The Effect Of Temperature On Hydrolysis Of Cellulose (Saw-Dust)

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ABSTRACT

The effect of concentration of hydrochloric acid on hydrolysis of cellulose (sawdust) to glucose was studied on this research project and the steps obtained to achieve this project involved treatment of saw-dust (cellulose) with different concentrations of the acid at constant temperature of 80°𝐶 (350k) for 30mins. This was followed by glucose analysis, some analysis or experiments were done on acid hydrolysis in order to study the effect of (HCL) acid on the hydrolysis of cellulose to glucose. The process used in this hydrolysis was acid hydrolysis in which HCL acid was used at constant temperature of 80oC and the saw-dust used [was obtained by grinding wood with saw] was weighed and mixed with water . Secondly, during this analysis/experiment, it was observed that hydrochloric acid hydrolyzed well from the readings gotten from each result that was carried out during the analysis. Then lastly, glucose analysis was carried out to determine the absorbance and glucose concentration. It was noticed that the best concentration of HCL acid during hydrolysis yields glucose concentration of 0.127g or 1.270%.

CHAPTER ONE

1.1 Introduction

Cellulose is the name given to a long chain of atoms consisting of carbon, hydrogen and oxygen arranged in a particular manner it is a naturally occurring polymeric material containing thousands of glucose-like rings each of which contain three alcoholic OH groups.
Its general each of which contain three alcoholic OH groups. Its general formula is represented as (C6H1005)n. the oh-groups present in cellulose can be esterifies or etherified, the most important cellulose derivatives are the esters.
Cellulose is found in nature in almost all forms of plant life’s, and especially in cotton and wood. A cellulose molecule is made up of large number of glucose units linked together by oxygen atom. Each glucose unit contains three(3) hydroxyl groups, the hydroxyl groups present at carbon-6 is primary, while two other hydroxyl are secondary. Cellulose is the most abundant organic chemical on earth more than 50% of the carbon is plants occurs in the cellulose of stems and leave wood is largely cellulose, and cotton is more than 90% cellulose. It is a major constituent of plant cell walls that  provides strength and rigidity and presents the swelling of the cell and rupture of the palms membrane that might result when osmotic conditions favor water entry into the cell. Cellulose is a fibrous, ought, water-insoluble substances, it can be seen in cell walls of plants, particularly in stalks, stems, trunks and all woody portions of the plant.
Cellulose is polymorphic, i.e there are number of different crystalline forms that reflect the history of the molecule. It is almost impossible to describe cellulose chemistry and biochemistry without referring to those different forms. Cellulose are gotten from cellulose, cellulose is also found in protozoa in the gut of insects such as termites. Very strong acids can also degrade cellulose, the human digestive system has little effect on cellulose. The world cellulose means β-1, 4- D glucan, regardless of source because of the importance of cellulose and difficulty in unraveling its secrets regarding structure, biosynthesis, chemistry, and other aspects, several societies are dedicated to cellulose, lignin, and related
molecues.

1.2 Definition of Terms

Hydrolysis: means hydro (water) lysis (splitting) or breaking down of a chemical bond by the addition of water (H2O), it is by the introduction of the elements that make up water hydrogen and oxygen. The reactions are more complicated than just adding water to a compound, but by the end of a hydrolysis reaction, there will be two more hydrogen’s and one more oxygen shared between the products, than there were before the reaction occurred. Hydrolysis of cellulose therefore is the process of breaking down the glucosidic bonds that holds the glucose basic units together to term a large cellulose molecule, it is a term used to describe the overall process where cellulsose is converted into various sweeteners. Sugar: is the generalized name for a class of chemically related sweet – flavored substances, most of which are used as food. They are carbohydrates, composed of carbon, hydrogen and oxygen  There are various sugar derived from different sources. Simple sugars are called monosaccharide’s and include glucose cellos known as dextrose, fructose and galactose. The table or granulated  sugar most customarily used as food is sucrose, a disaccharide other disacclarides include maltose and lacoose. Chemicallydifferent substances may also have a sweet taste, but are not classified as sugar but as artificial sweeteners.

1.3 STATEMENT OF THE PROBLEM

The new government policies and economy through low quality products has imposed motivated researchers to explore thenumerous domestic, industrial and economic importance of the Nigeria’s major waste product which is “cellulose” which forms the bedrock of this project. Sugar is a high demand for both domestic and industrial applications on daily basis in homes, small and medium scale industries etc this is why Nigeria government spends huge sums of money on importation of sugar and sugar products to meet the demand of citizens. Among the many processes of sugar production, is acid hydrolysis of (cellulose) has proved to be a process which encourages the production of high quality with minimum skill and  materials. This work is therefore an effort to encourage industrialist, researchers, and students to carry out more intensive studies on production of sugar from cellulose for production of sugar and enhanced economic resources for the nation.

1.4 SCOPE AND OF STUDY LIMITATIONS

This study is aimed at estimating the impact of some areas hindering the subject/project matter (disadvantages) the cellulose. It is obvious that cellulose materials have been used, including newspaper, carboard, cotton, straw, sawdust, hemp and corncob. Monticell was insulated with a form of cellulose. Modern cellulose insulation, made with recycled newspaper using grinding and dust removing machines and adding a fire retardant, began in the 1950s and came into general use in the U.S during the 1970s. The R value Rule” placed clear limitations on the claims that manufacturing and marketing firms can make about their product then also the effect of regulations by the CPSC put most of the small producers of cellulose insulation out of business. The costs  incurred by increasing fire testing made cellulose more expensive and the bad publicity helped decrease demand. Cellulose also has a few disadvantages. As compared to other insulation options, the R-value of 3.6 to 3.8 per inch is good but not the best. Many spray foams utilizes an environmentally harmful blowing agent, such as enovate HFC, cellulose does not. Dust: Cellulose contains some small particles which can be blown into the house through inadequate seals around fixtures or minute holes.
Wet-spray drying time: We-spray provides the moisture requires a longer drying time before the drywall/sheet-rock is applied to a newly insulation.

1.5 OBJECTIVES

The principal aim of undertaking this project is to determine the effect of concentration of acid on the yield of glucose production by acid hydrolysis of cellulose.
Hydrolysis of cellulose into glucose using different concentration of hydrochloric acid. Calculating and quantifying the yield of glucose from hydrolysis of cellulose using HCL acid. In the experiment, cellulose from variety of sources will be subjected to depolymerization conditions.

CHAPTER TWO

2.1 LITERATURE REVIEW

Cellulose is an organic compound with the formula (C6H1005)n, a polysaccharide consisting of a linear chain of several hundred to over ten thousand β (1-4) linked D-glucose units. It is the structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. Some species of bacteria secrete it to form biofilms. Cellulose is the most common organic compound on earth.
For industrial use, cellulose today is mainly used to produce paperboard and paper; to a smaller extent it is converted into a wide variety of derivative products such as cellophane and range. Converting cellulose from energy corps into biofuels such as cellulose ethanol is under investigation as an alternative fuel source, some animals, particularly ruminants and termites, can digest cellulose with the help of symbiotic micro-organisms that lives in there guts. Humans can also digest cellulose to some extents, however it mainly acts as a hydrophilic bulking agent for feces and is often referred to as “dietary fiber”,
I laboratory isolation of cellulose is used as the stationary phase fo thin layer chromatography cellulose is further used to make highly absorbent sponges. Cellulose fibers are also used in liquid filtration  It’s insulation male from recycled paper is becoming popular as an environmentally preferable material for building insulation. It can be treated with boric acid as a fire retardant.

2.2 HISTORY

Cellulose was discovered in 1838 by the French chemist anselme payen, who isolated it from plant matter and determined its chemical formula cellulose was used to produce the first successful thermoplastic polymer, celluloid, by Hyatt manufacturing company in 1870. Hermann Staudinger determined the polymer structure of cellulose in 1920. The compound was first chemically synthesized (without the use of any biologically derived enzymes) in 1992, by Kobayashi and shoda.
Discovery: the word “cellulose” was coined just 37 years before the American chemical society was formed. It appeared first in 1839 in  the French academy’s assessment on anselme payen’s research on “ligneous matter”, the then-current term for the combination of lignin and cellulose that forms the woody cell walls of trees and other plants. In 1838 payen reported that ligneous materials. One of these, the French chemist declared, had the same chemical composition as starch, but differed in structure and properties.

2.3 PRODUCTS

The kraft process is used to separate cellulose from lignin; another major component of plant matter cellulose is the major constituent of paper, paper board, and card stock and of textiles made from cotton, linen, and other plant fibers.
Cellulose can be converted into cellophane, a thin transparent film, and into rayon, an important fiber that has been used for textiles since the beginning of the 20thcentury. Both cellophane and rayon are known as “regenerated cellulose fibers”, they are identical to cellulose in chemical structure and are usually made from  dissolving pulp via viscose. A more recent method which is also friendly to the environment to produce a form of rayon is the lyoceu process. Cellulose is the raw material in the manufacture of nitrocellulose (cellulose nitrate) which is used in smokeless gunpowder and as the base material for celluloid used for
photographic and movie films until the mid-1930s. Cellulose is used to make water-soluble adhesives and binders such as methylcellulose and carbozymethyl cellulose, which are used in wallpaper paste. Microcystouline cellulose and powdered cellulose are used as inactive fillers in tablets and as thickeners and stabilizers in processed foods. Cellulose powder is for example used in kraft’s parmesan cheese to prevent caking inside the tube. Cellulose consists of crystalline and amorphous regions. By treating it with strong acid, the amorphous regions can be broken up, thereby producing nanocrystalline cellulose, a novel material with many desirable properties. Recently, nanocrystalline cellulose was used as the filler phase in bio-based polymer matrices to produce nanocomposites with superior thermal and mechanical properties.
Fig 4.2: Cellulose

2.4 CELLULOSE SOURCE AND ENERGY STORE OF CROPS

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The major combustible component of non-food energy crops is cellulose, with lignin second, non-food energy crops are more efficient than dibble energy crops (which have a large starch component), but still compete with food crops for agricultural land and water resources. Typical non-food energy crops include industrial hemp (though outlawed in some countries), switch grass, miscanthus, salix (willow),and populous (poplar) species. Some bacteria can convert cellulose into ethanol which can then be used as a fuel.

In photosynthesis, crops use light energy to produce glucose from carbondioxide. The glucose is stored mainly in the form of cellulose granules, in plastids such as chloroplasts and especially anyloplasts. Glucose is soluble in water hydrophilic, binds much  water and the takes much space. Glucose in the form of starch on the other hand, is not soluble and can be stored much more compactly.

Cellulose for industrial conversion comes from wood and scores of minor sources such as kenaf paper and rayon are now made mostly from wood pulp, cotton linters (short fibers are used to spin yams) are now used in high quality writing and currency papers. Cellulose forms very tightly packed crystallites, these crystals are sometimes so tight that neither water nor enzymes can penetrate them; cellulose consists of two cellulose molecules, crystalline and amorphous cellulose. The crystalline cellulose is insoluble because of the inability of water to penetrate cellulose, on the other hand amorphous cellulose allows the penetration of endogluconase, another subgroup of cellulose that catalyzed the hydrolysis of internal bonds. The natural consequences of this difference in the crystalline structure is that the hydrolysis rate is much faster for amorphous cellulose than crystalline cellulose. Some other cellulose comes from the hairs (trichomes) on seeds examples; cotton, kapo and milk weed. A commercial bacterial  cellulose produce (cellulon) was introduced by weyer haeuser for use in foods, the product is called primacel and is available from Nutrasweet kelco. Now as of reconnect, cellulose from sugar bet pulp and as a fat substitute.

2.5 STRUCTURE AND PROPERTIES

Cellulose has no taste, is odorless, is hydrophilic with the contact angle of 20-30°, is insoluble in water and most organic solvents, is chiral and is biodegradable it can be broken down chemically into its glucose units by treating it with concentated acids at high temperature.
Cellulose is derived from D-glucose units, which condense through β (1-4) – glycosidic bonds. This linkage motif contrasts with that for α (1-4) – glycosidic bonds present in starch, glycogen, and other carbohydrates cellulose is a straight chain polymer: unlike starch, no coiling or branching occurs, and the molecule adopts an extended and rather stiff rod-like conformation, aided by the equatorial conformation of the glucose from one chain form hydrogen bonds with oxygen atoms on the same or on a neighbor   chain, holding the chains firmly together side by side and forming microfibrils with high tensile strength. This strength is important in cell walls, where the microfibrils are meshed into a carbohydrate matrix, conferring rigidity to plant cells.
Fig:4.3 Strand of Cellulose.
A strand of cellulose (conformation 1ά), showing the hydrogen bonds (dashed) within and between cellulose molecules. Compared to starch, cellulose is also much more crystalline whereas starch undergoes a crystalline to amorphous transition when heated beyond 60-70oC in water (as in cooking),cellulose requires a temperature of 320oC and pressure of 25Mpa to become amorphous in water. Several different crystalline structures of cellulose are known, corresponding to the location of hydrogen bonds between and within strands. Natural cellulose is cellulose i, with structures iά and iβ, cellulose produced by bacteria and algae enriched in iά  while cellulose of higher plants consists mainly of iβ. Cellulose in regenerated cellulose fibers is cellulose ii. The conversion of cellulose i to cellulose ii is irreversible, suggesting that cellulose 1 is metastable and cellulose ii is stable with various chemical treatments it is possible to produce the structures cellulose iii and cellulose iv.
Many properties of cellulose depend on its chain length or degree of polymerization, the number of glucose units that make up onepolymer molecule. Cellulose from wood pulp has typical chain lengths between 300 and 1700 units; cotton and other plant fibers as well as bacterial cellulose have chain lengths ranging from 800 to 10,000 units. Molecules with very sall chain length resulting from the breakdown of cellulose are known as cellodextrins; in contrast to long-chain cellulose, cellodextrins are typically soluble in water and organic solvents. Plant derived cellulose is usually found in a mixture with hemicelluloses, lignin, pectin and other substances, while microbial cellulose is quite pure, has a much higher water content, and consists of long chains.
Cellulose is soluble in cupriethylenediamine (CED), cadmiumethylenediamine (cadoxen), N-methylmorpholien N-oxide and lithium chloride/dimethylformamide middle. This is used n the production of regenerated celluloses (such as viscose and cellophane) from dissolving pulp.
Fig. 4.4: Triple strand of Cellulose

A triple strand of cellulose showing the hydrogen bonds (cyan lines) between glucose strands.
Assaying a cellulose- containing material: given a cellulosecontaining material, the carbohydrate portion that does not dissolve in a 17.5% solution of sodium hydroxide at 200C is a cellulose, which is true cellulose. Acidification of the extract precipitates β cellulose. The portion tat dissolves in base but does not precipitate with acid is y cellulose.
Cellulose can be assayed using a method described by updegraff in 1969, where the fiber is dissolved in acetic and nitric acid to remove lignin, hemicelluloses, and xylosans. The resulting cellulose is  allowed to react with anthrone in sulfuric acid, the resulting coloured compound is assayed spectrophotometrically at a wavelength of approximately 635mm. in addition, cellulose is represented by the difference between acid detergent fiber (ADF) and acid detergent lignin (ADL).

2.6 BIOSYNTHESIS

In vascular plants cellulose is synthesized at the plasma membrane by rosette terminal complexes (RTCs). The RTCs are hexameric protein structures approximately 25nm in diameter, that contain the cellulose synthase enzymes that synthesise the individual cellulose chains. Each RTC flats in the cell’s plasma membrane and “spins” a microfibril into the cell wall.

RTCs contain at least three different cellulose syntheses, encoded by lesA genes, in an unknown stoichiometry. Separate sets of genes are involved in primary and secondary cell wall biosynthesis. Cellulose synthesis requires chain initiation and elongation, and the two processes are separate, CESA glucosyl transferase initiates cellulose polymerization using a steroid primer, sitosterol-beta- glucosides, and UDP-glucose. Cellulose syntheses utilizes UDP-Dglucose precursors to elongate the growing cellulose chain. A cellulose may function to cleave the primer from the mature chain. The concept of cellulose being synthesized by living organisms about “an acetic ferment which forms cellulose referring to the generation of cellulose by the gram negative rod, acetobacter xylinum, (Wikipedia,2013). Over the years, this microbe has led to the understanding of the structure and biosynthesis of cellulose. One of the earliest electrol microscopy studies of microbial cellulose was made in 1949 by Kurt muhlethaler. This led the way to numerous investigations of acetobacter in the 1960’s by J. Ross Colvin and colleagues from Canada. (Wikipedia,2013) In the late 1950’s reclofsen proposed the concept that cellulose microfibrils must “grow” by tips growth from enzyme complexes. (Wikipedia,2013). In 1964, Reginald preston proposed the “ordered  granule hypothesis” largely theoretical and based on the emerging freeze fracture technology at that time. It was not until 1976 that R. Malcolm Brown, Jr. and colleagues, then from the university of North Carolina at chapel Hill, discovered ordered membrane  associated aggregates associated with ends of developing cellulose microfibrils. This discovery was made in the green alga, oocystis apiculate. They named these putative enzyme complexes, TCs or “terminal complexes”.

Isolation and purification of cellulose syntheses has been very difficult. The first breakthrough in this area came from purification of a acetobacer cellulose synthase from the brown lab in 1989. Identification of catalytic sub-unit of bacterial synthase by azidoUDP-glucose binding led to the first sequencing of a cellulose synthase by the Brown laboratory in 1990. Cellulose synthase associated proteins involved in accelerating the in vitro reactions were discovered by the Benziman laboratory in Israel. The first in  vitro synthesis of cellulose came from work in the Brown laboratory(1993), and since this time, cellulose and callose biosynthesis have been physically separated; however, the purification of vascular plant cellulose synthase and its identification remain to this day as one of the mot difficult problems in plant biochemistry. (Young, 1986).

The genes for bacterial celluloses synthase were subjected to hydrophobic cluster analysis by a joint collaboration of the brown lab in Autin and the henrissat lab in Grenoble in 199s. using this information, delmer and her colleagues identified a cellulose synthase gene from cotton in 1996. Williamson and his lab in Australia also identified the gene for cellulose synthase in Arabidopsis in 1999. In the brown lab, the gene for cotton cellulose synthase was independently cloned and sequened. Then the DNA from this sequence was inserted into Escherichia coli which was coaxed into producing a polypeptide corresponding to the introduced gene. This polypeptide was extracted and purified and polyclonal antibodies produced to the recombinant cellulose sythnase catalytic subunit. In collaboration with the laboratory of Dr. Takao into in Kyoto, and using freeze fracture labeling, the two labs were able to prove unequivocally that the rosette subunit to cellulose synthase.

On a more novel approach, perfectly normal cellulose I has been synthesized synthetically in the brown lab (1994) using technology originally developed by Dr. kobayashi of Kyoto university in 1991.
Here, enzymatic polymerization utilizing β- cellobiosyl fluoride as the substrate monomer and celluloses of cellulose I in a reverse micelle, this very different approach may have some future industrial application.
On the other hand, focusing on native cellulose, the present time is very active in identifying cellulose syntheses genes from many plants and from various developmental stages. The next step will be the use of “antisense” to either halt, or the introduction of modified genes to literally modify the structure of cellulose synthesized by living organisms. Already transgenic plants with modified cellulose biosynthetic pathways are beginning to emerge in the literature as of 2000. Thus, the future for genetic modification of cellulose biosynthesis in plants and microbes promises a vast new future for the forest products and textile industries.

2.7 BREAKDOWN (CELLUCOLYSIS).

Cellulose is the process of breaking down cellulose into smaller polysaccharides called cellodextrins or complete into glucose units; this is a hydrolysis reaction. Because cellulose molecules bind  strongly to each other, cellulolyses is relatively difficult compared to the breakdown of other polysaccharides. Most mammals have only very limited ability to digest dietary fibres such as cellulose. Some ruminants like cows and sheep contain certain symbiotic anaerobic bacteria (like cellulomonas) n the flora of the rumen, and these bacterial produce enzymes called cellulose; the breakdown products are the used by the bacteria for proliferation. The bacterial mass is later digested by the ruminant in its digestive system (stomach and small intestine). Similarly, lower termites contains in their hindguts certain flagellate protozoa which produce such enzymes; higher termites contain bacteria for the job. Some termites may also produce cellulose of their own. Fungi, which in nature are responsible for recycling of nutrients, are also able to breakdown cellulose. The enzymes utilized to cleave the glycosidic linkage in cellulose are glycoside hydrolyses including endo-acting cellulose and exo-acting glycosidase. Such enzymes are usually secreted as part of multienzyme complexes that many include doctrines and
carbohydrate-binding modules.

2.8 HEMICELLULOSE

Hemicelluloses is a polysaccharide related is cellulose that comprises about 20% of he biomass of most plants. In contrast to cellulose, hemicelluloses is derived from several sugars in addition to glucose, especially Xylose but also including mannose, galactose, rhamnose, and arabinose. Hemi- cellulose consists of shorter chains-around 200 sugar units. Furthermore, hemicellulose is
branched, whereas cellulose is unbranched. (Wikipedia,2013)

 

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