified to lignin as compare to other polysaccharide which has

lignin/polysaccharide cross-linkage. Because of complex lignin structure and linkage to other

cell-wall polymers, analysis of lignin concentration in forages is difficult. The standard method


13

used in animal and agronomic sciences is the acid detergent lignin (ADL) method, of which there

are sulfuric acid hydrolysis and permanganate oxidation versions of the method (Van Soest 1967,

Goering and Van Soest 1970). The traditional lignin method used in wood chemistry, Klason

lignin, was long believed to give inaccurate results with forages because of perceived

contamination (Van Soest 1967). It has now been shown that the Klason lignin method does not

suffer from contamination when applied to forages and that the ADL method under-estimates

lignin concentration (Lowry et al. 1990).

Several mechanisms have been suggested for how lignin may inhibit cell-wall digestion,

however, it is now generally agreed that lignin simply acts as a physical barrier to the microbial

enzymes reaching their target polysaccharides (Chesson 1993, Jung and Deetz 1993). Questions

still remain as to how lignin can cross-linked to other cell-wall carbohydrate, and deposition and

distribution in the wall may modify the impact of lignin as a physical barrier to cell-wall

digestion. This is perhaps best illustrated by the fact that while the negative relationship of lignin

concentration is always observed when examined across forage samples of different maturities,

when plant maturity is similar large differences in lignin concentration and cell-wall digestibility

are observed but lignin and digestibility are often not correlated (Jung and Deetz 1993).

Obviously there must be modifying factors which influence the inhibition caused by lignin on the

digestion of cell wall, especially severe for grasses.

The relationship between lignin degradation and temperature was investigated. Horwath

and Elliott (1996) conducted an experiment using ryegrass for 45 days at 25°C and 50°C. The

amount of lignin degraded after compositing was 7% and 27%, respectively Klason method was

used. Although the elemental ratio will remain unchanged at 25°C and 50°C a change is

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observed in residual lignin and it was calculated that 6% of the residual Klason lignin was

remained unchanged after composting (Horwath and Elliott, 1996).

Silica/Lignin interaction

Primarily silica and lignin are the major drawback of rice husk. Silica is an inert element,

frequently found in earth crust. Silicon is a nutrient and plays variety of roles in rice formation

ranging from carbohydrate and phenol synthesis to cell wall protection. All these factors

ultimately affect the grain yield. The interactions between these factors minimize association of

digestibility between silica and lignin when different varieties were compared. Grain yield is

highly related to amount of silica in straw which is in turn associated with its availability in soil.

There are no thorough investigations on rice straw and rice husk lignin is conducted so far

especially in India and Pakistan. Lignin reported is actually acid-detergent, lignin the terms can

be used interchangeably, if estimated through the sulfuric acid or permanganate forms. Soluble

phenols in straw needs further study. It is reported that ammonia and urea can crack the silica

studded cuticle. Although silica shows solubility in sodium hydroxide but not in urea and

ammonia is does not get dissolved in these reagents in comparison to the action of sodium

hydroxide (Van Soest 2006).


15

Digestibility

Different treatments with rice straw and on other lingo cellulosic materials were reported

but very few studies were available on rice husk. Urea is used most frequently followed by

ammonia and sodium hydroxide. Steam and pressure treatments along with acid and white rot

fungi were also reported (Beg & Shah 1986). Feed supplementation and growth studies with

young animals were conducted. Traditionally use of urea in India and other Southeast Asian

countries is more popular than ammonia, although urea is less effective than ammonia (Hussain

1996). Farmer approval depends on expenses, manual labor, tools, health and safety. The various

studies involving animal digestion using in sacco, in vitro digestions techniques with rumen

microorganisms or cellulase, or in blend with pepsin has been reported. Gas formation during in

vitro rumen fermentation has also been used. Outcomes are stated in terms of total dry matter

rather on organic matter. It is hard to relate the results because techniques require

standardization. However, most of the treatments with ammonia and urea display little rise in

digestibility and feed consumption when determined in vivo trials. It is noticed that in vitro and

in sacco assessments show exaggerated enhancement in digestibility (Khanum 2010).

White- rot Fungi

The white-rot fungi are categorized in the major division named Eumycota means true

fungi first subdivision is Basidiomycotina, Hymenomycetes, Holobasidiomycetidae are class and

subclass respective(Rodr et al. 2011).This subclass encompasses almost all the wood-decay fungi

for example mycorrhizal, litter, mushrooms, puffballs, decomposer fungi, conks and other crust

like fungi. It is found that at one step below the subclass, the biological difference among

representatives is less, particularly in functional variability; nevertheless, at the level of family


16

occasional cases of functional variability have been recorded. Within a genus very less diversity

is found as far as biological function is concerned.

Fungi are the primary agents that degrade lignocellulose (Sanchez 2009) however

species in the genus can differ in their ability to perform specific functions. Additionally, not

only do they secrete enzymes that are vital for the decomposition of lignocellulose material, but

fungal growth using lignocellulose as a substrate is encouraged by the formation of structure

called mycelia that permit filamentous fungi to carry nutrients like nitrogen and iron, to the

polysaccharide portion of lingo-cellulosic substrate or rice husk (Hammel 1997).

Many fungi are also more resistant to wood-derived biocides that limit bacterial growth.

These compounds include tannins and various phenolic compounds (terpenes, stilbenes,

flavonoids and tropolones) that are particularly abundant in the hardwood of fallen trees. The

majority of wood-degrading fungi that have been characterized to date are members of the

phylum Basidiomycota and are characterized by either brown-rot or white-rot decay.

White-rot fungi secrete enzymes that breakdown lignin, hemicellulose and cellulose,

giving residual wood fiber a decolorized appearance. White-rot decay can follow two patterns of

decay have been differentiated by microscopic and structural investigations (Liese 1970). One

pattern is called simultaneous white-rot or “corrosion rot”. It causes a collective degradation of

polysaccharide and lignin at start and end of the wood decay process. Examples of fungi which

show simultaneous white-rot include Phellinus robustus, Fomesfomentarius, andTrametes

versicolor (Blanchette 1984, Blanchette 1994). By contrast selective (sequential) white-rot decay

follow the early degradation of lignin and hemicelluloses and then that of cellulose.

Ceriporiopsissubvermispora and Phlebiaradiata are perhaps the best studied fungi to elicit


17

selective white-rot decay (Blanchette, 1991)). Sequential white-rot fungi “selectively” degrade

lignin and hemicellulose in small tubules within a wood tissue such that decayed areas found

inside and intact tissues are found in periphery (Blanchette 1984). Wood acquires a fibrous

texture by progressive decay and delignification of primary cell wall (Schmidt 2006).

Importantly, whether it is simultaneous or sequential white-rot decay decisive factors will be

wood type, stage of wood decay, and fungal strain being used in the study (Messner and

Srebotnik, 1994). For example some strains of Phanerochaete chrysosporium (e.g. BKM-F-

1767), produce selective decay of major deciduous wood plant while few strains can cause

simultaneous wood decay (Blanchette 1992).

Lignocellulose-active enzymes that are produced by white-rot fungi are particularly

valuable for biomass conversion, since theycan be used to selectively transform both lignin and

polysaccharides (Kirk and Cullen 1998). The enzymes that contribute to this activity can be

broadly classified as Carbohydrate-Active enzymes (CAZymes) and oxidative fungal Lignin

enzymes (FOLymes) (Cantarel et al. 2009, Levasseur et al. 2008).

Carbohydrate Active Enzymes

A sequence-based classification scheme for carbohydrate-active enzymes was developed

in 1991, called the CAZy database (CArbohydrateen ZYme database) (Cantarel et al. 2009;

Henrissat 1991). At present, this database is comprised of 125 glycoside hydrolase families, 92

glycoside transfer families, 22 polysaccharide lyase families and 16 carbohydrate esterase

families. Glycoside hydrolases hydrolyze the glycosidic bonds between α-linked or β-linked

sugars, using a retaining or inverting mechanism (Davies and Henrissat 1995). Polysaccharide

lyases break polysaccharide form a double bond at non-reducing end by β-elimination whereas


18

carbohydrate esterases catalyze the deacetylation and demethylation of substituted

polysaccharides. Efficient disaggregation of carbohydrates requires collective interactions

between enzymes responsible for breaking the different linkages. Significant research has been

done to demonstrate and understand synergy between various isolated enzymes for degradation

of microcrystalline cellulose (Avicel) and commercial xylans (de Vries and Visser 2000). For

instance, hydrolysis of xylan by an Aspergillus xylanase was increased in the presence of

accessory enzymes that catalyze the hydrolysis of xylan side chains (Paszczynski et al. 1988).

Cellobiohydrolases, endoglucanases and β-glucosidases act on cellulose hydrolyze it and

form glucose molecules. All the three enzymes worked at different locations endo-cellulases act

on the internal position by hydrolyzing glycosidic cellulose molecules, Cellobiose is released

from the reducing or non-reducing end of cellulose by the action of cellobio-hydrolases and

further hydrolyzed into glucose by β-glucosidases. These enzymes when work together is

efficient enough to break amorphous and crystalline form of cellulose. To act upon polymeric

substrate two enzymes namely endo-cellulases or cellobiohydrolases are often linked with

cellulose-linking module to enhance their activity on polymeric substrates (Kirk and Cullen

1998). Of the 125 GH families, fungal cellulases belong to GH families 5, 6, 7, 9, 12, 44, 45, 48,

61 and 74 (Dashtban et al. 2009).

Oxidative enzymes also take part in cellulose degradation along with hydrolytic enzymes

(Kirk and Cullen 1998). Quinones radicals and phenoxy radicals reduces themselves in to

cellobiono-δ-lactone in the presence of cellobiose, enzyme is quinone oxidoreductase.

Respective acids were formed from cellobiose and longer cello-oligomers using molecular

oxygen and cellobiose oxidase. Wood hemicelluloses include xylan, (gluco) galactomannan, and

xyloglucan. Many polysaccharides have a linkage β-1-4 in its carbohydrate backbone with


19

acetylation or substitution in sugar branches. Given the diversity of hemicelluloses some

glycoside hydrolases and carbohydrate esterases participate in their disaggragation. (Scheller and

Ulvskov, 2010). For instance, xylan breaking that involves the activity of various enzymes for

example galactosidases, arabinofuranosidases, deacetylases, glucuronylesterases,

feruloylesterases and xylanases. Like cellulases, most of the actions work in collaborations

produces a collective effect associated with carbohydrate linkage modules that enhance enzyme

activity on substrates (de Vries et al. 2000; 14 Hervé et al. 2010). So far, fungal hemicellulases

were identified in nineteen GH families: 1, 2, 3,5, 10, 11, 26, 27, 36, 39, 43, 51, 53, 54, 62, 67,

74,115, and 116, and nine CE families: 1, 2, 3, 4,5, 6, 12, 15 and 16.

Fungal Oxidative Lignin Enzymes

Similar to carbohydrate-active enzymes, enzymes involved in lignin catabolic enzymes

can be divided into sequence-based classes, named the Fungal Lignin Oxidative enzymes

(FOLy) (Levasseur et al. 2008).Important enzymes for lignin degradation are Laccases and

peroxidases which are extracellular, lingo-lytic enzymes (ten Have and Teunissen, 2001). The

peroxidases include one is lignin peroxidase (LiPs) and second is manganese-dependent

peroxidase (MnP). Substrate is oxidized in two step reaction one electron oxidation step at a

time.The intermediate cation radical is formed. Enzymes for the reaction are Both LiP and MnP

(Sanchez 2009). LiP break down 90 % of non-phenolic lignin polymer whereas MnP produce

Mn3+, which can diffuse into phenolic and non -phenolic units of lignin. This diffusion is

facilitated by peroxidation of lipid (Cullen and Kersten 2004; Moen and Hammel, 1994).

Laccases are also called blue copper oxidases. These enzymes catalyze reduction of O2 to

H2O by oxidizing the phenols or aromatic amines or various electron-rich substrates. (d'Souza et


20

al. 1999). Laccases oxidize the phenolic units present in lignin and converting it into to phenoxy

radicals resulting into aryl-C cleavage (Kawai et al. 1988).Some phenolic substrates, xylan,

(galacto) glucomann, can also be oxidized by laccases but presence of some auxiliary substances

like 2,2´-azino-bis-3- ethylthiazoline-6- sulfonate is mandatory(Call and Muncke 1997).

We can categories fungal decay types according to their mode of degradation into three

groups. Namely brown rot, white rot and soft rot fungal decay. White rot is further divided into

two classes one is simultaneous rot and other is selective delignification whereas soft rot is

further divided into two classes’ types 1 and 2.White rot and brown rot can be differentiated on

the basis of their capacity to oxidize phenolic compounds extracellular.

During selective delignification, lignin is first to be degraded followed by cellulose and

hemicellulose degradation. As the hyphae grows into the cell luminary lignin oozes out of the

adjacent cell walls. Hyphae can also go into the cell walls separating the cells and then de-lignify

them. Selective delignification in some cases can decompose large volume of lignin leaving

cellulose almost unchanged, generally in the initial stages of decay white rot fungi that

selectively de-lignify the parenchyma tissue in contrast to brown rot (Schwarze 1995; Schwarze

et al. 2004).

A specific type of decay by fungi reported in chili named as “palopodrido’’where

residual lignin after decay is 0.9 % and cellulose is 97 %.So palopodrido became a general term

for all the selective and non-selective removal of lignin by white rot decay, however advanced

delignification is termed as paloblanco (Blanchette 1984). The structural changes in lignin during

white rot examined by thioacidolysis showed structural changes in lignin syringyl unit which

have ß-aryl ether-linkage.


21

Among the different types of the Genus Pleurotus, oyster mushrooms, consist of range of

edible mushrooms for example P.sajor-caju, P. citrinopileatus, P. ostreatus, 5and p. florida

available in the market believedto have some antitumor substances. The white oyster mushroom,

P. florida, is commercially available at the local markets during moderate temperature periods of

year. The analysis of fruiting body of P. florida show 37.19% protein, 3.72% fat, and 10.98%

ash on dry matter basis. This mushroom also contains neutral lipids, glycolipids, and

phospholipids making almost 0.5 % total lipids. Supplemented rice straw can act as a substrate

for fruit bodies of P. florida (Zhao, 2010).

Scanning Electron Microscopy

Scanning electron microscopy and other analytical techniques provided useful

information about husk surface features, organizational pattern and distribution of silica. It is

apparent that rice husk topology share a peculiar features with irregular boarders studded with

silica.

The outer and the inner epidermis of rice husk differ greatly in their topology and

composition which effect their interaction with each other as well as with reagents. From the

standpoint of topography and composition of cell walls the two surfaces of husk, i.e. the outer

epidermis and inner epidermis are likely to differ markedly in their interaction with each other as

well as with other reagents (Ang et al, 2012).

Rice husk has a complex structure in which outer epidermis and lignified fiber provide

stiffness strength and rigidity to husk. These properties help the husk to withstand harsh

environmental conditions like high wind. Treatments like grinding and steam explosion can

expose the husk internal tissues, although these tissues vary greatly in thickness and



22

composition. Thick walled fibers are less flexible than thin walled fibers. Thus, physical

properties and composition are important factors to be considered while designing treatment

strategies for rice husk (Schwarze, 2007).

Silica distribution in rice husk suggests that silica is either exposed on the surface or

embedded in the internal tissues. Surface silica require coupling agent like silane compounds,

however if internal silica is involved agents like maleic anhydride propylene will be used.

Fourier transform infrared spectroscopy (FT/IR) is a molecular vibrational spectroscopic

technique. This technique optically investigates the molecular changes of the substance. This

method provides a reliable and conservative way to investigate the bonding molecular

conformation and functional group presence in tissues cells and any other substance. That can be

used to optically probe the molecular changes associated with the tissues. The method is

employed to find more conservative ways of analysis to measure characteristics within tissue and

cells that would allow accurate and precise assignment of the functional groups, bonding types,

and molecular conformations. Specific bands in vibrational spectra are indicative of biochemical

composition. FTIR peaks express the vibration or bending of specific chemical bond of

functional group in a molecule (Coates, 2000). FTIR mainly used for dehydrated samples due to

strong absorption bands of water. It records the changes in dipole moment during molecular

vibration.


23

Treatments of rice husk

The composition of different lignocellulose material depends on its source. The lignin

and (hemi) cellulose component vary greatly with respect to its source.

Biochemical conversion of lignocellulose biomass into value added products is a subject

of great interest. To achieve this purpose it is required to modify the structure in such a manner

that accessibility of cellulose towards enzymes can be increased resulting in the conversion of

carbohydrate polymers into fermentable sugars (Mosier et al., 2005). Various chemical and

biological treatments involve the modification of lignocellulose biomass for rapid and easy

enzymatic hydrolysis of cellulose and hemicellulose.

Chemicals which can enhance the use of rice straw and rice husk can be either alkali, acid

or oxidative agents. Alkaline agents are most studied and enjoy greater acceptance at farm

level(Shreck 2013). The mechanism of alkaline action is the absorption of alkali through cell

wall breaking the ester bonding between cellulose/hemi cellulose will result in the swelling of

the tissues. This process facilitates the ruminal microflora action on polysaccharides enhancing

the digestibility and palatability. Alkaline agents of common use are ammonia, urea and sodium

hydroxide. (Prasad et al., 1998; Shen et al., 1998) There are several advantages of chemicals for

on farm use. Chemicals are cheap, easy to handle and no specialized equipment is required. This

advantage includes safety precautions and toxicity. Sodium hydroxide treatment is in practice

since a long time (Selim et al., 2004). Treated straws show remarkably increased digestibility

but for a small scale farmer, it’s still an expensive choice. High concentrations can cause

environmental hazards due to high discharge of sodium into the environment (Vadiveloo, 2000;

Sundstøl and Coxworth, 1984).


24

Biological treatment includes the use of fungi on their enzymes for the selective

delignification of ligno cellulose material that will enhance the digestibility. Although in the

developing country it is difficult to implement these strategies on farm level, however the

scenario is becoming bright day by day. The use of fungi and/or their enzymes that metabolize

lignocelluloses is a potential biological treatment to improve the nutritional value of agricultural

residues by selective delignification, as mentioned in the review by (Wang et al. 2014). The

Problems faced with use of fungi are toxic production and difficulty in optimizing the growth

conditions like temperature, pH, pressure, O2 and CO2 for the treatment of fodder (Schiere and

Ibrahim, 1989). Latest developments in fermentation technology and development of alternative

enzyme production system promises a key roles in future ruminant production systems(Soccol &

Vandenberghe 2003).

White-rot fungi treatment: White-rot fungi can be used to treat lingo cellulosic material.

(Eriksson et al., 1969).Its degradation potential was utilized to increase the nutritive value of

fodder for ruminant nutrition (Howard et a, 2003). Decomposition of free phenolic monomers

and degradation of lignin polysaccharides cross linkage in rice straw can be carried out white rot

fungi (Chen et al, 1996), enhancing IVDMD (Karunanadaa and Varga, 1996). Reports suggest

that 30 day incubation with white rot enhance the IVDMD of rice straw both for leaves and stem.

Comparative studies suggest highest IVDMD when treated with Cyathusstercoreus (Soest,

2006).The mechanism of action is same for almost all the species (Chen et al., 1996,

Karunanadaa and Varga, 1996a).

Crop residues usually have poor digestibility. This limit the use of crop residues as fodder

hence has a negative impact on animal production. Pond et al. (1980) Using white-rot fungi to

increase the degradability of straw is often at the expense of easy assessable carbohydrates, such


25

as cellulose and hemicellulose, resulting in less degradable feed for ruminant. Chemical

treatments can increase digestibility of poor quality crop (Anderson, 1978). Many studies are

conducted despite poor acceptability (Klopfenstein, 1978; Ben-Ghedalia, et al., 1983).

Cellulose is found in both primary and secondary cell walls. The monomeric unit is

glucose, which is linked by β1-4 glycosidic bonds in a linear polymer. Extent of polymerization,

or the measure of number of glucose molecule that make up one polymer molecule, ranges from

2,000 to 6,000 in the primary cell wall and can increase to greater than 10,000 in secondary cell

walls (Delmer, 1987). As a plant matures, the increase in polymerization leads to greater strength

and lower digestibility. In contrast to cellulose, xylans are more heterogeneous. In a forage cell

wall, polymers12 of linked β1-4 xylose are found, as well as arabinose, glucuronic acid, and

galactose residues (Wilkie, 1979). Bailey (1973) defined hemicelluloses as structural

carbohydrates that are not pectin or cellulose based. Determination of xylans and cellulose can

be determined by gravimetric, enzymatic-gravimetric, and enzymatic chemical methods. The

detergent fiber system, originally proposed by Van Soest (1963), is a gravimetric based analysis

that is the standard for fiber quantification for ruminants.

Neutral detergent fiber (NDF) solution solubilizes non -structural plant polysaccharides,

leaving hemicellulose, cellulose, and lignin. The disadvantages of NDF system include: all that is

solubilized is not structural carbohydrate, as well as interference from starch, fat (Buckner et al.,

2013), or protein. Acid detergent fiber (ADF) solution solubilizes hemicellulose and the

remaining residue is composed of lignin and cellulose.

Van Soest (1982) characterized polysaccharides in the plant cell wall as belonging to two

classes based on biological associations and nutrient availability. One class includes

polysaccharides covalently bonded to core lignin and partially fermented and other includes


26

those that are not bonded, soluble, and completely fermentable. The association of

polysaccharides with lignin was also considered as the primary factor limiting cell wall

digestibility. Additionally, it was proposed that the crystalline nature of cellulose and low surface

area for cellulose attachment as other impairments to fiber utilization (Sarnklong et al. 2010).

Forage digestibility

Silica can increase depression in digestibility and this phenomenon is studied in oat

plants grown hydroponically with different levels of silica (Van Soest and Jones 1968). Early

studies by (Smith et al, 1971) revealed that silica lowers organic matter by one unit. The study

was conducted using eight different species of grasses. A study was conducted using Bermuda

grass, reed canary grass, and rice straw with neutral detergent by Van Soest (1981) revealed that

removal of silica can increase NDF digestibility of organic matter per unit of silica removed.

Silica was dissolved in significantly high concentration and alfalfa was used as a negative

control. Two preliminary reports from the International rice institute (IRRI) in the Philippines

issued two preliminary reports indicated no effect of silica upon digestibility of rice straw (Lim

et al. 2012). Studies by Enishi (2002) showed that each unit of silica reduces organic matter

digestibility about one unit or more which is in agreement with NDF digestibility. The data was

not precise but showed a definite relationship between silica and digestibility

Studies by Hasan et al, 1993 compared straws with different lodging reaction in relation

to silica content and digestibility. In one study four varieties were studied and in another study

twelve varieties were studied.

Several studies report silica and digestibility on whole straws with little correlation. An

inverse relationship was found when rice was grown hydroponically with and without silica


27

addition. Silica with ash content was also directly proportional (Abou-El-Enin et al., 1999;

Agbagla-Dohnani et al, 2001).

Treatment with ammonia can form silicic acid but its polymerization and precipitation at

physiological pH is very slow (Van Soest et, 1971). Studies have investigated the inhibitory

effects of silicic acid upon cellulose and also on in vitro digestibility (Hartley, 1981; Smith and

Nelson, 1975; Smith and Urquhart, 1975).

Soluble silica whose nature is not well under stood can produce negative effects on

animal health. The intake of soluble silica has been linked with urinary siliceous stones mostly in

drier regions where water shortage may occur (Mahgoub et al, 2000). There have been no

definitive studies in India and Pakistan regarding the formation of urinary calculi

(Krishnamoorthy, personal communication). However, oxalate is known to be fermented in the

rumen and not thought to present a problem for ruminants. Vadivelloo and Fadel (1992) report

90 g/kg ytterbium which is a perceptible phenolic in rice straw. Ytterbium can also precipitate

oxalate.

Lignin contains guaiacyl groups which plays role in solubility. This cleaved lignin

although indigestible in ruminant but due to water solubility offers no resistance to digestion

(McBurney and Van Soest, 1984;Van Soest, 1994).Urea is also able to break lignin to

polysaccharide ester bond .It is different to show relation between lignin and digestibility in

untreated straw samples however Agbagla-Dohnani et al. (2003) conducted experiments with 14

samples and significant relationship between lignin and digestibility was found whereas Abou-

El-Enin et al. (1999) El emin with 53 samples showed non-significant results. It is postulated

that lignin is also exhibiting varietal variation just like silica.


28

The maturity of the plants is also found to be major player in determining the

digestibility. It is also emphasized that at maturity less variation is found (Lapierre et al., 1989).


29

STATEMENT OF PROBLEM

Using the crop residue like rice resources as a substrate to produce biomass through

chemical/biochemical treatments will substantiate fodder deficiency gap in Pakistan. Structural

modifications will pave the way for other value added products. The present study will be

conducted under the following main objectives:

1. To optimize the different conditions of hydrothermal treatment and its evaluation.

2. To improve the quality of rice husk by physical & chemical procedures.

3. Production of quality fungal protein using white rot fungus for quality fungal from rice

husk under standard laboratory conditions.

4. Production of different treated husk at large scale

To determine efficacy of different husks in small ruminants in terms of growth performance


30

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46

CHAPTER 3

EXPERIMENT 1

EFFECT OF VARIOUS TREATMENT ON CHEMICAL COMPOSITION OF RICE

HUSK

ABSTRACT

Rice husk obtained from the local threshing mill was first analyzed for total fiber contents, silica

and ash contents. Three reagents acid (sulphuric acid), alkali, (sodium hydroxide) and water were

selected. Three different concentrations of acid (0.1N,0.3N,0.5N) and alkali (2%,4%,6%) at three

different temperatures(25°C,70°C,100°C) were used having same reaction duration(24 hour).

Hydrothermal treatment was carried out with three different time duration (30 min, 60min, and

90min) and temperature (25°C, 70°C, 100°C).Reagent loading was optimized. All the samples of

rice husk after the acid, alkali and hydrothermal treatment were analyzed for fiber content, silica

and ash. The results show that temperature has a significant effect on the fiber content. After the

alkali treatment a significant reduction in the neutral detergent fiber (NDF)was observed (p ≤

0.005). All these treatments had little effect on lignin & silica content.

INTRODUCTION

Rice husk is mainly composed of cellulose, hemicellulose and lignin. In order to utilize

these carbohydrate moieties as fermentable sugars one need to breakdown or modify them. These

modifications can provide an easy access to reticulo-rumen microflora. Traditionally, rice husk

obtained from rice thresher has been used as ingredient in donkeys and horse feeds but the

problem of low nutrients digestibility, high silica/ash content and abrasive characteristics are

limiting factors in its utilization.

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Acid treatment can hydrolyze cell wall specially its hemicellulose component. This hydrolysis

facilitates the release of cellulose and lignin component. Alkalis have great potential to modify

the cell wall. Solvation and saponification are the main reactions involved.

Previously several studies have been reported on the physical and chemical properties

rice straw and rice husk (Shen et al., 1998). Moreover, various methods of physical, chemical

and biological treatments have been investigated to upgrade its utilization. These strategies

including supplementation with other feed stuffs or component (Selim et al., 2004). These three

components require cellulase, hemicellulase andligninase for their breakdown (Schiere and

Ibrahim, 1989). Animals cannot produce these enzymes but in the rumen they maintain the

population of microorganism which can produce cellulose and hemicellulose. Lignin will remain

undigested due to the lack of ligninase. Lignin impacts the livestock production through effects

on digestibility and feed intake. Lignin and its associated Phenolic monomers reduce microbial

carbohydrate degradation dramatically (Van Soest 1965, Jung & Deet3 93, Allen95). Both

concentration and composition of lignin appears to affect digest ability. Lignin can be classified

as core and noncore lignin. Core lignin is described as three dimensional structures composed of

condensed phenyl propanoid units that limit digestibility by encrustation. Non-core lignin, which

is the part of the total phenolic content, is soluble in alkali. A more likely factor reducing fiber

degrade ability is the stearic hindrance caused by lignin and polysaccharide linkages which limit

access of fibro lytic enzymes. As non-core lignin is soluble in alkali, the success of chemical

treatment by hydrolytic agents would be partially attributed to reducing polysaccharide-lignin

associations.

Silica is one of the elements of husk; present in relatively high concentrations can range

from 5% to 15%, depending on the variety of rice and type of soil (Agbagla-Dohnani et al.,

EXPERIMENT 1

48

2003). Silica acts as a barrier to the digestibility of rice straw or rice husk in the rumen by

reducing palatability.

Technology for the chemical treatment of crop residues likely comes from paper making

techniques which were introduced as early as 12th century. Since that time, a variety of methods

have been developed to improve the treatment through hydrolytic or oxidative means. Physical

treatments such as particle size reduction, steam treatment have also been used. A combination

of both is possible. Ideal chemical treatment must be asked to be increase intake or digestibility,

non-toxic or non-hazarders to handle and benefits that outweigh the costs associated with

treatment. Hydrolytic agents improve the digestibility by action of OH group disrupting cell wall

structure and increased swelling resulting in increased microbial attachment. Core lignin is

usually not affected by hydrolytic treatments but bonds between lignin and hemi-cellulose can be

broken and hemicellulose solubilization can likely to be occurred.

Treatment with water is also called Auto hydrolysis or hydrothermal processing. Water is

the cheapest reagent for these treatments and can be used under farm conditions easily. Only

hemicellulose degradation is expected resulting into sugar monomers and oligomers.

Alkali agents have the capacity to get absorbed making structural fibers to swell. This

process will chemically break down the ester bonds between lignin, hemicellulose and cellulose

(lanetal 2001). These processes provide enabling environment to rumen microflora to attach

them self easily with structural carbohydrates increasing digestibility and palatability of the

agricultural residues (Selim et al 2004).

Dilute acid hydrolytic is a common method used to treat lingo, cellulosic wastes. The

treatment with dilute (0.1-1%) H2SO4 at high temperature results degradation of hemicellulose,

while it has little effect on lignin. However treatment can disrupt interactions between lignin and

EXPERIMENT 1

49

cellulose resulting in the increased susceptibility for enzymatic hydrolysis. The main purpose of

the proposed study is to explore action of various treatments to the rice husk for enhancement of

its digestibility by ruminants.

MATERIAL AND METHOD

Raw material

Rice husk was obtained from local thresher rice mills and shifted to fermentation

laboratory of biochemistry department. Rice husk was kept in plastic container.

Acid Treatment

Rice husk was treated with three different concentrations of sulphuric acid 0.1N, 0.3N

and 0.5 N. 10 gram of rice husk was weighed and 50 ml of acid of each concentration was added.

All the samples were kept at 37°C, 70°C and 100°C for 24 hours. Treated husk will be analyzed

for ADF, NDF lignin and silica content. All the samples were run in triplicates.

Alkali Treatment

Rice husk was treated with three different concentrations of sodium hydroxide 2%, 4%

and 6%. 10 gram of rice husk was weighed and 80 ml of alkali of each concentration was added.

All the samples were kept at 37°C, 70°C and 100°C for 24 hours. Treated husk will be analyzed

for ADF, NDF lignin and silica content. All the samples were run in triplicates (Ebrahimet

al.2013).

Hydro thermal treatment

Rice husk will be treated by soaking in water with the ratio of 1; 8 .10 gram husk was

weighed and soaked into 80 ml of water and subjected to 37°C,100°C and 200°C for 30, 60 and

90 min. Degree of Treated husk will be analyzed for ADF, NDF lignin and silica content. All the

samples were run in triplicate (Hisaya et al. 2014).

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RESULTS AND DISCUSSION

Rice husk is predominantly composed of cellulose, hemicellulose lignin & silica. They

are amorphous in nature and connected with each other via 1-4 B bonding in cellulose. Lignin is

present between the cellulose chains making it more compact. Analysis results of husk as shown

in table 1 shows the cellulose content in rice husk is more than lignin and hemicellulose.

The results of acid treatments showed (Table 1.2) a significant change in percentage of NDF(p≤

.000),ADF(p≤.000) and ash(p≤.000) whereas non-significant results was shown with the

lignin(p≤.09) due to mild temperature conditions and silica(p≤.987) content.

This acid hydrolysis is in line with the other studies carried out earlier(Sun and Cheng,

2002).The sulphuric acid has dissolved most of the hemicellulose, increasing the susceptibility of

cellulose and no effect on lignin. Hydrolysis of cellulose and hemi cellulose would be converted

to their monomers. This ultimately would reduce the percentage of neutral detergent fiber (NDF)

and acid detergent fiber (ADF) as suggested by Bazargan and colleagues (2014).The major

disadvantage is the cost of the acid used and its corrosive character.

Effect of hydro thermal treatment on biochemical composition of rice husk was investigated.

Results showed (Table 1.4) a significant change in percentage of NDF (p≤ .000),ADF

(p≤.000),silica and ash(p≤.000) whereas non-significant results was shown with the

lignin(p≤.936) for time duration and (p≤0.008) for temperature. The decrease in silica content is

perhaps due to excessive washing by water. The results were in line with the results of previous

studies (Taherzadeh and Karim 2008).

Hydrothermal treatment can increase swelling of the cell wall resulting in the increased

microbial attachment. Core lignin is usually not affected by the treatment but bonds between

EXPERIMENT 1

51

lignin and hemicellulose can be broken. The mode of the action primarily related to the

solubilization of hemi cellulose. Hemicelluloses contain a variety of sugar units which can form

physical barriers surrounding the cellulose fibers Hence the removal of hemicellulose can

expose more hydrophilic hydroxyl groups of the cellulose. As treatment temperature is increased,

the removal of hydrophobic lignin can occur. This is accentuated by more exposure of cellulose

as suggested by Ndazi and colleagues in 2008.

Sodium hydroxide has been the principal base produced by the chemical industry. As a

chemical treatment agent, it is often the standard by which other treatments are compared to. In

the present study treatment of alkali showed significant change in the rice husk composition

(Table 1.3) as compared to untreated husk. NDF (p≤ .000), ADF (p≤.000), silica (p≤.131) and

lignin (p≤.003) showed significant whereas non-significant results as shown were shown with

the ash (p≤.880).The unusual high ash value was attributed at high alkali concentration (Detailed

results are shown in annexure 2).

There are various reasons which could be attributed to the change in the composition

after the alkali reaction. Rice husk is composed of lignin, cellulose, hemicellulose, silica and ash.

The outer layer of the husk is also covered with waxes and protective pectin and other impurities.

(Johar et al 2012) from among these components, the waxes and lignin are relatively

hydrophobic (Vanholme et al 2012). Hence the presence of lignin or waxes within the biomass

decreases hydrophobicity (Baumberger et al 1998) under moderate temperatures.

Alkali treatment can remove the protective waxes on the outer layer of the husk and thus

increase moisture content. This is counter-balanced by the removal of hemicellulose which is

composed of various matrix hydrophilic polysaccharides such as xylose and arabinose. The

hemicellulose polymers with more branches have stronger hydrophilic tendencies and are

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52

dissolved more easily in alkali media (Yang Het al 2013).. Moreover treating rice husk with

dilute solutions of NaOH will wash the impurities and also purifies the cellulose moiety present

in the rice husk. It has concluded that all the three treatments effect the composition of rice husk.

Hemicellulose is first to hydrolyze changing the percentage of NDF and ADF significantly.

However higher temperatures, longer treatment, and higher concentration of reagents can remove

some of the lignin content, whereas at lower temperatures the lignin will not degrade

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53

Table 1.1 Chemical composition of untreated rice husk

Dry Matter 95.492 ±1.91

Moisture 4.288 ±2.03

Crude protein 2.846667 ±0.65

Crude fiber 31.24196 ±0.335

Ether extract 2.72 ±0.13

ash 13.55167 ±1.8

ADF 53.66667 ±0.47

NDF 70 ±0.81

Cellulose 31.5 ±0.4

hemicellulose 17.66667 ±3.29

Lignin 10.06667 ±0.24

Silica 9.766667 ±0.169

Table 1.2 Effect of acid on rice husk composition.

Parameters Acid conc. 37°C 60°C 100°C

NDF 0.1N 68.74333 53.65667 70.94667

0.3N 71.08333 48.79 68.81

0.5N 59.9 60.73 47.48333

ADF 0.1N 54.25 54.94667 53.77667

0.3N 55.89 50.83667 50.78333

0.5N 50.69667 58.6 26.15667

lignin 0.1N 19.91 21.1 20.38667

0.3N 19.60667 19.58667 19.81333

0.5N 18.75333 18.50667 19.79667

silica 0.1N 10.96 10.67 10.71

0.3N 11.10 10.43 10.74

0.5N 12.55 11.18 9.50

ASH 0.1N 13.86 15.00 15.04

0.3N 14.07 15.23 15.75

0.5N 14.95 14.98 15.57

Acid treatment carried at three different temperatures and acid concentrations time duration

24 hour (all the values are average of three)

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Table 1.3 Effect on alkali on rice husk composition

Parameters Alkali Conc. 37°C 70°C 100°C

NDF 2% 70.93333 73 71.15

4 % 69.6 52.65667 66.56667

6 % 74.01333 70.33333 53.70667

ADF 2 % 55.44 41.41667 55.66667

4% 43.25667 39.05 43.45

6 % 58.11667 61.58333 39.82333

LIGNIN 2 % 18.875 18.675 15.175

4 % 18.19667 18.65 17.53667

6 % 18.88333 20.36667 15.93667

SILICA 2 % 10 9.5 12.175

4 % 9.256667 10.03333 11.08

6 % 10 10.20667 11.73

ASH 2% 14.33333 22.89 23.77667

4% 21.12 35.98333 33.71667

6% 44.75667 48.12333 44.86667

Alkali treatments carried at three different temperatures and alkali concentrations time duration

24 hour (all the values are average of three)

Table 1.4 Effect of hydrothermal treatment on rice husk composition.

Parameter Time (min) 37°C 180°C 200°C

NDF 30 71.18 76 54.47333

60 66.03667 67.46667 52.91667

90 69.34 56.45667 58.66667

ADF 30 54.86667 51.78333 57.82333

60 53.1 47.33333 58.6

90 55.6 42.35 58.85

Lignin 30 20.21667 19.70333 18.01333

60 20.13333 17.90667 19.91

90 20.35 18.15 21.5

silica 30 9.7 9.116667 8.423333

60 9.95 8.44 10.28333

90 9.25 7.6 12.45

Ash 30 14.91 14.04 11.48

60 15.28 12.37 11.28

90 15.23 11.80 10.45

Hydrothermal treatment carried at three different temperatures for three different time

duration pH of tap water 6.5 (all the values are average of three)

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REFERENCES

Agbagla, A.D. P. Noziere, G. Clement and M. Doreau, In sacco degradability, chemical and

morphological composition of 15 varieties of European rice straw, Anim Feed SciTechnol,

94, 15 (2001).

AOAC. 1990. Official Methods of Analysis. Association of Analytical Chemists (15th Ed.)

Artington Virginia, USA.

Baumberger S, Lapierre C, Monties B, Della Valle G. 1998. Use of kraft lignin as filler for starch

films. Polymer Degradation and Stability, 59(1), 273-277.

Bazargan A, Gebreegziabher T, C. Hui. 2014. The effect of alkali treatment on rice husk

moisture content and drying kinetics. Biomass and Bioenergy. 70: 468-475.

Bioethanol (2010) http://bluefirethanol.com

Chung YY. 2011. Production of higher heating values of biomass from proximate and ultimate

analyses, Fuel. 9(3): 1128-1132

Johar N, Ahmad I, Dufresne A. 2012. Extraction, preparation and characterization of cellulose

fibers and nanocrystals from rice husk. Industrial Crops and Products, 37(1), 93-99.

Ju X, Engelhard M, Zhang X. 2013. An advanced understanding of the specific effects of xylan

and surface lignin contents on enzymatic hydrolysis of lignocellulosic

biomass. Bioresource technology, 132: 137-145.

Kim TH, Kim JS, Sunwoo C, Lee YY. 2003. Pretreatment of corn stover by aqueous ammonia.

Bioresource Technology, 90(1): 39-47.

Ndazi BS, Nyahumwa CW, Tesha J. 2008. Chemical and thermal stability of rice husks against

alkali treatment. Bio Resources, 3(4), 1267-1277.

http://bluefirethanol.com/

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Pasha TN. 1998. Feed resources for livestock and poultry in Punjab, Punjab, Pakistan. Germany

agency for technical cooperation (GTZ), Livestock and Dairy Development Department,

Government of the Punjab, Lahore.C.

Sarnklong, Cone JW, Pellikaan W, Hendriks WH. 2010. Utilization of rice straw and different

treatment to improve its feed value for ruminants.A review.Asian.Aust J Anim. 23(5):

680-692.

Selim ASM, Pan J, Takano T, Suzuki T, Koike S, Kobayashi Y, Tanaka K. 2004. Effect of

ammonia treatment on physical strength of rice straw, distribution of straw particles and

particle-associated bacteria in sheep rumen. Anim Feed Sci Technol. 115:117-128.

Srubar WV, Frank CW, Billington SL. 2012. Modeling the kinetics of water transport and

hydroexpansion in a lignocellulose-reinforced bacterial copolyester. Polymer, 53(11),

2152-2161.

Taherzadeh MJ, Karimi K. 2008. Pretreatment of lignocellulosic wastes to improve ethanol and

biogas production: a review. Int J Mol Sci. 9 (9): 1621–1651

Vanholme R, Morreel K, Darrah C, Oyarce P, Grabber JH, Ralph J, Boerjan W. 2012. Metabolic

engineering of novel lignin in biomass crops.New Phytologist, 196(4): 978-1000.

Vimal K, Tripathi L, Iatinder P, Sehgal, Puniya AK, Singh K. 2007. Effect of administration of

anaerobic fungi isolated from cattle and wild blue bull (Boselaphustragocamelus) on

growth rate and fiber utilization in buffalo calves. Arch AnimNutr. 61: 416- 423.

Yang H, Chen Q, Wang K, Sun RC. 2013. Correlation between hemicelluloses-removal-induced

hydrophilicity variation and the bioconversion efficiency of lignocelluloses. Bioresource

technology, 147: 539-544.

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Yu J, Zhang J, Liu Z, Yu Z. 2009. Combinations of mild physical or chemical pretreatment with

biological pretreatment for enzymatic hydrolysis of rice hull. Bioresource Tech. 100,

903–908.

H.S. Shen, F. Sundstol and D.B. Ni, Studies on untreated and urea-treated rice straw from three

cultivation seasons, Evaluation of straw quality through in vitro gas production and in

sacco degradation measurements, Anim. Feed Sci. Technol, 74, 193 (1998)

S. Van and P.J, Use of detergents in analysis of fibrous feeds. III. Study of effects of heating and

drying on yield of fiber and lignin in forages. J. Assoc. Off. Anal.Chem, 48,785 (1965)

H.G. Jung, and D.A. Deetz, Cell wall lignification and degradability, In H.G. Jung, D.R. Buxton,

R.D. Hatfield, and J. Ralph, eds. Forage Cell Wall Structure and Digestibility. ASA-CSSA-

SSSA, Madison, WI, USA, 315 (1993)

Y. Sun and J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production, Department

of Biological and Agricultural Engineering, North Carolina State University, Bioresource

Technology 83, 1 (2002)

58

CHAPTER 4

EXPERIMENT 2

Nutritive enhancement of Rice husk with Pleurotusflorida through Solid state fermentation

ABSTRACT

Rice husk is one of the abundantly available agricultural wastes in Pakistan. It is always

desirable to use rice husk in ruminant or poultry feed but low protein content and high degree

lignifications are major hazards. In this study we will try to overcome these barriers. Solid state

fermentation (SSF)was the technique employed to ferment rice husk. The strain of white rot

fungi, Pleurotus florida, was provided by fermentation lab of institute of Biochemistry and

Biotechnology (IBBt). The Tien and Kirk basal media was used and sample were taken after

7,14,21 and 30 days. Other experimental conditions like temperature (28C), pH(5.4),water

substrate ratio(35%) were pre-optimized. Analysis for crude protein (CP), neutral detergent fiber

(NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) was carried out.All the

samples were run in triplicate. An increase of 400% in protein content and 70 % decrease in

lignin was achieved after 21 days of incubation.

INTRODUCTION

Rice paddy (Oryza sativa) contains a rough and hard outer covering, called rice husk

which is inedible for human consumption and is also not being fully incorporated in livestock

feeding (Kumar et al 2010).


donkey feeds but the problem of low nutrients digestibility, high silica/ash content and abrasive

characteristics are limiting factors in its utilization. Rice husk is underutilized and highly

available resource. The composition of rice residues including straw and husk has 32-47%, 19-

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27% and 5-24% cellulose, hemicellulose and lignin respectively (Beg et al, 2010). It also have

considerable high amount of silica almost 80% in addition to that 5% of K2O & P2O5 , 4% of

CaO and small amounts of Mg, Fe, and Na is present (Mansary&Galy 1999). The highly

complex nature of rice husk is a barrier for its potential usage.

The feeding value of the poor quality rice husk can be improved through various

biotechnological methods (Selimet al., 2004). Biotechnological methods are simple and specific.

Hence they are methods of choice for improving the quality of poor agricultural residues (Mosier

et al, 2005). The use of appropriate microbe using Solid State Fermentation (SSF) is one of them.

Microbes like fungi are able to degrade lignin, hence increasing rumen microbial accessibility

which in-turn enhance the digestibility. Fermented rice husk can be used for the production of

cheap and good quality protein for poultry/livestock. At present, efforts are being made all over

the world by this method. The modern technological information regarding their culture,

inoculation & harvest has provided sufficient understanding and working tools for the mass

production of biomass protein for poultry and livestock.

White rot fungi are composed of a group of fungus that are capable of degrading

heterogeneous polyphenolic compound called lignin, which is present in huge amount within the

lignocellulose wastes including rice husk (Hendriks 2008). The degeneration of lignin does not

provide net energy so the polysaccharides and carbohydrates complexes present in lignin are

generated during secondary metabolism, which are inaccessible to organisms (Sanchez 2009).

White rot fungi secrete one or more three extracellular enzymes namely manganese peroxidase,

lignin peroxidase and laccase that are fundamental for degradation of lignin, and they are

generally mentioned as lignin modifying enzymes LMEs (Hammel 1997).

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Solid state fermentation (SSF) can use rice husk and straw, wheat bran, fruit and

vegetable wastes, bagasse, synthetic media, coconut coir and paper pulp as substrate. It requires

low water content than the submerged fermentation (Ikram, 2003). All the substrates which can

be utilized are nutrient rich waste materials. So this slow and steady technique not only utilize

rice husk well, but also made it possible to release the nutrients in a controlled manner.

Fungi are the best choice for solid state fermentation due to less requirement of water as

compared to bacteria, which require high water activity (Jahromi, 2010).

The aim of the present study is to explore the rice husk nutritional enhancement attained

through solid state with white rot fungus.

MATERIALS AND METHODS

2.1 Source of microorganism:

Pleurotus florida strain of white rot fungus was obtained from Fermentation Laboratory

of IBBt UVAS, Lahore.

2.2 Maintenance of organism:

The fungus was maintained on Kirks and Tein media slants.

2.3 Inoculum preparation:

The inoculum prepared for the fermentation media was through inoculation of Pleurotus

florida from a slant into autoclaved 100mL Kirk’s and Tein liquid broth. The inoculated flask

was placed on shaker at 130 rmp at 28°C till the OD reached 1.6 at 620 nm. It was used further

to inoculate fermentation batch (Bakkiyaraj et al. 2013).

The fiber and crude protein analysis of substrate

The proximate analysis (crude protein, ash content, NDF, ADF and ADL) of rice husk

was carried out according to AOAC (1990) methods.

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2.4. Solid state fermentation

Basal media was prepared by adding components enlisted in table 1. The procedure was

carried out by optimizing various components of the fermentation process. 100 grams of rice

husk was added in all the twelve flasks. Flasks were sealed with cotton plugs and aluminum foil.

The flasks were autoclaved for 15 min at 121ºC. After autoclaving the flasks were cooled down

and inoculums was added under sterilized conditions Fermentation was carried out using pre

optimized conditions of temperature 28°C and pH 5.5 with 35 % water substrate ratio. The

samples were collected after 7, 14, 21, and 30 days and subjected to crude protein and fiber

analysis.


The experiment conducted showed mycelium covered the husk after 10 days. Fruiting

bodies were not observed after 27 days which is in line with the previous studies.Highest CP

content was after 21 days which showed a decreased trend onward due to the utilization of

available sugars. Fungal treatment reduces the NDF, ADF non-significantly and ADL

significantly. The percentage of NDF and ADF is not the direct measure of digestibility. The

voluntary intake of feed decreased as the percentage of hemicellulose increased.

Up to 27 days very small change in total ash content was observed, which shows little organic

matter loss. Organic matter loss was observed in other studies when rice straw, Sago fiber or

sawdust was fermented using white rot fungus (Vadiveloo 2003a). However this loss is attributed

due to the Primary metabolism and secondary metabolism of the soluble and structural

carbohydrates respectively. Studies with rice straw using Pleurotussajor-caju, (Cohen et al.

2002) suggested that microorganism got accessible to the hemicellulose as the fungus degrade

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lignin and facilitate the digestion (Karunanandaa et al. 1992),significant increase in crude protein

is due to fungal biomass (Hadar et al. 1992) which can be a good source of protein, if

incorporated in lambs diet (Cobos et al., 2002).

White-rot fungi (WRF) are capable of degrading lignin without affecting much of cellulose and

hemicelluloses (Vadiveloo et al 2009) thus causing decayed residue to turn white. WRF attack

unaltered lignin polymers causing cleavage of inter lignol bonds and aromatic ring cleavage,

which ultimately results in an increase in in vitro digestibility (Vadiveloo et al 2009). They

mainly degrade poly-saccharides by hydrolytic enzymes like cellulases and xylanases, and lignin

by oxidative ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP)

and laccase. White-rot fungi are the most efficient degraders of lignocellulose as they can

degrade cellulose and hemicellulose, as well as lignin (Schmidt 2006).

Lignocellulose-active enzymes that are produced by white-rot fungi are particularly

valuable for biomass conversion, since they can be used to selectively transform both lignin and

polysaccharides (Kirk and Cullen 1998). Using ligninolytic fungi, including their enzymes, can

be a potential alternative to provide more practical and environmental-friendly approach for

enhancing the nutritive value of rice husk.

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Table 2.1 Composition of Basal media for fungus growth.

Sr. No Components (g/100 ml)

1 KH2PO4 0.21

2 CaCl2 0.04

3 MgSO4 0.03

4 Glucose 1.5

5 (NH4)2SO4 0.2

6 Yeast Extract 0.4

Table 2.2 Rice husk composition after 7, 14, 21 and 30 day

of solid state fermentation (SSF) with Pleurotus florida

Day CP NDF ADF ADL

0 3.11± 0.02 69.89±0.84 54.68±0.50 9.73±0.32

7 5.4±0.12 68.62±0.86 53.54±0.33 8.93±0.06

14 7.11±0.09 66.07±0.20 52.65±0.51 7.24±0.2

21 12.98±0.08 63.96±0.61 50.9±0.85 6.87±0.13

30 9.25±0.28 67.13±1.27 51.06±0.62 6.52±0.34

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Graph.2.1 Chemical composition of untreated basmati rice husk

Graph2.2 Effect of incubation period on crude protein content of rice husk at 28°C pH 5.5

0

20

40

60

80

100

120

Series1

0

2

4

6

8

10

12

14

0 day 7 day 14 day 21 day 30 day

%

CP

CP

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Graph2.3.Effect of incubation period on NDF content of rice huskrice husk at 28°C pH5.5

60

61

62

63

64

65

66

67

68

69

70

71


%

NDF

NDF

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Graph2.4 Effect of incubation period on ADF content of rice huskrice husk at 28°C pH 5.5

Graph2.5 Effect of incubation period on ADL content of rice huskrice husk at 28°C pH 5.5

49

50

51

52

53

54

55


%

ADF

ADF

0

2

4

6

8

10

12


%

ADL

ADL

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REFERENCES

Akinfemi A, Adu OA, Doherty F. 2010. Conversion of sorghum stover into animal feed with

white-rot fungi: Pleura/us ostreatus and Pleura/us pulmonarius. Afr J Biotechnol.

9: 1706-1712.

AOAC. 1984. Official Methods of Analysis, 14th ed.Washington, DC.

Beg S, Zafar SI, Shah FH. 1986. Rice husk biodegradation by Pleurotusostreatusto produce a

ruminant feed. Agric.Wastes 17:15–21.

Bakkiyaraj S, Aravindan R, Arrivukkarasan S, Viruthagiri T. 2013. Enhanced laccase production

by Trameteshirusta using wheat bran under submerged fermentation.Int J Chem

Tech Res. 5: 1224-1238.

Cohen R, Persky L, Hadar Y. 2002. Biotechnological applications and potential of wood-

degrading mushrooms of the genus Pleurotus. ApplMicrobiol. Biotechnol. 58:

582–594.

Cobos MA, Garcia LE, Gonzalez SS, Barcena JR, Hernandez DS, Perez-Sato M. 2002.The effect

of shrimp shell waste on ruminal bacteria and performance of lambs.Anim Feed

Sci Technol. 95, 179–187.

Hadar Y, Kerem Z, Gorodecki B, Ardon O. 1992. Utilization of lignocellulosic waste by the

edible mushroom, Pleurotus. Biodegradation 3: 189–205.

Ikram UH, Barque. 2003. Optimization of growth conditions of Arachniotus species on rice

polishings for its protein enrichment. JAnimPlant Sci. 13: 73-77.

Karunanandaa K, Fales SL, Varga GA, Royse DJ. 1992. Chemical composition and

biodegradability of crop residues colonized by white-rot fungi. J Sci Food Agric.

60, 105–112

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Kirk, T. K. & Cullen, D. (1998).Enzymology and molecular Genetics of Wood degradation by

White-Rot Fungi. Environmentally Friendly Technologies for the Pulp and Paper

lndustry. Hoboken, NJ: John Wiley & Sons

Hendriks ATWM, Zeeman G. 2008. "Pretreatments to enhance the digestibility of lignocellulosic

biomass."Bioresource Technology 100(1): 10-18.

Hammel KE (1997) Fungal Degradation of Lignin. In: Cadisch G, Gillier KE (eds) Driven by

Nature: Plant litter quality and decomposition. CAB International, pp 33-45

Jahromi MF, Liang JB, Rosfarizan M, Goh YM, Shokryazdan P, Eo YW. 2010. Effects of

Aspergillus Niger on nutritive value of rice straw African J Biotech. 9: 7043-7047

Mansaray, K.G. &Ghaly, a. E., 1997. Physical and Thermochemical Properties of Rice

Husk.Energy Sources, 19(9), pp.989–1004.

Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M. 2005.Features of

promising technologies for pretreatment of lignocellulosic biomass.Bioresource

Technology 96 (6):673-686

Park BD, Wi SG, Lee KH, Singh AP, Yoon TH, Kim YS. 2003. Characterisation of anatomical

features and silica distribution in rice husk using microscopic and micro-

analytical techniques. Biomass Bioenergy 25: 319–327.

Ramos LP. 2003. The chemistry involved in the steam treatment of lignocellulosic materials.

Quim. Nova 26, 863–871.

Selim, A.S.M., J. Pan, T. Takano, T. Suzuki, S. Koike, Y. Kobayashi and K. Tanaka

(2004).Effect of ammonia treatment on physical strength of rice straw, distribution of

strawparticles and particle-associated bacteria in sheep rumen.Anim Feed Sci

Technol.115: 117–128.

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Sanchez C (2009) Lignocellulosic Residues - Biodegradation and bioconversion by fungi.

Biotechnology Advances 27:185-194

Schmidit O (2006) Wood and Tree Fungi. Biology, Damage, Protection and Use. Springer-

Verlag, New York

Vadiveloo, J. (2003a). Solid-state fermentation of fibrous residues. J Anim Feed Sci. 12, 665–

676.

Vadiveloo, J., B. Nurfariza and J. G. Fadel (2009). Nutritional improvement of rice

husks. Animal Feed Sci Tech. 151(3): 299-305.

70

CHAPTER 5

EXPERIMENT 3

Effect of acid and alkali on surface modification of rice husk

ABSTRACT

Rice husk is a notable fraction of lingo-cellulosic waste, abundantly available in rice producing

countries. Its utilization in ruminant feed is always center of interest in agricultural communities.

Various methods of its utilization are adopted in far and near past. In the present study the acid

(H2SO4) and alkali (NaOH) were used to treat the rice husk. The dissolved sugars were not

recovered. The objective of the study was to explore implication of the acid and alkali treatments

on rice husk composition and structure that will ultimately have an impact on the rice husk

digestibility. The treatment was carried out using two reagents: sulphuric acid (H2SO4) and

sodium hydroxide (NaOH), in three different concentration (0.1N, 0.3N, 0.5N and 2%,4%,6%

respectively) and temperature ranges(25°C,70°C,100°C). Compositional analysis of untreated

and treated rice husk was carried out. Surface modification was assessed using Fourier transform

infrared spectroscopy (FT/IR) and Scanning electron microscopy (SEM). The results showed that

even a mild treatment with acid and alkali produces visible changes in the topology. Certain

bond vibrations were also demonstrated, although little variation was observed in compositions.

This study showed that a mild treatment, which is otherwise harmless for ruminant consumption,

can be used to alter the topology and bond vibrations of rice husk, making it an easy target for

ruminal micro flora.

INTRODUCTION

Rice husk is one of the important agricultural wastes, abundantly available and has the

potential to be converted into various value added products. As the production of rice is

increasing in developing industries, the amount of husk is also increasing causing disposal

problem for farmers. (Lim et al. 2012).Both acid and alkali act as a reagent of choice for treating

lingo-cellulosic residues for the simple reason of being cheap and easy. Plant cell wall is

susceptible to acid particularly their hemicellulose component. To attain the desirable hydrolysis

it is important to determine the optimum concentration, temperature and amount of reagent used.

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Alkali has also the potential of modifying cell wall, which is well documented. Alkali acts by

two mechanisms, one is solvation and other is saponification. These two process results in the

swelling of biomass, which make inner space, access an easy task. (Bazargan 2014)Alkali

treatments also reduce lignin partially. The other constituents of cell wall namely xylan and

lignin supports the cellulosic backbone of cell wall, forming a stable matrix. As xylan and lignin

support the cellulose back bone that constitutes the cell wall biomass. Although ammonia has

been widely used leading to environmental issues. In the present study we used NaOH which is a

cheaper source than ammonia. (Selim et al 2002).

The composition of rice husk is 32.24%, 21.44% and 21.34% of cellulose, hemicellulose and

lignin. Although composition varies from one variety to another, and even seasonally in a single

variety (Isa et al 2011) the cellulose and hemicellulose and lignin are associated with each other

forming a very stable matrix structure. The structure of rice husk reveals the presence of wax and

natural fat on its inner surface. Their presence on the other hand provides protection for the

grain, but also effect quality of husk both physically and biologically (Agbagla-dohnani et al

2001).

Chemical modifications of rice husk surface can upgrade or improve the rice husk quality

utilization (Vadiveloo 2009) Treatment of rice husk with organic and inorganic acids has been

established to improve its quality by delignification and reducing the silica content. The present

work focuses on the development of low cost acid, alkali treatments to change the surface

properties of rice husk for their subsequent use in animal feed stuffs, energy production and as

adsorbents. The effect of treatments on surface function groups were also investigated, along the

topology of rice husk (LiC 2010, LiQ 2009).

Material and methods

Treatment of rice husk with acid and alkali

The rice husk was obtained from local threshing mill, it was washed using distilled water and

then air dried. To modify the surface characteristic chemical treatment was carried out using acid

(H2So4) and alkali (NaOH). The two different concentrations of sulphuric acid (0.3, and 0.5N)

were used. The rice husk was treated for 15 minutes at 100°C. Reagent loading was optimized as

10 gram husk was soaked is 50ml of acid. There different cone of alkali NaOH (2%, 4%, and

6%) were used. Treatment duration and temperature was 24 hours and 70C0the pre-optimized

reagent loading was 10 gram husk to 80ml of NaOH. All the five samples along with one control

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untreated sample was subjected to scanning electron microscopy and FT/IR for the study of

structural and functional group modifications.

Fourier Transform Infrared (FT/IR) Spectroscopy Procedure

KBr salt was mixed with sample in 3:1 ratio. Mixture was grinded to get homogenous

powder.Small amount placed in pellet assembly and pressed very firmly to form transparent

pellet. Fourier transform infrared spectroscopy (Midac Corporation Model 2000) was done by

giving command of 32 scans. Structural changes were analyzed in the region of 600-400cm-1.

Scanning Electron microscopy (SEM)

SEM microscopy was done using electron microscope JSM 648 operated at 10 KV under low

vacuum. Samples were devoid of any moisture content.

Results and discussion

The treated rice husk was characterized in terms of compositional analysis and morphological

characteristic by FT/ IR and scanning electron microscopy.

Structural characterization

FT-IR and SEM analysis

Rice husk is predominantly composed of cellulose hemicellulose and lignin. It also has a

significant percentage of silica. Cellulose chains cemented by lignin and reinforced by silica

make it a harder choice for digestion, but if these linkages were hydrolyzed up-till certain extent

digestibility can be increased. The effect of various concentrations of acid and alkali on the

dissolution of rice husk was investigated in this experiment, results showed that acid and alkali

treatments did not completely dissolute the rice husk. Alkali treatment at 70C0 for 24 hours

dissolves hemicellulose more effectively with 4% alkali. As the concentration of alkali increases

no further degradation takes place rather the increased concentration of alkali leads to the

increased ash content. However it is concluded acid and alkali treatments did not show many

changes in lingo cellulosic composition of rice husk.

Analysis by FT-IR and SEM was done for the closer look on the effect of acid and alkali on rice

husk. Two techniques were employed for the detailed impact of chemical treatment. Analysis

was done by FT/IR spectroscopy by using the range of 600-4000cm-1. The structural changes in

the treated rice husk and untreated rice husk were studied. The reagent showed higher intensity

band at 799 cm-1 indicated our productivity. Treated samples shows reduced absorbance at

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1033cm-1 (graph 1).The absorption bands at 794, 1033, 1457, 1511, 1635, 2916, 3312, 3748

798, 1035, 1457, 1513, 1637, 2919, 3312, and 3750 Cm-1 in the untreated rice husk samples

corresponds to lignocellulosic region. The group frequencies are shown in Table 3.1, along with

their respective role. The banding present shows the presence of strong lignin carbohydrate

matrix in all the treated samples corresponds to asymmetric bending present of CH3 and OCH3

present in lignin which is in line with the studies carried by Angand colleagues in 2012.The

spectra of treated and untreated husk samples are shown in Graph 3.1.

The results also showed that treatment also reduces -H stretching at 2896cm-1 and OH stretching

at 3312. The disappearance of absorption bands at 1457cm-1alsosuggested some removal of

lignin reported by Labbe 2005. The chemical composition of rice husk mainly corresponds in the

region ranges from 800-1513cm-1 and 2995-4000. The most distinct peak at 1388 in our study

corresponds to asymmetric bonding present in lignin, emphasizing the fact as rice husk has the

highest lignin contact in agriculture residues. The peaks lies in the region corresponding to C-O

stretch vibration in cellulose, hemicellulose and lignin of untreated husk showed a shift after

the treatment, 617, 725, 1157, 1141, 2800, 2969, has been shifted to disappearance in treated

samples to 717, 1133, 1419, 1434, 2784, and 2946 repetitively as depicted in Table 3.2.The

shifting banding pattern also showed the structural disruption.

Images taken from SEM are shown in fig 3.1The images show significant surface modifications

of rice husk residue. The image of untreated rice husk show a smooth and even texture, on the

other hand the topology of the treated husk changed. The SEM image shows that the surface

appeared to be swollen comparatively to untreated. Epidermis cracks are clearly visible in alkali

treated husk. In acid treated swelled husk is visible along with shiny silica particles which are in

line with previous studies (Coates 2000).

This study found that mild treatment of rice husk with acid and alkali (NaOH) can alter the

surface structure significantly, although little changes in chemical composition are recorded. The

suggested treatment is beneficial for the conversion of rice husk into value added products.

EXPERIMENT 3

74

Table 3.1 Reference table for group frequencies of respective function groups

Cm-1 Functional

group

Corresponding bonding Ref

800-950 OH Free and hydrogen bonded OH-stretching Labbe et al 2005

950-

1035

C-H Stretching in cellulose rich material Guo et al 2008

1035-

1457

OH O-H bonding vibration of observed water

molecule

Hurtubise&Krassing

1960

1457-

1513

C=C-cx Aromatic skeleton stretch in lignin Hsu.T et al 2010

1513-

1637

C-H Asymmetric bending present in CH3 and

O-CH3 present in lignin

Liu et al 2007

1637-

2919

C-O C-O stretch vibration in cellulose hemi

cellulose and OH in lignin

Lee et al 2009

2919-

4000

C-H Deformation vibration in cellulose Ang et al 2012

EXPERIMENT 3

75

Table 3.2 Absorbance of treated and untreated samples (SS1=untreated husk SS2=

2%NaOH treated, SS3 =.5 N H2SO4 treated, SS4 = 4%NaOH treated husk)

Absorbance

band

Absorbance

SS1 SS2 SS3 SS4

794.53 70.8 60.48 62.99 57

1033 61.5 47.49 52.73 49.38

1457 41.6 25.9 28.9 21.36

1511 49.05 39.18 42.17 34.44

1635 51.98 41.96 43.4 42.3

2916 39.65 38.9 35.7 36.7

3312 45.39 31.38 33.45 33.83

3748 46.29 .0003 17.09 69.009

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Figure 3.1 FT-IR spectra of treated and untreated rice husk 1= untreated husk, 2 =2%

NaOH 3=.5%H2SO4, 4= 4% NaOH

Figure 3.2 Electron micrograph of rice husk treated with 0.3 N sulphuric acid

EXPERIMENT 3

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Figure 3.3 Electron micrograph of rice husk treated with 0.5 N sulphuric acid

EXPERIMENT 3

78

Figure 3.4 Electron micrograph of rice husk treated with 2 % sodium hydroxide

Figure 3. 5 Electron micrograph of rice husk treated with 4 % sodium hydroxide

EXPERIMENT 3

79

Figure 3.6 Electron micrograph of untreated rice husk

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80

REFERENCES

Ang TN, Ngoh GC, Chua ASM, Lee MG. 2012.Elucidation of the effect of ionic liquid

pretreatment on rice husk via structural analyses.Biotechnol Biofuels5, 67-77.

Ang TN, Ngoh GC, Chua ASM. 2009. Pre-treatment of rice husks for fungalfermentation. Int J

Chem Eng. 2:197–202.

Bazargan A, Gebreegziabher T, Hui CW, McKay G. 2014. The effect of alkali treatment on rice

husk moisture content and drying kinetics. Biomass and Bioenergy. 70: 468-475.

Chung YY. 2011. Production of higher heating values of biomass from proximate and ultimate

analyses, Fuel. 9(3): 1128-1132

Coates J. 2000.Interpretation of infrared spectra, a practical approach.Chichester:John Wiley &

Sons Ltd.

Guo GL, Chen WH, Chen WH, Men LC, Hwang WS.2008. Characterization ofdilute acid

pretreatment of silvergrass for ethanol production. BioresourceTechnol. 99:6046–

6053.

Hsu TC, Guo GL, Chen WH, Hwang WS.2010. Effect of dilute acid pretreatmentof rice straw on

structural properties and enzymatic hydrolysis.Bioresource Technol. 101:4907–

4913.

Hurtubise FG, Krassig H. 1960.Classification of fine structural characteristics incellulose by

infrared spectroscopy. Anal Chem. 32:177–181.

Kuo CH, Lee CK. 2009.Enhancement of enzymatic saccharication of celluloseby cellulose

dissolution pretreatments.Carbohydr Polymer. 77:41–46.

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Labbe N, Rials TG, Kelley SS, Cheng ZM, Kim JY, Li Y. 2005.FTIR Imaging andpyrolysis-

molecular beam mass spectrometry: new tools to investigatewood tissues. Wood

Sci Tech. 39:61–77.

Lee SH, Doherty TV, Linhardt RJ, Dordick JS.2009. Ionic liquid-mediated selectiveextraction of

lignin from wood leading to enhanced enzymatic cellulosehydrolysis.

BiotechnolBioeng. 102:1368–1376.

Li C, Knierim B, Manisseri C, Arora R, Scheller HV, Auer M, Vogel KP, Simmons BA, Singh

S. 2010. Comparison of dilute acid and ionic liquid pretreatment of switchgrass:

biomass recalcitrance, delignification and enzymatic saccharification. Bioresource

Technol. 101:4900–4906.

Li Q, He Y, Xian M, Jun G, Xu X, Yang J, Li L. 2009.Improving enzymatic hydrolysis of wheat

straw using ionic liquid 1-ethyl-3-methyl imidazolium diethyl phosphate

pretreatment.Bioresource Technol. 100:3570–3575.

Liu CF, Sun RC, Zhang AP, Ren JL.2007. Preparation of sugarcane bagassecellulosic phthalate

using an ionic liquid as reaction medium. CarbohydrPolymer 68:17–25.

Mansaray, K.G. &Ghaly, a. E., 1997. Physical and Thermochemical Properties of Rice

Husk.Energy Sources, 19(9), pp.989–1004.

Selim ASM, Pan J, Takano T, Suzuki T, Koike S, Kobayashi Y, Tanaka K. 2004. Effect of

ammonia treatment on physical strength of rice straw, distribution of

strawparticles and particle-associated bacteria in sheep rumen.Anim Feed Sci

Technol.115, 117–128.

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Vadiveloo J, Nurfariza B, Fadel JG. 2009. Nutritional improvement of rice husks. Ani Feed

SciTech.151 (3-4): 299-305

83

CHAPTER 6

EXPERIMENT 4

To investigate the feeding value of processed rice husk in growing Lohi sheep

ABSTRACT

Rice husk was subjected to different processing techniques using acid, alkali, water treatment

and fermentation to prepare various processed moiety on pilot scale. All these processed

materials were included in experimental ration up to 20 % level. All iso-caloric and iso-

nitrogenous rations were offered to experimental animals having almost uniform body weight

(BW). During 60 days of trial, weekly weight gain daily feed intake and weekly FCR was

calculated. Result revealed maximum weight gain and best feed conversion ratio (FCR) was of

group consuming fermented husk. No mortality was observed.

Key words: Fermentation, FCR, Acid, Alkali, crude protein.

INTRODUCTION

Rice is the staple food for more than half of the world population. Pakistan produces

6748 million tons rice annually, almost 20 % of which is husk Pakistan economic survey (2014).

Pakistan is an agricultural country and its economy is mainly based on agriculture and livestock

production. Green and dry roughages are the most important livestock feed in the country. The

dry roughages are comparatively much cheaper as compared to green fodders and are usually the

cereal crops residues in the form of straws, stover and husk. The present forage production is not

coping with the feeding requirements of livestock in Pakistan. Pakistan is deficient by 40% in

forages and 80% in concentrate feed Pasha (1998). Stresses for the exploitation of new feed

resources and the interest for utilization of low quality crop residues like rice husk as an animal

feed have been increased.

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84

This crop residues have poor nutritive value (low CP, high lignin content), characterized

by poor digestibility and hence their intake is also low. Energy is limited due to lignocellulosic

bonds Sarwaret al. (1994). The silica present in the rice husk is a major barrier to its digestion,

Agbagla-Dohnaniet al. (2003) explained that silica prevent parenchymal tissue from degradation.

The rice husk has 20% silica presents in outer epidermal wall, which increases the abrasive

character of rice husk Park et al. (2003), silica exists as highly compact carbon-silicon

composite. Lignin has bonding with cellulose which further makes it hard for digestion. The

higher lignin content further makes it a harder choice. Although rice husk is potential animal

feed very few studies on its varietal differences, chemical treatment effects, response to solid

state fermentation and in vivo feeding trials were conducted in Pakistan.

Biochemical conversion of lingo-cellulosic biomass into value added products is a

subject of great interest. To achieve this purpose it is important that structure of the cellulosic

biomass should be modified in such a manner, that they become accessible to enzymatic

degradation Mosier et al. (2005). Various chemical and biological treatments involve these

structural changes, which result in high yields in less time.

Physical and chemical treatments with acid, alkali and steam under pressure break

covalent and non-covalent bonds between lingo-cellulosic components; it has the capacity to

hydrolyze hemicellulose and to some extent de-lignified the rice husk. Biological treatment of

lingo-cellulosic material, solid state fermentation using white fungi improve its protein content

and digestibility. This degradation is mentioned by the enzymes secreted by white rot fungi

mainly consist of lignin degrading enzymes and auxiliary enzymes Ang et al. (2009)

The livestock of Pakistan is producing below their optimum potential because of poor

availability of fodder in terms of quantity as well as quality. By using the value added crop

EXPERIMENT 4

85

residues in the diet of cattle, it will enhance the production (milk/ meat) of animals resultantly a

solution to overcome the shortage of green fodder in lean periods and ensure consistent supply of

feed for the livestock, consequently improved health of the under nourished people Sarnklonget

al. (2010).

The objective of the present studies is to explore the prospectus of chemical and

biologically treated husk as a small ruminant feed.

MATERIAL AND METHODS

Alkali and acid treatment

The husk of Basmati rice (Oryza sativa) was procured from local threshing mill and

brought to fermentation lab of Biochemistry at UVAS. Alkali treatment was carried using 4 %

NaOH for 24 hours at the ambient temperature of 25°C. Husk to NaOH pre-optimized ratio was

2:1. After 24 hours no residual alkali was present in mixture.

In the second treatment rice husk was treated with 0.5N H2SO4 for 24 hours at same

temperature as that of alkali treatment. The pre-optimized ratio of husk to acid was 3:1. Both

acid and alkali treated samples were dried and analyzed for the fiber content, CP, and total Ash

content AOAC 1990.

Hydro thermal treatment

For this application water was added to the husk. pre-optimized husk to water ratio was

1:3.Husk was kept boiling for 60 minutes Very little residual water was present after the boiling,

which was then air dried for two days at 25°C (room temp).

Solid state fermentation

Untreated rice husk was fermented by Pleurotus florida at 28°C in the dark for 21 days

without supplementation. Conditions for fermentation were pre-optimized. A mixed mixture

EXPERIMENT 4

86

(turn which basal media) was added to ensure moisture content of 35%. The fermentation was

carried out in wide mouth plastic containers with once in 24 hour agitation. Containers were

sterilized using ethanol before the commencement of fermentation process. Fermentation

duration in this study was 30 days.

All treated samples were subjected to chemical composition for CP, NDF, ADF, TDN (total

digestible nutrients), E,E (ether extract).and ash contents and large scale preparation of total

mixed ration was carried out.

To determine feeding value of all above treated rice husk, a biological trial of 60 days

was conducted at Ravi campus Pattoki. Before the trial 10 days were given as preliminary

period.

Feeding trials of Lohi sheep

The objective of this study was to use treated rice husk as a cheap source of dietary ingredient of

small ruminant feed. As rice husk has poor nutritive value and abrasive texture leading to limited

utility as a ruminant feed. All processed rice husks were included in concentrate ration to

determine nutritive ability and digestibility. For this purpose 36 sheep of mixed sexes but of

uniform weight and age were randomly divided into 6 groups each having three replicates.

Experimental animals and their management

All the experimental lohi sheep with 8-9 months age and average body weight (24±5) kg

at the beginning of the experiment. Sheep were fed ad libitum twice daily (09.00 and 16.00

hours) in cemented managers. After the morning feeding all sheep were let loose in open

paddock throughout the experimental period except during extreme weather. Sheep were housed

and managed in replicates in well ventilated shed with concrete floor. Daily sweeping and

cleaning of floors was practiced to provide good hygienic environment. Fresh water was

EXPERIMENT 4

87

provided. Replicate feed intake was recorded daily by measuring the amount of refused before

the morning feeding. Composition of experimental rations is depicted in table 1.

Parameters studied

Daily Feed intake was recorded daily. This was done by subtracting the amount of refusal

from the feed offered before the morning feeding. Sheep were weighed weekly to monitor the

growth rate after restriction of feed and water intake for 16 hours throughout the experimental

period by using an electronic scale. Data thus collected were used to calculate feed efficiency.


Weekly intake and weight gain of Lohi sheep is illustrated in Table 3.The total DM

intake of all the six groups ranged between 975± 68to 1092±77 grams per day and weekly

weight gain ranges from 0.7±0.1 to 0.977±.052 Kg/week. Highest intake was found with the

water treated husk and lowest with the untreated husk. This intake is in line with the results

obtained earlier fattening trials of lohi sheep Jabbar and Anjum (2008). Although for some

foreigner breeds an exceptional high intake of 6.4±7.4% of body weight was reported El Hag and

Al-Shargi (1998). Initial weight gains of animals were significantly high but the subsequent

weight gain followed the uniform pattern as shown in graph 1. This is perhaps due to the shifting

of animals from grazing and consequently average daily gains follow the pattern which is in line

with previous studies. Growth rate followed the linear pattern for all the six groups. Lambs

growth rate depends upon feed intake rather than the duration of the intake Butter®eld (1988). In

the present study growth rates obtained were almost the same as that of tropical breeds Gatenby

(1986); Kusinaet al. (1991), although they are slightly lower than those using conventional

rations for the same breed Jabbar and Anjum (2008).

EXPERIMENT 4

88

It is also found that high energy diets were more efficient, as efficiency is the function of

weight gain and dry matter intake. Interpretation is difficult as energy used for maintenance is

not distinguished from energy used for weight gain. It is found that increased protein content

results in the better values of feed conversion ratio (FCR), supported by the studies of Kusina et

al. (1991); Butter®eld (1988). The FCR found in the current study were better than previously

reported, for the same breed. The treatments increased the digestibility and palatability of rice

husk which is proved in in vitro digestibility trials, although DM intake was reduced. Previous

study using agricultural by products showed 11.5 and 12.4 FCR in sheep El Hag and Al-Shargi

(1995). Whereas diets based on Rhodes grass hay Chesworth et al. (1996) showed a remarkably

good FCR of 5.48 and 6.02.This study found significant difference between the groups shown in

table 3. As far as the economy of rice husk was concerned it is comparable to the conventional

ration. Water treated husk due to large intake and poor FCR placed it most expensive in the

group .Current study fully support the use of rice husk as a convenient replacer of conventional

wheat straw.

EXPERIMENT 4

89

Table 4.1 Ration formulations of various groups

Ingredients (%)

positive

Control

(PC)

Alkali

treated

(B T)

Acid

treated

(AT)

Hydrothermal

Treated

(HT)

Fermented

(FT)

Negative

Control

(NC)

Corn, Grain 21.00 21.00 22.00 22 22.00 20.00

Wheat Bran 14.00 15.00 15.00 15 15.00 16.00

Rice Polish 10.00 11.00 11.00 10 12.00 10.00

Molasses (S Cane) 7.00 7.00 7.00 7.00 7.00 7.00

Canola Meal 8.00 8.00 8.00 8.00 8.00 8.00

Sunflower Ml 8.00 8.00 8.00 8.00 7.00 8.00

Corn Gluten Ml

30%

10.00 8.00 7.00 8.00 7.00 9.00

Cmn Salt NaCl 0.50 0.50 0.50 0.50 0.50 0.50

Sodium Bi

Carbonate

0.50 0.50 0.50 0..50 0.50 0.50

Min Mix

(Ruminants)

1.00 1.00 1.00 1 1.00 1.00

Wheat Straw 20.00 20.00(RH) 20.00

(RH)

20 (RH) 20.00

(RH)

20.00

(RH)

Table 4.2: Nutritional Profile of various Rations

Parameter PC NC AT B T HT Fermented

husk

CP 14.95 14.99 15.22 15.28 15.31 15.36

C.F 14.77 13.03 13 11.08 12.20 12.83

NDF 14.59 14.75 9.57 10.41 14.54 14.06

ADF 9.45 11.29 5.30 7.66 11.87 12.22

TDN 64.65 62.72 64.75 65.15 65.02 66.07

E.E 3.91 3.77 3.91 3.95 3.88 4.04

M.E 2.30 2.21 2.26 2.28 2.15 2.29

ASH 6.57 7.06 7.41 7.60 7.05 7.23

EXPERIMENT 4

90

Table 4.3: Growth performance and economics of Lohi sheep fed on differently treated rice

husk a wheat straw replacer

PC FT NC BT AT HT

Daily feed

intake g

1040.91±91

7

1061.08±9

6

975.68±68.

7

1058.66±7

1

1040.91±9.

7

1092.61±7.

5

weekly

weight

gain Kg

0.977±.052 0.972±0.18 0.729±0.11 0.922±0.18 0.935±0.19 0.7±0.10

Feed

conversio

n ratio

11.69±1.401 7.679±0.89 9.924±1.2 8.09±1.26 8.008±1.5 11.131±1.4

4

EXPERIMENT 4

91

Graph 4.1. Weekly Weight Gain

Graph 4.2 weekly average feed intake

0.00

10.00

20.00

30.00

40.00

week1

week2

week3

week4

week5

week6

week7

week8

week9

Bo

dy

Wei

ght(

Kg)

Weekly Weight Gain

Control

Husk

Fermented

Alkali

Acid

Water

EXPERIMENT 4

92

Table 4.4 No. Daily Feed intake per week

1st Week 2nd Week 3rd Week 4rth Week 5th Week 6th Week 7th Week 8th Week 9th Week

C 871.5 975.9167 984.0833 941.5 1059.917 1098.417 1080.333 1150.917 1174.833

H 884.3333 1061.667 999.25 943.25 1087.917 1120 1139.833 1143.917 1206.917

F 792.1667 888.4167 896 842.9167 984.0833 1044.167 1015.583 1044.167 1157.333

B 903.5833 1024.333 957.8333 964.25 1094.333 1180.667 1171.333 1162.583 1269.333

A 866.8333 987 1008.583 936.8333 1071.583 1148 1129.333 1162 1235.5

W 926.9167 1041.25 1036.583 1020.25 1123.5 1229.667 1257.083 1288.583 1331.167

EXPERIMENT 4

93

REFERENCES

Agbagla- D., Noziere P., Gaillard.M.,Puard.M., and Doreau. M (2003). Effect of silica on rice

straw ruminal degradation The.JAgriSci 140(2) 183-192

Ang TN, Ngoh GC, Chua ASM. 2009. Pre-treatment of rice husks for fungal fermentation. Int J

Chem Eng. 2:197–202.

AOAC. 1990. Official Methods of Analysis. Association of Analytical Chemists (15th (Ang et

al. 2013)(Ang et al. 2013)Ed.) Artington Virginia, USA.

Butter®eld, RM. 1988.New Concepts in Sheep Growth. University of Sydney, Australia, 168.

Chesworth JM, Byerley DJ, Mahgoub O. 1996. Evaluation of whole date palm frond as a forage

source for sheep, Anim. Sci 62, 664, (Abstr.).

El-Hag MG, Al-Shargi KM. 1998. Comparative performance of goats and sheep fed on high-

®ber pelleted diets supplemented with different nitrogen sources. J ApplAnim

Res. 13, 179±184.

Gatenby RM. 1986.Sheep Production in the Tropics and the Sub- Tropics. Longman, New York,

USA, p. 351

Jabbar MA. Anjum1 MI. 2008.Effect of diets with different forage to concentrate ratio

forfattening of lohi lambs. Pak Vet J. 28(3): 150-152.

Kusina NT, Hale DH, Chesworth JM, Mutisi C. 1991.Effect of the amount of dietary energy on

growth and body composition of Sabi lambs. In: Isotope Aided Studies on Goat

and Sheep Production in the Tropics. International Atomic Energy Agency,

Vienna, pp. 13±27.

EXPERIMENT 4

94

Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M. 2005. Features of

promising technologies for pretreatment of lignocellulosic biomass.Bioresource

technology, 96(6), 673-686.

Park, B. D., S. G. Wi, K. H. Lee, A. P. Singh, T. H. Yoon and Y. S. Kim (2003).

Characterisation of anatomical features and silica distribution in rice husk using

microscopic and micro-analytical techniques. Biomass Bioenergy 25: 319–327.

Pasha TN. 1998. Feed resources for livestock and poultry in Punjab, Punjab, Pakistan. Germany

agency for technical cooperation (GTZ), Livestock and Dairy Development

Department, Government of the Punjab, Lahore.

Sarnklong C, Cone JW, Pellikaan W, Hendriks WH. 2010. Utilization of Rice Straw and

Different Treatments to Improve Its Feed Value for Ruminants: A Review. Asian-

Australasian J Anim J Anim Sci.23(5), 680–692. doi:10.5713/ajas.2010.80619.

Sarwar M, Iqbal MA, Ali CS and Khaliq T. 1994. Growth performance of buffalo male calves as

affected by using cowpeas and soybean seeds as a source of urease during urea

treated wheat straw ensiling process. Egyptian J Anim Prod.2: 179.

Van-Soest, P. J., J. B. Robertson and B. A. Lewis (1991).Method for dietary fiber, neutral

detergent fiber and non-starch polysaccharides in relation to animal nutrition.J Dairy Sci.

74, 3583–3597.

95

CHAPTER 6

SUMMARY

Pakistan is mainly an agriculture country. Livestock is an integral part of the Agricultural

economy unfortunately Livestock in Pakistan is producing for below its full potential. The main

reason this poor performance is the shortage of fodder quantitatively and qualitative. Therefore,

to overcome the shortage of green fodder, we have to export the conventional and non-

conventional sources of feed. Pakistan is one of the major producer of rice in Asia, that’s shy rice

husk which is a by-product of rice threshing process in frequently and cheaply available. In this

study we study this underutilized source, by applying various treatments, evaluating their impact

on husk topology and itsimpart on husk topology and its impact on ruminant feeding.

In the first two experiments, the rice husk was subjected to acid, alkali, and water

treatment at various temperature and concentration, we found a significant effect of temperature

on rice husk (P>0.001). Whereas concentration in case of alkali treatment produced significant

results (P?0.001). Topology of rice husk 4 bond specific bond vibrations was studied with the

help of FT/IR and scanning electron microscopy.

Electron micrographs showed swelling and even cracking of the epidermises. FT/IR results

demonstrate a change in the absorbance in the polysaccharide bonding region.

The third experiments deals with the enhancement of nutritive value of rice husk by SSF

using Pleurotusflonda. Experiment shows significant increase in the CP and decrease in the total

lignin content (P> 0001.

Finally in the fourth experiment a ration having 20% differently procured husk were

feeded to the small ruminant (Lohi Sheep) and the an average weekly weight gain comparable

feed intake and FCR were found comparable with control.

SUMMARY

96

The data was analyzed using Mintab version 20:01. It is concluded that improving the

quality of certain under utilized agricultural residue can minimize the feed gap in Pakistan. Rice

husk is a valuable and acceptable source of fed for ruminant and there is need to educate the

small farmer to break this myth.

97

Annexure 1

Table 1. Projections of Population in Major Rice Producing and Consuming Countries in Asia, 1995 to 2025

Country Population (mill.) 1995

Annual Growth Rate (% per year)

Projected Population

(mill.) in 2025

Percent Increase

1995-2025 1995-2000 2020-2025

China 1199 0.9 0.5 1471 23

India 934 1.7 1.0 1370 47

Indonesia 192 1.4 0.8 265 38

Bangladesh 121 1.8 1.1 182 50

Vietnam 74.1 2.0 1.2 117 58

Thailand 60.5 1.3 0.7 80.8 34

Myanmar 46.8 2.1 1.1 72.9 56

Japan 125 0.3 -0.3 124 -1

Philippines 69.2 2.2 1.2 115 66

Rep. of Korea 44.8 0.8 0.3 52.9 18

Pakistan 130 2.7 1.6 243 87

Asia (excluding China) 2244 1.8 1.1 3389 51

ANNEXURES

98

Annexure 2 Multilevel Factorial Design Factors: 2 Replicates: 3

Base runs: 9 Total runs: 27

Base blocks: 1 Total blocks: 1

Number of levels: 3, 3

Design Table

Run Blk A B

1 1 1 1

2 1 1 2

3 1 1 3

4 1 2 1

5 1 2 2

6 1 2 3

7 1 3 1

8 1 3 2

9 1 3 3

10 1 1 1

11 1 1 2

12 1 1 3

13 1 2 1

14 1 2 2

15 1 2 3

16 1 3 1

17 1 3 2

18 1 3 3

19 1 1 1

20 1 1 2

21 1 1 3

22 1 2 1

23 1 2 2

24 1 2 3

25 1 3 1

26 1 3 2

27 1 3 3

General Linear Model: NDF versus Time, Temprature Factor Type Levels Values

Time fixed 3 30, 60, 90

Temprature fixed 3 37, 100, 200

Analysis of Variance for NDF, using Adjusted SS for Tests

Source DF SeqSS Adj SS Adj MS F P

Time 2 177.13 177.13 88.56 51.49 0.000

Temprature 2 943.73 943.73 471.87 274.36 0.000

Time*Temprature 4 492.67 492.67 123.17 71.61 0.000

Error 18 30.96 30.96 1.72

Total 26 1644.49

S = 1.31145 R-Sq = 98.12% R-Sq(adj) = 97.28%

ANNEXURES

99

Unusual Observations for NDF

Obs NDF Fit SE Fit Residual St Resid

14 65.1500 67.4667 0.7572 -2.3167 -2.16 R

R denotes an observation with a large standardized residual.

Least Squares Means for NDF

Time Mean SE Mean

30 67.22 0.4371

60 62.14 0.4371

90 61.49 0.4371

Temprature

37 68.85 0.4371

100 66.64 0.4371

200 55.35 0.4371

Time*Temprature

30 37 71.18 0.7572

30 100 76.00 0.7572

30 200 54.47 0.7572

60 37 66.04 0.7572

60 100 67.47 0.7572

60 200 52.92 0.7572

90 37 69.34 0.7572

90 100 56.46 0.7572

90 200 58.67 0.7572

Residual Plots for NDF

210-1-2

99

90

50

10

1

Residual

Pe

rce

nt

7570656055

2

1

0

-1

-2

Fitted Value

Re

sid

ua

l

210-1-2

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

2

1

0

-1

-2

Observation Order

Re

sid

ua

l

Normal Probability Plot Versus Fits

Histogram Versus Order


ANNEXURES

100

General Linear Model: ADF versus Time, Temprature Factor Type Levels Values

Time fixed 3 30, 60, 90


Analysis of Variance for ADF, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

Time 2 66.773 66.773 33.387 21.55 0.000

Temprature2 266.001 266.001 133.000 85.85 0.000

Time*Temprature4 227.380 227.380 56.845 36.69 0.000

Error 18 27.888 27.888 1.549

Total 26 588.041

S = 1.24471 R-Sq = 95.26% R-Sq(adj) = 93.15%

Unusual Observations for ADF

Obs ADF Fit SE Fit Residual St Resid

4 49.6000 52.1000 0.7186 -2.5000 -2.46 R

13 54.5000 52.1000 0.7186 2.4000 2.36 R


Least Squares Means for ADF

Time Mean SE Mean

30 56.20 0.4149

60 54.14 0.4149

90 52.36 0.4149

Temprature

37 55.27 0.4149

100 49.98 0.4149

200 57.46 0.4149

Time*Temprature

30 37 59.47 0.7186

30 100 54.44 0.7186

30 200 54.71 0.7186

60 37 52.10 0.7186

60 100 51.50 0.7186

60 200 58.83 0.7186

90 37 54.23 0.7186

90 100 44.00 0.7186

90 200 58.83 0.7186

ANNEXURES

101

Residual Plots for ADF

210-1-2

99

90

50

10

1

Residual

Pe

rce

nt

60555045

2

1

0

-1

-2

Fitted Value

Re

sid

ua

l

2.41.20.0-1.2-2.4

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

2

1

0

-1

-2

Observation Order

Re

sid

ua

l




General Linear Model: lignin versus Time, Temprature Factor Type Levels Values

Time fixed 3 30, 60, 90


Analysis of Variance for lignin, using Adjusted SS for Tests

Source DF Seq SS AdjSS Adj MS F P

Time 2 0.0407 0.0407 0.0203 0.07 0.936

Temprature 2 4.7791 4.7791 2.3895 7.86 0.004

Time*Temprature4 29.8207 29.8207 7.4552 24.54 0.000

Error 18 5.4689 5.4689 0.3038

Total 26 40.1093

S = 0.551207 R-Sq = 86.36% R-Sq(adj) = 80.30%

Unusual Observations for lignin

Obs lignin Fit SE Fit Residual St Resid

21 17.1500 18.1300 0.3182 -0.9800 -2.18 R


Least Squares Means for lignin

Time Mean SE Mean

30 19.49 0.1837

ANNEXURES

102

60 19.43 0.1837

90 19.52 0.1837

Temprature

37 20.07 0.1837

100 19.19 0.1837

200 19.17 0.1837

Time*Temprature

30 37 19.30 0.3182

30 100 21.03 0.3182

30 200 18.13 0.3182

60 37 20.82 0.3182

60 100 18.70 0.3182

60 200 18.76 0.3182

90 37 20.10 0.3182

90 100 17.84 0.3182

90 200 20.62 0.3182

Residual Plots for lignin

1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

21201918

1.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

1.00.50.0-0.5-1.0

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l



Residual Plots for lignin

General Linear Model: Si versus Time, Temprature Factor Type Levels Values

Time fixed 3 30, 60, 90


Analysis of Variance for Si, using Adjusted SS for Tests


Time 2 12.5988 12.5988 6.2994 12.45 0.000

Temprature 2 6.4692 6.4692 3.2346 6.40 0.008

Time*Temprature4 20.6625 20.6625 5.1656 10.21 0.000

Error 18 9.1039 9.1039 0.5058

ANNEXURES

103

Total 26 48.8345

S = 0.711178 R-Sq = 81.36% R-Sq(adj) = 73.07%

Least Squares Means for Si

Time Mean SE Mean

30 10.510 0.2371

60 8.841 0.2371

90 9.780 0.2371

Temprature

37 10.161 0.2371

100 9.030 0.2371

200 9.940 0.2371

Time*Temprature

30 37 11.117 0.4106

30 100 10.450 0.4106

30 200 9.963 0.4106

60 37 9.950 0.4106

60 100 8.450 0.4106

60 200 8.123 0.4106

90 37 9.417 0.4106

90 100 8.190 0.4106

90 200 11.733 0.4106

Residual Plots for Si

10-1

99

90

50

10

1

Residual

Pe

rce

nt

12111098

1.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

1.00.50.0-0.5-1.0

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l




General Linear Model: Ash versus Time, Temprature Factor Type Levels Values

Time fixed 3 30, 60, 90

ANNEXURES

104


Analysis of Variance for Ash, using Adjusted SS for Tests


Time 2 5.3305 5.3305 2.6652 13.82 0.000

Temprature2 68.1867 68.1867 34.0933 176.74 0.000

Time*Temprature 4 5.9868 5.9868 1.4967 7.76 0.001

Error 18 3.4721 3.4721 0.1929

Total 26 82.9761

S = 0.439200 R-Sq = 95.82% R-Sq(adj) = 93.96%

Unusual Observations for Ash

Obs Ash Fit SE Fit Residual St Resid

27 9.7500 10.6333 0.2536 -0.8833 -2.46 R


Least Squares Means for Ash

Time Mean SE Mean

30 13.70 0.1464

60 13.08 0.1464

90 12.62 0.1464

Temprature

37 15.13 0.1464

100 13.02 0.1464

200 11.24 0.1464

Time*Temprature

30 37 15.00 0.2536

30 100 14.32 0.2536

30 200 11.78 0.2536

60 37 15.05 0.2536

60 100 12.87 0.2536

60 200 11.31 0.2536

90 37 15.35 0.2536

90 100 11.87 0.2536

90 200 10.63 0.2536

ANNEXURES

105

Residual Plots for Ash

1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

16141210

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

0.60.40.20.0-0.2-0.4-0.6-0.8

6.0

4.5

3.0

1.5

0.0

Residual

Fre

qu

en

cy

2624222018161412108642

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l




ANNEXURES

106

Multilevel Factorial Design Factors: 2 Replicates: 3




Design Table

Run Blk A B

1 1 1 1

2 1 1 2

3 1 1 3

4 1 2 1

5 1 2 2

6 1 2 3

7 1 3 1

8 1 3 2

9 1 3 3

10 1 1 1

11 1 1 2

12 1 1 3

13 1 2 1

14 1 2 2

15 1 2 3

16 1 3 1

17 1 3 2

18 1 3 3

19 1 1 1

20 1 1 2

21 1 1 3

22 1 2 1

23 1 2 2

24 1 2 3

25 1 3 1

26 1 3 2

27 1 3 3

General Linear Model: NDF versus Concentration, Temprature Factor Type Levels Values

Concentration fixed 3 0.1, 0.3, 0.5




Concentration 2 360.53 360.53 180.27 651.23 0.000

Temprature 2 690.29 690.29 345.14 1246.87 0.000

Concentration*Temprature4 1074.42 1074.42 268.60 970.36 0.000

Error 18 4.98 4.98 0.28

Total 26 2130.22

S = 0.526125 R-Sq = 99.77% R-Sq(adj) = 99.66%

ANNEXURES

107



4 72.0000 71.0833 0.3038 0.9167 2.13 R

9 48.3500 47.4833 0.3038 0.8667 2.02 R



ConcentratioMean SE Mean

0.1 64.45 0.1754

0.3 62.89 0.1754

0.5 56.04 0.1754

Temprature

37 66.58 0.1754

70 54.39 0.1754

100 62.41 0.1754

Concentratio*Temprature

0.1 37 68.74 0.3038

0.1 70 53.66 0.3038

0.1 100 70.95 0.3038

0.3 37 71.08 0.3038

0.3 70 48.79 0.3038

0.3 100 68.81 0.3038

0.5 37 59.90 0.3038

0.5 70 60.73 0.3038

0.5 100 47.48 0.3038


1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

7065605550

1.0

0.5

0.0

-0.5

Fitted Value

Re

sid

ua

l

0.80.40.0-0.4-0.8

4.8

3.6

2.4

1.2

0.0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

Observation Order

Re

sid

ua

l




ANNEXURES

108

General Linear Model: ADF versus Concentration, Temprature Factor Type Levels Values





Concentration 2 424.56 424.56 212.28 510.84 0.000

Temprature 2 684.41 684.41 342.21 823.49 0.000

Concentration*Temprature4 1086.53 1086.53 271.63 653.66 0.000

Error 18 7.48 7.48 0.42

Total 26 2202.98

S = 0.644636 R-Sq = 99.66% R-Sq(adj) = 99.51%



19 53.1800 54.2500 0.3722 -1.0700 -2.03 R




0.1 54.32 0.2149

0.3 52.50 0.2149

0.5 45.15 0.2149

Temprature

37 53.61 0.2149

70 54.79 0.2149

100 43.57 0.2149


0.1 37 54.25 0.3722

0.1 70 54.95 0.3722

0.1 100 53.78 0.3722

0.3 37 55.89 0.3722

0.3 70 50.84 0.3722

0.3 100 50.78 0.3722

0.5 37 50.70 0.3722

0.5 70 58.60 0.3722

0.5 100 26.16 0.3722

ANNEXURES

109


1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

60504030

1.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

0.750.500.250.00-0.25-0.50-0.75-1.00

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l




General Linear Model: ADL versus Concentration, Temprature Factor Type Levels Values



Analysis of Variance for ADL, using Adjusted SS for Tests


Concentration 2 9.4501 9.4501 4.7250 12.61 0.000

Temprature 2 1.4931 1.4931 0.7465 1.99 0.165

Concentration*Temprature 4 3.5670 3.5670 0.8918 2.38 0.090

Error 18 6.7471 6.7471 0.3748

Total 26 21.2573

S = 0.612239 R-Sq = 68.26% R-Sq(adj) = 54.15%

Unusual Observations for ADL

Obs ADL Fit SE Fit Residual St Resid

2 20.0000 21.1000 0.3535 -1.1000 -2.20 R

15 18.7800 19.8133 0.3535 -1.0333 -2.07 R


Least Squares Means for ADL


ANNEXURES

110

0.1 20.47 0.2041

0.3 19.67 0.2041

0.5 19.02 0.2041

Temprature

37 19.42 0.2041

70 19.73 0.2041

100 20.00 0.2041


0.1 37 19.91 0.3535

0.1 70 21.10 0.3535

0.1 100 20.39 0.3535

0.3 37 19.61 0.3535

0.3 70 19.59 0.3535

0.3 100 19.81 0.3535

0.5 37 18.75 0.3535

0.5 70 18.51 0.3535

0.5 100 19.80 0.3535

Residual Plots for ADL

1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

212019

1.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

1.00.50.0-0.5-1.0

6.0

4.5

3.0

1.5

0.0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

lNormal Probability Plot Versus Fits


Residual Plots for ADL

General Linear Model: Si versus Concentration, Temprature Factor Type Levels Values





Concentration 2 52.995 52.995 26.498 3.25 0.062

Temprature 2 1.815 1.815 0.907 0.11 0.895


ANNEXURES

111

Error 18 146.807 146.807 8.156

Total 26 204.248

S = 2.85586 R-Sq = 28.12% R-Sq(adj) = 0.00%

Unusual Observations for Si

Obs Si Fit SE Fit Residual St Resid

17 18.4500 12.9333 1.6488 5.5167 2.37 R

18 19.8900 13.2967 1.6488 6.5933 2.83 R




0.1 10.397 0.9520

0.3 10.757 0.9520

0.5 13.532 0.9520

Temprature

37 11.919 0.9520

70 11.456 0.9520

100 11.311 0.9520


0.1 37 10.623 1.6488

0.1 70 10.667 1.6488

0.1 100 9.900 1.6488

0.3 37 10.767 1.6488

0.3 70 10.767 1.6488

0.3 100 10.737 1.6488

0.5 37 14.367 1.6488

0.5 70 12.933 1.6488

0.5 100 13.297 1.6488

ANNEXURES

112


5.02.50.0-2.5-5.0

99

90

50

10

1

Residual

Pe

rce

nt

1413121110

5.0

2.5

0.0

-2.5

-5.0

Fitted Value

Re

sid

ua

l

6.04.53.01.50.0-1.5-3.0-4.5

16

12

8

4

0

Residual

Fre

qu

en

cy

2624222018161412108642

5.0

2.5

0.0

-2.5

-5.0

Observation Order

Re

sid

ua

l




General Linear Model: Ash versus Concentration, Temprature Factor Type Levels Values





Concentration 2 1.7568 1.7568 0.8784 5.41 0.014

Temprature2 11.8303 11.8303 5.9151 36.44 0.000


Error 18 2.9215 2.9215 0.1623

Total 26 20.2460

S = 0.402869 R-Sq = 85.57% R-Sq(adj) = 79.16%



11 15.7200 15.0267 0.2326 0.6933 2.11 R

20 14.1400 15.0267 0.2326 -0.8867 -2.70 R

21 14.0000 14.7100 0.2326 -0.7100 -2.16 R



ANNEXURES

113


0.1 14.52 0.1343

0.3 15.04 0.1343

0.5 15.08 0.1343

Temprature

37 14.02 0.1343

70 15.00 0.1343

100 15.62 0.1343


0.1 37 13.82 0.2326

0.1 70 15.03 0.2326

0.1 100 14.71 0.2326

0.3 37 13.68 0.2326

0.3 70 15.21 0.2326

0.3 100 16.23 0.2326

0.5 37 14.55 0.2326

0.5 70 14.77 0.2326

0.5 100 15.93 0.2326


1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

16.015.515.014.514.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

0.60.40.20.0-0.2-0.4-0.6-0.8

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l




Multilevel Factorial Design Factors: 2 Replicates: 3


ANNEXURES

114



Design Table

Run Blk A B

1 1 1 1

2 1 1 2

3 1 1 3

4 1 2 1

5 1 2 2

6 1 2 3

7 1 3 1

8 1 3 2

9 1 3 3

10 1 1 1

11 1 1 2

12 1 1 3

13 1 2 1

14 1 2 2

15 1 2 3

16 1 3 1

17 1 3 2

18 1 3 3

19 1 1 1

20 1 1 2

21 1 1 3

22 1 2 1

23 1 2 2

24 1 2 3

25 1 3 1

26 1 3 2

27 1 3 3

General Linear Model: NDF versus Concentration, Temrature Factor Type Levels Values

Concentration fixed 3 2, 4, 6

Temrature fixed 3 37, 70, 100



Concentration 2 354.93 354.93 177.47 70.17 0.000

Temrature 2 299.96 299.96 149.98 59.30 0.000

Concentration*Temrature 4 899.89 899.89 224.97 88.95 0.000

Error 18 45.52 45.52 2.53

Total 26 1600.31

S = 1.59031 R-Sq = 97.16% R-Sq(adj) = 95.89%



13 64.8500 69.6000 0.9182 -4.7500 -3.66 R

22 73.9500 69.6000 0.9182 4.3500 3.35 R

ANNEXURES

115




2 71.69 0.5301

4 62.94 0.5301

6 66.02 0.5301

Temrature

37 71.52 0.5301

70 65.33 0.5301

100 63.81 0.5301

Concentratio*Temrature

2 37 70.93 0.9182

2 70 73.00 0.9182

2 100 71.15 0.9182

4 37 69.60 0.9182

4 70 52.66 0.9182

4 100 66.57 0.9182

6 37 74.01 0.9182

6 70 70.33 0.9182

6 100 53.71 0.9182


5.02.50.0-2.5-5.0

99

90

50

10

1

Residual

Pe

rce

nt

7570656055

5.0

2.5

0.0

-2.5

-5.0

Fitted Value

Re

sid

ua

l

420-2-4

20

15

10

5

0

Residual

Fre

qu

en

cy

2624222018161412108642

5.0

2.5

0.0

-2.5

-5.0

Observation Order

Re

sid

ua

l




General Linear Model: ADF versus Concentration, Temrature Factor Type Levels Values



ANNEXURES

116



Concentration 2 635.21 635.21 317.61 497.00 0.000

Temrature 2 182.36 182.36 91.18 142.68 0.000

Concentration*Temrature4 1074.66 1074.66 268.67 420.42 0.000

Error 18 11.50 11.50 0.64

Total 26 1903.74

S = 0.799403 R-Sq = 99.40% R-Sq(adj) = 99.13%



22 41.6500 43.2567 0.4615 -1.6067 -2.46 R




2 50.84 0.2665

4 41.92 0.2665

6 53.17 0.2665

Temrature

37 52.27 0.2665

70 47.35 0.2665

100 46.31 0.2665


2 37 55.44 0.4615

2 70 41.42 0.4615

2 100 55.67 0.4615

4 37 43.26 0.4615

4 70 39.05 0.4615

4 100 43.45 0.4615

6 37 58.12 0.4615

6 70 61.58 0.4615

6 100 39.82 0.4615

ANNEXURES

117


210-1-2

99

90

50

10

1

Residual

Pe

rce

nt

6055504540

1

0

-1

Fitted Value

Re

sid

ua

l

1.00.50.0-0.5-1.0-1.5

4.8

3.6

2.4

1.2

0.0

Residual

Fre

qu

en

cy

2624222018161412108642

1

0

-1

Observation Order

Re

sid

ua

l




General Linear Model: Lignin versus Concentration, Temrature Factor Type Levels Values



Analysis of Variance for Lignin, using Adjusted SS for Tests


Concentration 2 6.887 6.887 3.443 8.07 0.003

Temrature 2 56.696 56.696 28.348 66.43 0.000


Error 18 7.682 7.682 0.427

Total 26 110.956

S = 0.653265 R-Sq = 93.08% R-Sq(adj) = 90.00%

Least Squares Means for Lignin


2 17.38 0.2178

4 18.52 0.2178

6 18.37 0.2178

Temrature

37 18.67 0.2178

70 19.51 0.2178

100 16.10 0.2178


2 37 18.62 0.3772

ANNEXURES

118

2 70 18.45 0.3772

2 100 15.07 0.3772

4 37 17.87 0.3772

4 70 18.98 0.3772

4 100 18.70 0.3772

6 37 19.50 0.3772

6 70 21.08 0.3772

6 100 14.52 0.3772

Residual Plots for Lignin

1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

2220181614

1.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

1.00.50.0-0.5-1.0

4.8

3.6

2.4

1.2

0.0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l



Residual Plots for Lignin

General Linear Model: Si versus Concentration, Temrature Factor Type Levels Values





Concentration 2 1.8978 1.8978 0.9489 2.44 0.115

Temrature2 16.8074 16.8074 8.4037 21.61 0.000


Error 18 6.9997 6.9997 0.3889

Total 26 28.8811

S = 0.623598 R-Sq = 75.76% R-Sq(adj) = 64.99%

Unusual Observations for Si

ANNEXURES

119

Obs Si Fit SE Fit Residual St Resid

10 9.0000 10.0433 0.3600 -1.0433 -2.05 R




2 10.569 0.2079

4 10.039 0.2079

6 10.629 0.2079

Temrature

37 9.736 0.2079

70 9.982 0.2079

100 11.519 0.2079


2 37 10.043 0.3600

2 70 9.583 0.3600

2 100 12.080 0.3600

4 37 8.963 0.3600

4 70 10.073 0.3600

4 100 11.080 0.3600

6 37 10.200 0.3600

6 70 10.290 0.3600

6 100 11.397 0.3600


1.00.50.0-0.5-1.0

99

90

50

10

1

Residual

Pe

rce

nt

1211109

1.0

0.5

0.0

-0.5

-1.0

Fitted Value

Re

sid

ua

l

1.00.50.0-0.5-1.0

8

6

4

2

0

Residual

Fre

qu

en

cy

2624222018161412108642

1.0

0.5

0.0

-0.5

-1.0

Observation Order

Re

sid

ua

l




ANNEXURES

120

General Linear Model: Ash versus Concentration, Temrature Factor Type Levels Values





Concentration 2 915.1 915.1 457.6 3.19 0.065

Temrature 2 392.9 392.9 196.4 1.37 0.279


Error 18 2578.6 2578.6 143.3

Total 26 4053.1

S = 11.9690 R-Sq = 36.38% R-Sq(adj) = 8.10%



25 9.8500 33.2667 6.9103 -23.4167 -2.40 R

26 10.1200 35.7467 6.9103 -25.6267 -2.62 R

27 10.8900 33.4633 6.9103 -22.5733 -2.31 R




2 20.33 3.990

4 30.27 3.990

6 34.16 3.990

Temrature

37 22.91 3.990

70 31.54 3.990

100 30.32 3.990


2 37 14.33 6.910

2 70 22.89 6.910

2 100 23.78 6.910

4 37 21.12 6.910

4 70 35.98 6.910

4 100 33.72 6.910

6 37 33.27 6.910

6 70 35.75 6.910

6 100 33.46 6.910

ANNEXURES

121


20100-10-20

99

90

50

10

1

Residual

Pe

rce

nt

3530252015

10

0

-10

-20

-30

Fitted Value

Re

sid

ua

l

100-10-20

20

15

10

5

0

Residual

Fre

qu

en

cy

2624222018161412108642

10

0

-10

-20

-30

Observation Order

Re

sid

ua

l




Documents

PHYSICAL, CHEMICAL AND BIOLOGICAL TREATMENT OF RICE HUSK