Dissertation on "Biofuels to Dissect Locust Guts to Culture"

Dissertation 10 pages (3671 words) Sources: 30 Style: Harvard

[EXCERPT] . . . .

Biofuels

To dissect locust guts

To culture anaerobic microorganisms (bacteria) from locust guts on cellulose

containing media and screen for microbes with Cellulolytic activity.

To extract DNA from Cellulolytic microorganisms

To do PCR of 16s rDNA to identify the bacteria.

To clone and sequence PCR product.

The study discusses the aims and objective to enhance greater understanding on the strategy the locust gusts could be used to produce cost effective and clean biofuels for energy use.

Locusts are the species specially found in desert, and they are short-homed grasshoppers that could form the mobile swarms, which could be spread to the 20% of the landmass. Typically, the swarm could comprise of 30 million locusts and could travel at 80 miles per day. Locust swarm has ability to eat the same amount of food that several thousands of people could eat per day. Illustration in Fig 1 shows a sample of locust.

Fig 1: A Sample of Locust

Source: U.S. Department of Energy (2008).

Ability of the locusts to break down the plant mass rapidly makes this study to investigate the impact of the locust guts in breaking down the lignocelluloses, which could lead to the discoveries of new microbes in the locust gusts for commercial production of biofuels. (U.S. Department of Energy 2008). With the identification of microorganism inside the locust guts, the aim of this study is to culture the bacteria inside the locust guts and use the microbes for celluloytic activity. Shi et al. (2010) argue the symbiotic microbiota of the locust guts could be exploited for biotechnological purpose. Typically, locust guts harbor abundance of microorganisms that play important roles in the development and resistance of pathogens, and loss of these bacteria could lead to the reduction of insect hosts. The role of microorganisms inside the locust guts could enhance the "lignocelluloses digestion, methanogenesis, acetogenesis from H2 and CO2, nitrogen fixation, maintenance of a low redox potential, and prevention of entry of foreign microorganisms." (Feng, Wang. Zhou et al. 2011 P. 2). Cacemier, (1969) reveals that the bacteria counts inside the locust guts are estimated to reach between 70 and 80 million in the foregut, and 11 million in the midgut and 33 and 44 million in the hindgut. Culture of the bacteria inside the locust guts will provide a greater understanding of the possibility of these microorganisms to produce digestive enzymes that could assist in the mass production of biofuels.

The DNA metabolism could also be extracted from the locust guts to evaluate their microorganisms. Proper methods for DNA extraction from the locust guts are the key method to achieve unbiased evaluation of metagenomes for the unculturable microorganisms. The study implements metagenomic DNA that is free of contamination with downstream DNA processing which polymerase chain reaction (PCR), vector litigation and enzyme digestion. Direct analysis methods and cell recovery method are two strategies, which are very common for metagenomic DNA isolation. However, there are commercial kits recently available for DNA extraction and this kit simplified the cultivation of independent analysis methods. Commercial kits that could be used for the DNA extraction are "GenElute bacterial genomic DNA kit, Qiagen DNeasy Tissue kit, QIAamp DNA Mini Kit, WizardTM Genomic DNA Purification Kit from Promega, and PowerSoilTM DNA isolation kit." (Shi, Syrenne, Sun, et al. 2010 P. 2002). Despite the importance of these kits for the extraction, these kits are not designed for metagenomic DNA isolation from the locust guts. Traditional approach technique plays important role in understanding the function and composition of the locust guts symbionts. The extraction of the DNA from the locust guts will be achieved by boiling bacteria cell suspension using the sterile distilled water. The extraction of DNA is also done in the total volume of 50 ?L which contain 5 ?L DNA extract. Additionally, 2 ?L of 10 ?mol/L is used for each primer. (Stokes, Holmes, Nield et al. 2001).

Additional aim and objective of the study is to implement PCR of 16s rDNA in order to identify the bacteria. Gene targeting technique plays important role in conserving 16S rRNA gene and identify a specific function of the metagenomic DNA of locust guts. The approach has been applied for the understanding of the symbiotic microbiota analysis of the locust guts. The study of 16S rRNA gene is also very important in understanding the phylogenetic diversity of bacteria in the locust guts. The 16S rRNA is isolated from the locust guts and analysis of the 16S rRNA will produce bacteria strain, which is classified into Brevibacterium, Corynebacterium, Stenotrophomonas, Staphylococcus, Bacillus, and Klebsiella.

More importantly, cloning and sequence PCR (polymerase chain reactions) product is very importantly in understanding the gene discovery and the locust guts microbial communities. The PCR as well as analysis of microbial diversity provides understanding of locust guts species. Although, PCR is very effective for the microbial diversity analysis and understanding of gene discovery, however, its effectiveness depends on the PCR amplification that could greatly limit the application. Additionally, PCR technique could only be used for partial cloning and full length cloning through PCR-based chromosome application. (Cowan, Meyer, Stafford 2005).

Understanding of cellulose, hemicelluloses and lignin is very important in understanding the metabolism of the locust guts and the method could assist in the mass production of biofuels.

Fig 2: Processing of Cellulose through Bacteria activity

Cellulose, Hemicelluloses and Lignin

Cellulose is an insoluble, linear biopolymer that composes repeating ? -- D-glucopyranose residues which is linked to the ?-1, 4 glycosidic bonds. Cellulose is not glucose but a disaccharide and cellobiose. Cellulose could exhibit high degree polymerization and individual glucan chains could reach the lengths greater than 25,000 glucose residues. Cellulose is most resistance to degradations, and cellulose produced by plants contains large voids, which compose of highly amorphous regions. Typically, cellulose primarily comes from plant, however, some bacteria, alga species, and animals, could produce the polymer. Under aerobic systems, cellulose could degrade into H2O and CO2 while under anaerobic systems, cellulose is degraded into Chapter 4 and H2. Cellulolytic species could be found within the Proteobacteria, Actinobacteria, phyla Thermotogae, Bacteroids, Spirochaetes, Fibrobacteres and Firmicutes. From these species, almost 80% are found within the Firmicutes and Actinobacteria. (Bergquist, Gibbs, Morris, 1999).

"Cellulose is the most abundant biopolymer in the world. Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation is accomplished in a single process step by cellulolytic microorganisms." (Carere, Sparling, Cicek et al. 2008 P. 1342-1343).

Discarded cellulose biomass could be derived from agriculture, forestry and municipal sources, which include feedstocks for the biofuels synthesis. Typically, the estimation of the cellulose produced on earth annually is about 7.5 x 1010 tons, which is produced through photosynthetic processes. Cellulose could also be found in the plant cell wall and compose of the lignin, hemi-celluloses and xylan.

Biofuels could be produced from different sources such as food crops and cellulosic substrates. Food crops such as sugar beets and sugarcane provide 60% of total global bioethanol production. Other food crop such as wheat, cereal crops and corn could be converted to glucose during fermentation.

Hemicelluloses

"The term hemicellulose refers to a group of homo- and heteropolymers consisting largely of anhydro-b-(1-4)-D-xylopyranose, mannopyranose, glucopyranose, and galactopyranose main chains with a number of substituents." ( Jeffries, 1994 P. 234). Generally, Hemicelluloses is always found in association with cellulose and hemicelluloses is primarily present in the cell wall of plants such as the component of softwoods, glucomannan and hardwood hemicelluloses. (Ratledge, 1994).

Hemicelluloses are an important heteropolymers or arabinoxylans, which is present along with cellulose. Hemicelluloses could be derived from the plant and wood cell. Additionally, Hemicelluloses, polysaccharides could be found in the cell walls of plants, which are not cellulose. Various varieties of hemicelluloses include

Pentoses such as xylose, arabinose

Hexoses, which include glucose, mannose, galactose, rhamnose and corresponding uronic acids.

Major industrial application of Hemicelluloses is its conversion into biofuels. Despite its importance in the production of biofuels, its industrial application is not yet being exploited in a mass scale. Jeffries, (1994) argues that composition of Hemicelluloses depends on the plant species. Its composition also depends on the age, stage of growth of plants and environmental conditions under which plants grow.

Lignin

Lignin is a chemical compound commonly derived from wood, cells, fibres and vessels and lignified element of plant. After cellulose, lignin is the most abundant renewable product and between 40 and 50 million lignin are produced annually. Lignin composes of building blocks such as coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol. The inter-monomer linkage of lignin is composed of hardwood, softwood, and grass lignins. Meanwhile, Lignin degradation could assist in understanding of the enzymatic mechanisms and various pulp bleaching reactions. (See Fig 2). Despite the abundance of lignin globally, the lignin is poorly degraded by most microorganisms due to its complex structure. Based on the large production of Cellulose, Hemicelluloses and Lignin globally, and their importance in the production of biofuels, the locust could be used to break the complex structure of cellulolytic material which could enhance the large production of… READ MORE

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