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Bioremediation of Agricultural Waste: Lemongrass

1.0 Introduction

Lignocellulosic waste is generated in large amounts by various sectors of economy which include forestry and agricultural practices, paper-pulp industries, timber industries and many other growing industries(D. Akin, 2007). Globally, 998 million of agricultural waste is produced every year. In Malaysia, around 1.2 million tonnes of agricultural waste is produced every year. Malaysia is the fourth largest producer of agricultural waste in Asia, with a generation rate of 0.122 kg/capita/day (Agamuthu, 2009) (Table 1).

Country

Agricultural

Waste

Generation (kg/capita/day) Japan Singapore R. Korea Malaysia Chine Thailand Nepal Bangladesh Table 1: 0.17 0.165 0.15 0.122 0.12 0.096 0.06 0.04

Comparison of agricultural waste generation rates between countries in Asia (Agamuthu, 2009)

Currently, much of the lignocellulose waste produced is disposed by mass burning (Leving, 1996). However, with increasing emphasis in research, various applications have been discovered for lignocellulose waste, the most noted of which is the production of biofuel. In addition, lignocellulose waste can also be converted into a panoply of value-added products including chemicals, cheap energy sources for fermentation, improved animal feeds and human nutrients. Lignocellulytic enzymes also have significant potential applications in various

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Bioremediation of Agricultural Waste: Lemongrass

industries including chemicals, fuel, food, brewery and wine, textile and laundry, pul and paper and agriculture (Howard R.L. et al, 2003).

Lignocellulose is a macromolecular complex of lignin and the polysaccharides cellulose and hemicellulose (holocellulose). A number of enzymes are required in order to completely breakdown lignocellulose, as its constituent polymers are physically intertwined and chemically bound. The enzymes that degrade cellulosic material have been classified into three major groups: endoglucanases, exoglucanases and B-glucosidases.

In natural decomposition of lignocellulosic matter, both fungi and aerobic bacteria play an important role in degrading holocellulose and lignin to lower- molecular-weight products, some of which are then further metabolized by facultative and obligate anaerobic soil nacteria and actinomycetes(Benner et al, 1984). In practical use, both fungi and bacteria have been heavily exploited for their abilities to produce a wide variety of cellulases and hemicellulases. Most emphasis has been placed on the use of fungi because of their capability to produce copious amounts of cellulases and hemicellulases which are secreted to the medium for easy extraction and purification (Maki et al, 2005).

Lemongrass (Cymbopogon) is an essential oil crop native to Southern India and Sri Lanka, Indonesia and Malaysia. The plant in its raw form is most commonly used as an herb, while its oil which is extracted through steam distillation has many applications in perfumery, medicine and cosmetics. As a practice, lemongrass waste obtained after steam distillation is partially dried then burned to generate steam, while the remainder is relinquished in the fields for natural biodegradation. (Rolz et al, 1986). Based on the production of essential oils, the total world production of lignocellulosic residues of all species of lemongrass is about 30,000,000 tonnes per annum. While marginal if compared to waste produced by other agricultural sectors like oil palm, wood, coconut and rice, the management of lemongrass waste is required in order to create a sustainable production practice in the sector itself, besides reducing the overall waste produced by the agricultural sector.

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Bioremediation of Agricultural Waste: Lemongrass

Cellulase-producing bacteria and fungi have been isolated from many sources such as composting heaps, decaying plant material, feces of ruminants, soil and organic matter, and extreme environments to name a few. In the present study, we have attempted to isolate and characterize the bacteria and fungi from the residues of a native species of lemongrass obtained from a farm in Semenyih, Selangor. The cellulose degrading capability as well as the morphology of the bacterial and fungal isolates was observed.

2.0 Literature Review 2.1 Lignocellulosic materials All the plants as well as the lemongrass consist of lignocellulosic which is made up of cellulose, lignin and hemicelluloses. Thus, agricultural residue also can be called as lignocellulosic biomass. Lignocellulosic biomass (plant biomass), is a great potential resource for the production of biofuels because it is largely abundant, inexpensive and production of such resources is environmentally sound. Agricultural residues are a great source of lignocellulosic biomass which is renewable, chiefly unexploited, and inexpensive. Such resources include: leaves, stems, and stalks from sources such as corn fibre, corn stover, sugarcane bagasse, rice hulls, woody crops, and forest residues. Also, there are multiple sources of lignocellulosic waste from industrial and agricultural processes, e.g., citrus peel waste, sawdust, paper pulp, industrial waste, municipal solid waste, and paper mill sludge. In addition, dedicated energy crops for biofuels could include perennial grasses such as Switchgrass and other forage feedstocks such as Miscanthus, Bermuda grass, Elephant grass, etc. Approximately 70% of plant biomass is locked up in 5- and 6-carbon sugars. These sugars are found in lignocellulosic biomass, which is comprised of mainly cellulose (a homologous polymer comprised of long chains of glucose); less so, hemicelluloses (heterologous polymer of 5- and 6-carbon sugars); and least of all lignin (a complex aromatic polymer). The major component cellulose, is a homopolysaccharide comprised of glucose units, linked by -(14) glycosidic bonds. Cellobiose is the smallest repeating unit of cellulose and can ultimately be converted into glucose. Hemicellulose is a heterogeneous polymer, which varies in composition from plant to plant and within different parts of the same plant. It is made up of mainly pentoses (D-xylose, D-arabinose), hexoses (D-mannose, D-glucose, D-galactose) and sugar acids. In hardwoods hemicellulose contains mainly xylans, while in softwood mainly glucomannans are present. Hydrolysis of hemicelluloses requires various types of enzymes.
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Bioremediation of Agricultural Waste: Lemongrass

Briefly, xylan degradation requires endo-1-4,--xylanase, -xylosidase, -glucuronidase, -Larabinofuranosidase, as well as acetylxylan esterases. In glucomannan degradation mannanase and -mannosidase are required to cleave the polymer backbone. The major structural component of lemon grass is lignocellulose, a matrix of crosslinked polysaccharide networks, glycosylated proteins, and lignin. Lignocellulose consists of lignin, hemicellulose and cellulose. While the content of each component differs between species to species, on average the lignocellulosic content of grasses are as follows: cellulose 25-40%, hemicellulose 25-50%, lignin 10-30%. 2.1.1 Cellulose Cellulose is a homogenous polymer of glucose. It consists of a linear chain of several hundred to more than 10,000 D-glucose units linked by B-1,4-bonds. Cellulose chains are crystalline in structure and assemble into cable like bundles of crystalline fibrils. Cellulose chains in the crystals re stiffened by inter and intra chain hydrogen bonds and the adjacent sheets which overlie one another are held together by weak Van der-waals forces. This crystalline array accords relative impermeability of not only large molecules like enzymes but in some cases even small molecules like water. The polymeric structure contains crystalline and amorphous regions; in addition there exists several types of surface irregularities. This heterogeneity makes the fibers capable of swelling when partially hydrated, with the result that the micropores and cavities become sufficiently large enough to allow penetration of larger molecules including enzymes. 2.1.2 Hemicellulose Hemicellulose macromolecules is typically made up of chains of xylose interspersed with side chains containing arabinose, galactose, mannose, glucose, acetyl, and other sugar groups. Hemicellulose also includes a small amount of pectin, with which it forms a crosslinked network.

2.1.3 Lignin Lignin is a cross-linked macromolecule composed of three types of phenylpropanoids, ro aromatic alcohols (coniferyl alcohol, sinapyl and p-coumaryl). In addition, grass and dicot
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Bioremediation of Agricultural Waste: Lemongrass

lignin also contain large amounts of phenolc acids such as p-coumaric and ferulic acid, which are esterified to alcohol groups of each other and to other alcohols such as sinapyl and pcoumaryl alcohols. Lignin is linked to hemicellulose and cellulose by covalent bonds, and forms a physical seal around the two components that becomes a barrier which prevents the penetration of solutions and enzymes.

2.2 Lemongrass Cymbopogon (lemongrass) is a genus of about 55 species of grasses, native to warm temperate and tropical regions of the Old World and Oceania. Lemongrass belongs to the grass or Poaceae family (formerly known as Gramineae). Lemon grass is tall, perennial sedge throwing up dense fasciclles of leaves from a short rhizome. The culm is stout, erect, up to 1.8 meter high. Leaves are long, glaucous, green, linear tapering upwards and along the margins; ligule very short; sheaths terete, those of the barren shoots widened and tightly clasping at the base, others narrow and separating. It is a short day plant and produce profuse flowering in South India. The inflorescence is a long spike about one metre in length. Flowers borne on decompound :spatheate ; panicles 30 to over 60 cm long. Common names include lemon grass, lemongrass, barbed wire grass, silky heads, citronella grass, cha de Dartigalongue, fever grass, tanglad, hierba Luisa or gavati chaha amongst many others. Most of the species of lemon grass is native to India, Sri Lanka, Malaysia and Indonesia. The so called East Indian lemon grass (Cymbopogon flexuosus) , also known as Malabar or Cochin grass is native to India , Sri Lanka, Burma and Thailand ; for the related West Indian lemon grass (Cymbopogon citratus), a Malaysian origin is generally assumed. Both the species are today cultivated throughout tropical Asia. Both of the common species are fast growing perennials that grow to 3 to 6 feet tall and 3 feet wide.

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Bioremediation of Agricultural Waste: Lemongrass

Figure 1:

Lemongrass plant

One of the main constituents of the many different species of lemongrass (genus Cymbopogon) is citral (3,7-dimethyl-2,6-octadien-1-al). Lemongrass oil has been found to contain up to 75-85% citral. Lemongrass also contains z-citral, borneol, estragole, methyleugenol, geranyl acetate (3,7-dimethyl-2,6-octadiene-1-ol acetate), geraniol (some species higher in this compound than citral), beta-myrcene (MYR, 7-methyl-3-methylene-1,6 octadiene), limonene, piperitone, citronellal, carene-2, alpha-terpineole, pinene, farnesol, proximadiol, and (+)-cymbodiacetal. The volatile oil from the roots contains 56.67% longifolene-(V4) and 20.03% selina-6-en-4-ol. In particular, a study of Cymbopogon martinii isolated fatty acids, common sterols, and 16-hydroxypentacos-14(z)-enoic acid. Lemongrass requires a warm and humid climate with plenty of sunlight and rainfall, ranging from 250 cm to 300 cm, uniformly distributed over the year. The plant is hardy and resistant to drought. High temperature and sunshine are conducive to the development of oil in the plant. It grows luxuriantly in hilly places receiving heavy rainfall and is harvested more frequently; its oil and citral contents are less compared to the plants growing in regions receiving less rainfall. Lemongrass grows well at altitudes between 100 and 1,200 m above sea level, generally on poor soils with hilly slopes but flourishes on a wide variety of soils ranging from rich loam to poor laterite. It grows best on well-drained sandy loam soil; it even thrives on

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Bioremediation of Agricultural Waste: Lemongrass

light sandy soils, provided they are well- manured. Plants from such a sandy soil yield relatively more oil with higher citral content. Lemongrass oil is one of the most important essential oils produced in the world. The oil is produced because of its high citral content, which is used as a basic raw material to synthesize bionones which is used to synthesize a number of aromatic useful compounds and Vitamin A. The oil is distilled from leaves and flowering tops of lemongrass. The Citral rich oil has germicidal, medicinal and flavouring properties. West Indian lemon grass (C.citratus) has low citral content in the oil and has meager trade in the country. Lemongrass oil consists of 65 to 85 percent Citral as it is the main component in lemongrass oil. Citral is a pale yellow liquid, often colorless, with a strong fresh lemon smell. Citral is extracted from fresh leaves by using steam distillation. It can be blended with other ingredients as well, including Bergamot, Cardomon and Petigrain Rose.

Figure 2:

Products of lemongrass oil

Normally, lemongrass oil is extracted by various ways such as the solvent, accelerated solvent, Soxhlet (Sargenti and Lancas., 1997), dense carbon dioxide (Carlson et al., 2001), solid-phase matrix (Pham-Tuan et al., 2001), and supercritical fluid (Schaneberg and Khan, 2002) extraction methods. However, the common procedure of extracting essential oil is by the hydrodistillation method (Kulkarni et al., 2003). Gas chromatography-mass spec-trometry (GC-MS) has been the most applied analytical techniques for essential oil analysis (Masada,1976) followed by the supercritical fluid extraction-gas chromatography (Liu et al., 1993). Due to the complexity of essential oil compositions, sophisticated instruments such as high performance liquid chromato-graphy in combination with gas chromatography (HPLCGC) (Mondello et al., 1996) is the preferred analysis. HPLC is effective for a broad class
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Bioremediation of Agricultural Waste: Lemongrass

separation of a sample, which can be introduced into a GC for further high resolution separation.

Table 2:

Composition of lemongrass essential oil (taken from

http://www.scielo.br/scielo.php?pid=S0104-66322004000200019&script=sci_arttext#tab03) Citral, an ingredient of lemongrass oil, has numerous uses, and can be further processed to possess a violet-like fragrance for perfumery, and as a source of vitamins A and E. Lemongrass oil is also used for deodorants, waxes, polishes, detergents and insecticides where its low cost is attractive. Lemongrass oil is used as a fragrance component in soaps, detergents, and cosmetics. It is also used in aromatherapy, and it improves circulation and muscle tone. Astringent and toning, a lemongrass facial is administered through steam inhalation. It tightens, refines and firms a sluggish, lacklustre, oily skin. In aromatherapy it is used as tissue toner. Lemongrass oil, used as a pesticide and preservative, is put on the ancient palm-leaf manuscripts found in India as a preservative. It is used at the Oriental Research Institute Mysore, the French Institute of Pondicherry, the Association for the Preservation of the Saint Thomas Christian Heritage in Kerala and many other manuscript collections in India. The lemon grass oil also injects natural fluidity into the brittle palm leaves and the hydrophobicnature of the oil keeps the manuscripts dry so that the text is not lost to decay due to humidity. The grass is considered a diuretic, tonic, antiseptic and stimulant. It promotes good digestion, and a preparation of lemongrass with pepper is used for relief of menstrual troubles and nausea. It induces perspiration, cools the body and reduces a fever. It is used to treat
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Bioremediation of Agricultural Waste: Lemongrass

diarrhoea, stomach-ache, headaches, fevers, and flu. It is helpful in treating muscular pain, poor circulation, and muscle tone and slack tissue. The antiseptic oil treats athletes foot and acne. Lemongrass fights airborne bacteria when used in a vaporiser for relief of symptoms of colds and flu. A lemongrass foot bath scent refreshes sweaty feet while its antibacterial properties prevent fungal infections. Dilute lemongrass oil and apply directly on topical areas affected by ringworm. In veterinary use it appears in dog and cat shampoos as repellent for fleas, lice and ticks. Lemongrass oil has been found to have anticancer properties when tested on animal subjects. Researchers at the Indian Institute of Integrative Medicine in Jammu, India, studied the effects of lemongrass oil on several different types of cancer cells by injecting the oil into the body of mice that were afflicted with the cancer. Published in the May 15, 2009 issue of Chemico-biological Interactions, the results of the study indicated that lemongrass oil exhibited anticancer activity by initiating apoptosis, or cellular death of the cancer cells. Lemongrass oil was most effective against colon cancer cells and neuroblastoma cancer cells. 2.2.1 Lemongrass Waste As the lemongrass provided many kinds of uses to us, they also contribute to the agricultural waste production in Malaysia. Most people use the lower part of the lemongrass but seldom use the leaves of the upper part of the lemongrass. Many farmers just use it as a natural fertilizer. After the above part of the lemongrass is cut off, some of them were just thrown away on the ground. Besides that, there are a few farmers use them as the animal feedstock. The leaves are not totally can be label as a waste because some of the farmers have found the way to reduce the waste by utilizing them, but not all the farmers are being exposed by the way of utilizing the leaves of lemongrass. So, that is why there are still lots of leaves of lemongrass which are just being tossed away. Despite of all this, the leaves of the lemongrass definitely contributed to the total of the agricultural waste production in Malaysia.

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Bioremediation of Agricultural Waste: Lemongrass

Figure 3 :

The figure shows the bottom half of lemongrass plants which is used the

upper half is discarded and becomes waste

The diagram below shows the proportions of waste that Malaysia generates from the production of rice, palm oil, rubber, coconut and forest products (ESCAP 1997). Even though the waste from lemongrass plant is not stated because of its minority, but the lemongrass wastes are still acknowledged by people.

Diagram 1 :

Proportionate Annual Production of Agricultural Waste in Malaysia

2.2.2 Application of the Lemongrass waste

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Bioremediation of Agricultural Waste: Lemongrass

Normally the lemongrass wastes are come from the process of extracting the lemongrass oil. In order to get the lemongrass oil, the leaves of lemongrass will get through the steam distillation process, producing the unused spent lemongrass leaves. The spent grass can be used as cattle feed fresh or after ensilaging. It can be used for mulching or manuring crops as such or after composting. In some plantations in India, spent lemongrass after drying is used as a fuel for distillation. It is also a cheap packing material. All of these are some of the example of the spent lemongrasss example. Not all the farmers or the researchers took all the lemongrass leaves in the process of extracting the oil. There are also plenty of unused lemongrass leaves that already being toss away from the beginning. As mentioned earlier, some of the educated farmers use the unused lemongrass leaves as the natural fertilizers and animal feedstock. Unfortunately, the knowledge of this application is not known worldwide. So that is why the unused lemongrass leaves are being left accumulated and become a waste. Notwithstanding, all of the unused leaves and spent leaves of lemongrass have contributed to the amount of waste from the lemongrass plant, especially in Malaysia.

2.3 Degradative enzymes Degradative enzyme is the cellulolytic enzymes that act synergistically ti hydrolyze cellulose or its modified polymer. Conversion of abundant cellulose waste materials such s straw, husk, sawdust and agriculture biomass. Degradative enzymes are also defined as the protein that hydrolyse cellulosic materials; cellulose and hemicellulose that made up plant fibre. Microbial degradation of lignocellulosic waste and the downstream products resulting from it is accomplished by a concerted action of several enzymes.

Figure 4: Model of cellulose enzyme Cellulolytic enzymes are the main enzymes needed in the degradation process of lignocellulosic compounds of the biomass. Cellulase refers to a class of enzymes produced
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Bioremediation of Agricultural Waste: Lemongrass

chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis. In general the enzyme catalyse the hydrolysis of 1,4- - D-glycosidic linkages in cellulose, lichenin and cereal - Dglucans. In many bacteria, cellulases in-vivo is complex enzyme structures organized in supramolecular complexes, the cellulosomes. They contain roughly five different enzymatic subunits representing namely endocellulases, exocellulases, cellobiases, oxidative cellulases and cellulose phosphorylases wherein only endocellulases and cellobiases participate in the actual hydrolysis of the (1 4) linkage.

Diagram 2:

Mechanism of cellulolysis

The three types of reaction catalyzed by cellulases: 1. Breakage of the non-covalent interactions present in the crystalline structure of cellulose (endocellulase) 2. Hydrolysis of the individual cellulose fibres to break it into smaller sugars (exocellulase) 3. Hydrolysis of disaccharides and tetrasaccharides into glucose (- glucosidase). A typical free cellulase is composed of a carbohydrate binding domain (CBD) at the Cterminal joined by a short poly-linker region to the catalytic domain at the N-terminal. CBD is the most common accessory module of cellulases and there are 54 distinct families. The major function of CBDs is to deliver its resident catalytic domain to crystalline cellulose. Binding brings the catalytic domain into close contact with the crystalline cellulose for efficient hydrolysis. Binding of the cellulase via CBD is extremely stable, yet still allows the enzymes to
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Bioremediation of Agricultural Waste: Lemongrass

diffuse laterally across the surface of the substrate and in some cases CBD has also been shown to catalyze the disruption of noncovalent interactions between cellulose chains of crystalline cellulose. Some other CBDs are preferential to binding noncrystalline cellulose (Maki et al, 2009). 2.3.1 Exoglucanase 1, 4- - D-glucan cellobiohydrolase catalyze the removal of cellobiose units from non reducing ends of cellulose chains. Other names that used for these types of enzymes are cellobiohydrolase, exoglucanse, exocellulase, cellobiosidase and avicellase .Simply, the enzyme hydrolyses -1, 4-D-glycosidic bonds of cellulose and cellodextrins into mainly cellobiose and the reaction catalyzed make the enzyme exo-acting. It reacts with the products of endoglucanase reaction since it requires non reducing ends to react with. Exoglucanase requires a non-reducing end of a broken amorphous region of reactive cellulose to react. It has the ability to also hydrolyse the crystalline region. May achieve >80% hydrolysis depending on the source of exoglucanase. Exoglucanase is more active than endoglucanase when cellulose is highly amorphous. 2.3.2 Endoglucanase 1, 4- - glucan 4-glucanohydrolase hydrolyse the internal 1.4- - D-glycosidc linkage in cellulose, lichenin, and cereal - D- glucans. Common names are endoglucanase, endo-1, 4- glucanse and CM cellulose. In general, The enzyme hydrolyses the - 1, 4- D- glycosidic bonds of cellulose to form oligosaccharides. It is endo- acting where it broke the internal bond of cellulose.

2.3.3 - glucosidase - D- glucoside glucohydrolase hydrolyze cellobiase and involves in removal of glucose from non reducing ends of cello-oligosaccharode and glycosyl transfer of cellobiase.
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Bioremediation of Agricultural Waste: Lemongrass

In the end of this catalytic reaction, -glucose is formed. This enyme is also commonly known as cellobiase and - glucosidase. This enzyme is produced by both fungi and bacteria. The difference is that the enzyme in bacteria exists as an intracellular enzyme. Therefore the hydrolysis of cellobiose by bacteria takes place at the cell wall.

Diagram 3: 2.4 Cellulolytic microorganisms

Mechanism of - glucosidase activity

Regardless of source, lignocellulosic materials contain cellulose, hemicelluloses and lignin as major components. Lignocellulose is the predominant component of woody plant and dead plant materials (Ohkuma et al.). In nature, dead plant will undergo biological degradation by microorganism. In natural decomposition of lignocellulosicmatter, both fungi and aerobic bacteria play an important role in degrading holocelluloseand lignin to lower-molecular-weight products (Rolz et al.). Cellulose is degraded by many heterotrophic bacteria and fungi into soluble saccharides for use in growth and energy production resulted as the stable form of products. There are several bacteria that have the enzymatic potential to degrade proteins into amino acids which then enables the assimilation process of other organisms. On the other hand, there are also many fungi that can produce extracellular enzymes that enable them to break them cellulose. Cellulolytic microorganisms are primarily carbohydrate degraders and generally derive energy sources for growth from lipids and proteins. Cellulolytic microbes, notably the bacteria Cellulomonas and Cytophaga and most fungi can utilize a variety of other carbohydrates in addition to cellulose, while the anaerobic cellulolytic species have a restricted carbohydrate range, limited to cellulose and or its hydrolytic products. Fungi characteristically secrete large amounts of extracellular proteins and such strains are most suited for the production of higher levels of extracellular cellulases. One of the most extensively studied fungi is Trichoderma reesei, which converts native as well as derived cellulose to glucose.
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Bioremediation of Agricultural Waste: Lemongrass

Other commonly studied cellulolytic organisms include: Trichoderma, Humicola, Penicillum, Aspergillus (fungi); Bacilli, Pseudomonas, Cellulomonas (bacteria); Streptomyces, Actinomucor, and Streptomyces (actinomycetes). 2.4.1 Fungi Among the microorganisms, fungi are usually faster lignin degraders, particularly the so-called white-rot fungi, which completely metabolize the complex polymer, exhibit the highest reported rates, and have been the most studied (Rolz et al.). White-rot fungi can degrade cellulose whereas most brown-rot fungi exhibit sparse growth on pure cellulose despite their rapid degradation.

Aspergillus niger Aspergillus niger (Figure 5) is a haploid filamentous fungi. A. niger produce colonies that are composed of white or yellow felt that is covered by dark asexually produced fungal spores. The fungi are most commonly found in mesophilic environments such as decaying vegetation or soil and plants. A. Niger produces acetyl- xylan esterase to degrade lignocelluloses. A. niger has a metabolic system which is composed of the cytoplasm, mitochondria, and peroxisome. Incorporated in this system are carbohydrate metabolism and amino acid metabolism which take place in both anabolic and catabolic reactions. Other properties of this species include pathogens that cause the spoilage of food and production of secondary metabolites, such as aflatoxin. This species of filamentous fungi produces several secondary metabolites, one of the most important being ochratoxin A. Ochratoxin A is an abundant food-contaminating mycotoxin. The productioin of ochratoxin A from A. niger, is liable to cause immunotoxcitiy in animals. Furthermore, A. niger is relatively harmless compared to other filamentous fungi. In addition, one of the most common ways that A. niger gains energy is through bioleaching. A. niger gains its energy by breaking down the minerals into its most basic element. The fungi specifically is able to break down copper, tin, aluminum, nickel, and lead, and with this energy is able to produce oxalic acid, gluconic acid, and citric acid. One of the most important products that A. niger produces is citric acid. Many of the enzymes produced by A. niger rather than citric acid are amylases, lipases, cellulases, xylanases and proteases. A. Nigerhas also been used for waste treatment and chemical modifications known as biotransformations.
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Bioremediation of Agricultural Waste: Lemongrass

Figure 5: Trichoderma reesei

Aspergillus niger

Trichoderma reesei (Figure 6), also known as Hypocrea jecorina, is a mesophilic fungus which grows in the moderate temperature range, about 20oC up to 45oC. It is a filamentous ascomycete that has the ability to produce extracellular lignocellulose-degrading hydrolases in large amount(Arja, 2004).It is an asexually reproducing filamentous fungus derived from from a population of the tropical saprophytic Ascomycete Hypocrea jecorina (Kuhls et al., 1996). The cellulolytic system of T. reesei can be divided into three major enzyme classes which are exoglucanases, endoglucanases and -glucosidase. T. reesei has a cellulolytic system composed of at least eight endoglucanases and and two cellobiohydrolases.Structurally, the active site of cellobiohydrolases is located in a thunnel, whereas the endoglucanases have the active site in an open cleft.

Figure 6:

Trichoderma reesei

T. reesei is considered to be a save production organism, because it is non-pathogenic to healthy human and does not produces mycotoxins or antibiotics under the condition used for
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enzyme production (Nevalainen et al., 1994, Nevalainen & Neethling, 1998). Industrial strain of T. reesei can achieve production levels up to 100g/L (Cherry & Fidantsef, 2003). The consideration regarding the production influence is the morphological and physiological changes of this fungus. It has been reported that only the second fungal state of T. reesei can produce enzymes and the primary mycelium does not efficiently secrete enzyme (Velkovska et al., 1997). 2.4.2 Bacteria While several organisms can metabolize cellulose as an energy source, only a few strains can produce a complex of cellulose enzymes, which have a practical application in the enzymatic hydrolysis of cellulose. The microbes commercially exploited for cellulose preparations are mostly limited to T. Reesei, H. Insolens, A. Niger, Thermomonospora fusca, Bacillus sp, and a few other organisms (Table 3). Bacillus subtilis Bacillus subtilis (Figure 7) cells are rod-shaped, Grampositive bacteria that are naturally found in soil and vegetation. B. subtilis grow in the mesophilic temperature range. The optimal temperature is 25-35 degrees Celsius (Entrez Genome Project). Stress and starvation are common in this environment; therefore, B. subtilis has evolved a set of strategies that allow survival under these harsh conditions. One of the strategies is the formation of stress-resistant subtilis has endospores. only one B. DNA Table 2: Commercially used cellulolytic organisms(R.K. Sukumaran 2005)

molecule which is a circular chromosome. B. subtilis bacteria Table 3:

17

Microorganisms commonly used for the commercial production of cellulases

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Bioremediation of Agricultural Waste: Lemongrass

are capable of secreting antibiotics in great numbers to the exterior of the cell. Five signal peptidase genes were found to be important for this secretion function. Many of B. subtilis cells' genes are responsible for antibiotic synthesis. B. subtilis bacteria have been considered strictly aerobic, meaning that they require oxygen to grow and they cannot undergo fermentation. However, recent studies show that they can indeed grow in anaerobic conditions making them facultative aerobes. B. subtilis is different from other facultative aerobes in that it undergoes fermentation without external acceptors of electrons. During fermentation, the regeneration of NAD+ is chiefly mediated by lactate dehydrogenase, which is found in the cytoplasm. Lactate dehydrogenase converts pyruvate to lactate. B. subtilis is most commonly found in soil environments and on plant undergrowth. B. subtilis concurrently produces antibiotics and spores.

Figure 7:

Bacillus subtilis

Antibiotic production increases B. Subtilis's chances at survival as the organism produces spores and a toxin that might kill surrounding gram positive microbes that compete for the same nutrients. . When the nutrients required for the bacteria to grow are abundant, they exhibit metabolic activity. These organisms can produce antibiotics during sporulation. Examples of the antibiotics that B. subtilis can produce include are polymyxin, difficidin, subtilin, and mycobacillin. B. subtilis bacteria are non-pathogenic. The bacteria used on plants as a fungicide. They are also used on agricultural seeds, such as vegetable and soybean seeds, as a fungicide. B. subtilis bacteria secrete enzymes such as amylase, protease, pullulanase, chitinase, xylanase and lipase. These enzymes are produced commercially and this enzyme production represents about 60% of the commercially produced industrial enzymes. Clostridium acetobutylicum
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Bioremediation of Agricultural Waste: Lemongrass

Clostridium acetobutylicum(Figure 7) is a gram-positive bacterium and categorised under saprophytes microorganism.This bacterium is a chemoorganotroph where it obtains energy via substrate phosphorylation by fermentation. This bacterium is a rod-shaped, motile by peritrichous flagella.C. acetobutylicum is capable of fixing atmospheric nitrogen, and some strains produce inducible carboxymethyl cellulase and cellobiase enzymes (Cato et al., 1986).Clostridium genus is known pathogens, including Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringen. Although other members of the genus produce some of the most lethal neurotoxins known, C. acetobutylicum is considered a benign microorganism. C. acetobutylicum accumulates acids acetate and butyrate in the early stage. Once the cells reach stationary phase, these acids are converted to acetone and butanol, respectively.It has the capabilities of naturally produce acetone, butanol and ethanol. Throughout its long history of use for production of acetone and butanol, there have been no reports of adverse effects to human health or the environment. It is not pathogenic or toxigenic to humans, animals, or plants.C.acetobutylicum is able to ferment several pentose and hexose sugars (Liu et al., 1999). Glucose was found to be preferred carbon source for C. acetobutylicum with the total biomass compared to xylose substrate. C. acetobutylicum is able to use a number of different fermentable carbohydrates as an energy, as well as carbon source.In addition, C. acetobutylicum is an obligate anaerobe. It can only survive hours in an aerobic environment before undergoing sporulation producing endospores as a means to survive for much longer periods of time in the aerobic environment.This mature spore is resistant to high temperature, chemicals and many types of radiation allowing it to survive for extraordinary number of years.

Figure 8:

Clostridium acetobutyricum

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Bioremediation of Agricultural Waste: Lemongrass

2.5 Degradation of lignocelluloses Of the three components of lignocellulose, lignin is the most recalcitrant to degradation whereas cellulose, because of its highly ordered crystalline structure, is more resistant to hydrolysis than hemicellulose. On this note, suitable pretreatments resulting in the disruption of lignin structure and increase accessibility of enzymes have been shown to increase the rate of its biodegradation. Alkaline and acid hydrolysis methods have been used to degrade lignocellulose. Weak acids tend to remove lignin but result in poor hydrolysis of cellulose whereas strong acid treatment occurs under relatively extreme corrosive conditions of high temperature and pH which necessitate the use of expensive equipment. Also, alkaline and acid hydrolysis yield non-specific by-products which may promote glucose degradation and therefore reduce its yield. Meanwhile, there are other more environmentally- friendly possibilities. White-rot fungi are recognized as the msot active lignin-degrading microorganisms. Oxidative enzymes produced by the fungi, along with catalysts, produce free radicals in the aromatic moieties, which result in degradation of aromatic compounds.. For many processes enzymes are preferred to acid or alkaline processes since they are specific biocatalysts, can operate under much milder reaction conditions, do not produce undesirable products and are environmentally-friendly. Once the lignin portion of lignocelluloses has been degraded, the cellulose portion is now open to enzymatic action. There are only two modes of action for the hydrolysis of cellulose by cellulases, either inversion or retention of the configuration of the anomeric carbon. At least two amino acids with carboxyl groups located within the active site catalyze the reaction by acid-base catalysis. The commonly described mode of action for cellulases on polymers is either exo- or endo-cleavage, and all cellulases target the specific cleavage of 1,4-glycosidic bonds 12. Using this classification system, cellobiohydrolases were classified as exo-acting based on the assumption that they all cleave -1, 4-glycosidic bonds from chain ends. As well, those enzymes truly exo-acting often have a tunnel-shaped closed active site which retains a single glucan chain and prevents it from re-adhering to the cellulose crystal 1315. While endoglucanases on the other hand, are often classified as endo-acting cellulases because they are thought to cleave -1, 4-glycosidic bonds internally only and appear to have cleft-shaped open active sites. Endoglucanase are active on amorphous regions of cellulose and thus their activity can be assayed using soluble cellulose substrates. However, there is now supporting evidence that some cellulases display both modes of action, endo- and exo- 16.
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Thus classification has changed; cellobiohydrolases are described as active on the crystalline regions of cellulose; whereas, endoglucanases are typically active on the more soluble amorphous region of the cellulose crystal. There is a high degree of synergy seen between cellobiohydrolases (exoglucanases) and endoglucanases, and it is this synergy that is required for the efficient hydrolysis of cellulose crystals. References Challenges and Opportunities in Agro-waste Management: An Asian Perspective Prof. Dr. P. Agamuthu, Institute of Biological Sciences, Faculty of Science, University of Malaya, Inaugural Meeting of First Regional 3R Forum in Asia, 2009 Ara, K., et al. "Bacillus minimum genome factory: effective utilization of microbial genome information." Biotechnol. Appl. Biochem.. 2007 March; 46(Pt 3):169-78. Bandow, J.E., H. Brtz, M. Hecker. "Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the B-Dependent General and Multiple Stress Response". Journal of Bacteriology. 2002 January; 184(2): 459-467.

Chemical composition and citral content in lemongrass (Cymbopogon citratus) essential oil at three maturity, Accessed 10/3/2012 (http://www.academicjournals.org/AJB/PDF/pdf2012/7Feb/Tajidin%20et %20al.pdf)

D.E. Akin, Grass Lignocellulose: Strategies to Overcome Recalcitrance, Humana Press Inc., 2007 Howard R.L., Abotse E., Jansen van Rensbur E.L., Howard S., Lignocellulose Biotechnology: Issues of Bioconversion and Enzyme Production African Journal of Biotechnology, 2003, Volume 2(12) pp. 602-619

Kuhad R.C., Singh A., and Eriksson L., (1997) Microorganisms and Enzymes Involved in the Degradation of Plant Fiber Cell Walls Advances in Biochemical Engineering/Biotechnology, Vol.57, Springer-Verlag Berlin Heidelberg.

Miranda Maki, Kam Tin Leung, Wensheng Qin, The prospects of celluloseproducing bacteria for the bioconversion of lignocellulosic biomass International Journal of Biological Sciences, 2009, Vol. 5(5), pp 500-516
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Bioremediation of Agricultural Waste: Lemongrass Pekarek, E., Jacobson, K., Donovan, A. High Levels of Genetic Variation Exist in Aspergillus niger Populations Infecting Welwitschia mirabilis Hook. Journal of Heredity. 2006. Volume 97. p. 270-278.

R.K. Sukumaran, T.T. Singhania, A. Pandey, Micobial Cellulases-Production, applications and challenges Journal of Scientific & Industrial Research Vol. 64, November 2005, pp832-844

Ronald Benner, S.Y. Newell, A.E. Maccubbin, Robert E. Hodson, Relative Contributions of Bacteria and Fungi to Rates of Degradation of Lignocellulosic Detritus in Salt- Marsh Sediments Applied and Environmental Microbiology, 1984, pp 36-40

Schuster, E., Dunn-Coleman, N., Frisvad, J., van Dijck, P. On the safety of Aspergillus niger a review. Applied Microbiology and Biotechnology. 2002. Volume 59. p. 426-435.

Utilization of Organic Wastes and Natural Systems in Malaysian Agriculture Accessed 10/3/2012 (http://www.infrc.or.jp/english/KNF_Data_Base_Web/PDF %20KNF%20Conf%20Data/C1-4-010.pdf)

William F. Anderson, Danny E. Akin, Structural and chemical properties of grass lignocelluloses related to conversion for biofuels Journal of Industrial Microbiology and Biotechnology, 2008, Vol. 35, pp 355-366

Agricultural Waste and Residues Accessed 10/3/2012 (http://www.unescap.org/esd/environment/soe/2000/documents/CH08.PDF) Components of Lemongrass Oil Accessed 10/3/2012 (http://www.ehow.com/list_6886947_components-lemongrass-oil.html) Lemon Grass Accessed 10/3/2012 (http://nhb.gov.in/report_files/lemongrass/LEMON%20GRASS.htm) Lemon Grass Oil Accessed 10/3/2012 (http://www.livestrong.com/lemongrass-oil/) Aromatic Plants Accessed 9/4/2012 (http://books.google.com.my/books? id=e68EbGOCayAC&pg=PA102&lpg=PA102&dq=spent+lemongrass&source =bl&ots=OewHmFdbz&sig=SrZ5QNMRVZjwDB_cvsd1DdSuTxs&hl=en&sa=X&ei=KBN7TSfMYjQrQeRwP2OAg&ved=0CEAQ6AEwBA#v=onepage&q=spent %20lemongrass&f=false)
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Bioremediation of Agricultural Waste: Lemongrass Lemongrass Production Guide Accessed 9/4/2012 (http://www.agribusinessweek.com/lemongrass-production-guide/) NCBI: Aspergillus niger, Accessed , Accessed 9/4/2012 (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? mode=Info&id=380704&lvl=3&p=mapview&p=has_linkout&p=blast_url&p=g enome_blast&lin=f&keep=1&srchmode=1&unlock)

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