Division of City Schools City of Mandaluyong Science High School E. Pantaleon St.

, Hulo, Mandaluyong City

CHITIN FROM SHRIMP SHELLS AS SOIL CONDITIONER A Science Investigatory Project submitted to Mr. Benjamin Ola Research Adviser In Partial Fulfillment for the Requirements in Research II Prepared by: Diano, Jessieca B. Deynata, James Dean I. Escabusa, John Vincent D. Gacura, Mathew A. Magno, Ron Matthew C. Neria, Khyle Tim A.

Acknowledgement First, we would like to thank God for his gift of knowledge, patience and perseverance. Next, the researchers would like to extend their gratitude to Mr. Benjamin G. Ola, the subject adviser, who have patiently guided them in every part of accomplishing this investigatory project. For his unending support and guidance throughout the whole execution of the investigation in this study. And, For giving suggestions and tips for an effective way on how we can make our investigatory become systematic and reliable

ABSTRACT This investigatory project determines the possibility of making an effective soil conditioner using chitin extracted from shrimp shells to reduce the development of Streptomyces scabies in potatoes (Solanum tuberosum). It also aims to reduce the water pollution caused improper disposal of shrimp shells.

The severity of infection in potatoes by Streptomyces scabies causing corky lesions formed on tuber and root crops as well as decreasing the growth of seedlings was reduced by application of chitin from shrimp shells as soil conditioner. The chitin was extracted from the shrimp shells through boiling it in a dilute solution of lye, and soaking it in a dilute solution of muriatic acid. It is also soaked in hypochloric acid for decolorization. There were four setups of potato plants made. The first one containing 15 g of chitin, the second setup with 20 g, the third setup with 25 g of chitin and the last setup as the controlled or independent setup. The results of the crops from the setups were compared with its russet, pitted and erumpent lesions. It is found out that the third setup has the least lesions and has the most favorable appearance. Therefore the researchers conclude that the chitin can be extracted from shrimp shells and it can help reduce the infection of Streptomyces Scabies in potato plants.

CHAPTER I INTRODUCTION The potato is a starchy, tuberous crop from the perennial Solanum tuberosum of the Solanaceae family. The potato was first domesticated in the region of modern-day southern Peru and extreme northwestern Bolivia between 8000 and 5000 BCE. It has since spread around the world and become a staple crop in many countries. Today, the demand for potato is increasing rapidly. During the past twenty years, the production of potatoes had increased more than two times in rich countries. Production of potato is limited due to the unavailability of disease free plant materials and disease reduction procedures in planting the potatoes. Shrimp shell wastes were also becoming one of the problems in our environment. After meals, people threw wastes of shrimp shells into trashes which later on, go into our oceans. The shrimp shells were now considered as one of the water pollutants in our oceans. It had been a contributor in the destruction of the marine habitat. Instead of being thrown into the water, shrimp shells were being studied as an alternative soil conditioner because shrimp shells contain chitin. Chitin is a polysaccharide found in the outer skeleton of insects, crabs, shrimps, and lobsters and in the internal structures of other invertebrates. Chitin is composed of (1-4) linked units of the amino sugar N-acetyl-glucosamine, and is the main source of production of chitosan, which is used in a number of applications, such as a flocculating agent, a wound healing agent, a sizing and strengthening agent for paper, and a delivery vehicle for pharmaceuticals and genes.

Background of the Study Potato (Solanum tuberosum) is the third most important food crop in the world after rice and wheat in terms of human consumption. More than a billion people worldwide eat potato, and global total crop Production exceeds 300 million metric tons. But theres a problem in the skin of these plants, it can be easily infected by some kinds of fungi. One of these is the common scab of potatoes, a soil-borne disease which is caused by the bacteria-like organism Streptomyces scabies. In the search of preventing these kinds of diseases, the researchers had discovered a way to reduce the development of Streptomyces Scabies in potato plants. Most of us recognize that the shrimp shells are often removed before cooking or serving. Instead of disposing these, the researchers found out that their shells contain a natural fiber called chitin which has a potential in agriculture with regard to controlling plant diseases They were reported to be active against viruses, bacteria and other pests. Recent studies have shown that shrimp have 25 to 35 percent of chitin in its shells. The researchers then decided to extract chitin from the shrimp shells. There are several treatments such as temperature, duration, concentration of the chemicals, concentration and size of the crushed shells. Shell also contains lipids and pigments as a decolorizing is needed which can be done by soaking in organic solvents or in a very dilute solution of sodium hypochlorite.

Statement of the Problem 1. Is it possible to extract chitin from shrimp shell? 2. Is it possible to use chitin from shrimp shells as soil conditioner?
3. Does the use of shrimp shells as soil conditioner prevent infection from Streptomyces

scabies in potatoes? Significance of the Study This study was conducted to reduce the Streptomyces scabies with the use of extracted chitin from the shrimp shells. The researchers believe that the problem regarding the infection of potato plants could be lessened by the research proposal. Streptomyces scabies or Streptomyces scabiei is a streptomycete bacterium species found in soils around the world. Unlike most of the 500 or so Streptomyces species it is a plant pathogen causing corky lesions to form on tuber and root crops as well as decreasing the growth of seedlings. Along with other closely related species it causes the potato disease common scab, which is an economically important disease in many potato growing areas.This organism attacks the stems, stolons and roots of the potato, and more importantly, young, rapidly growing tubers, stimulating the growth of unsightly corky tissue. A severe infection can reduce the marketable yield and can damage the eyes of seed potatoes. It can also greatly reduce the market value of the crop. With this study, the researchers can make use of disposable shrimp shells and lessen the bacteria that attack the skin of the potato plant. It can also help in yielding of more crops and a better quality of plants.

Scope and Limitation The duration of this period is between the months of October and December 2012. The focus of the investigatory project is the extraction of shrimp shells as a soil condition that aims to reduce Streptomyces Scabies. The purpose of this study is to benefit from waste materials such as the shrimp shells that are usually taken off. The limitation of this study is that there is a limited percent of chtin in shrimp shells, so a large amount of shrimps is needed to conduct the research. It also takes a lot of time for the potato plant to sprout. Definition of Terms Chitin - A polymer of nitrogen-containing polysaccharide rendering a tough, protective covering or structural support in certain organisms. Demineralization - the process of removing minerals, in the form of mineral ions Potato- a starchy, tuberous crop from the perennial Solanum tuberosum of the Solanaceae family (also known as the nightshades). Streptomyces scabies or Streptomyces scabiei - A streptomycete bacterium species found in soils around the world; it is a plant pathogen causing corky lesions to form on tuber and root crops as well as decreasing the growth of seedlings Soil Conditioner - A product which is added to soil to improve the soils physical qualities, especially its ability to provide nutrition for plants. Deproteinization - the process of removing protein

Review of Related Literature A study of the complete genome sequence of Streptomyces scabies 87.22, a common causative agent of scab disease of tubers including potato (Solanum tuberosum), is described. This work includes annotation of the genome and in-depth description of gene clusters likely to encode biosynthetic pathways for complex natural products and not also found in either Streptomyces coelicolor A3(2) or Streptomyces avermitilis MA-4680. Twenty-eight gene clusters were identified as likely to encode enzymes for the biosynthesis of complex natural products. Substances predicted by this work, not previously known to be made by S. scabies 87.22, were confirmed by collaborators as products - desferrioxamines, germicidins, and hopene. Of the clusters identified, fourteen gene clusters are not conserved in the other two streptomycete genome sequences for which comparisons have been undertaken. The Streptomyces genus is a reservoir of producer organisms from which many complex natural products of therapeutic importance have been isolated. These findings suggest that the cargo of cryptic and silent gene clusters amongst other members of this genus may add significantly to previous estimates of undiscovered bioactive natural products. Methods developed in this work could enable other researchers to rapidly identify gene clusters likely to encode enzymes involved in biosynthesis of complex natural products from complete genome sequences. De-replication is a problem for approaches to drug discovery based on activity screening and isolation of wild producer organisms. Computational methods in this work allow rapid de-replication of gene clusters following sequencing which may lead to discovery of many new natural products with therapeutic benefit. Sequences predicted to be involved in scab disease pathogenicity are not found in only one pathogenicity island location as expected, but at several loci. Two possible mechanisms were identified from sequence data which it is suggested could be involved in

regulation of pathogenicity traits: an MbtH-like protein family and an iron box sequence likely to be triggered response to low iron conditions.
(Wellington, E. M. H. June 2009)

CAMPBELL, L. L. JUN. 8n WILLIAMS, 0. B. (1951). J. gen. Microbiol. 5, 894r905. A Study of Chitin-decomposing Micro-organisms of Marine Origin \BY L. L. CAMPBELL, JUN. AND 0. B. WILLIAMS Department of Bacteriology, University of Texas, Austin, Texas, U.S.A. SUMMARY : Twenty strains of aerobic, facultatively anaerobic, chitinoclastic bacteria have been isolated from marine mud by enrichment cultures. Each was able to derive its full carbon and nitrogen requirements from chitin. None was an obligate chitinovor. These cultures comprised four new species of Achromobacter, two new species of Pseudomas, one new species of Flavobacterium and one new species of Micrococcus. Detailed descriptions for each species are appended. Each organism was able to liberate ammonia and reducing sugar from the chitin molecule, Glucosamine and acetic acid were not detected in the cultures, possibly because of their ready availability as supplementary nutrients.

CHAPTER II METHODOLOGY This section includes the details how the study was conducted. It includes the plans for each stage, the actual experimentation with corresponding tools and equipments the researchers used in performing the experiment and special procedures or techniques they have used to produce a desired output. Materials used with corresponding measurements and equipments are listed below: Materials Used (for extracting chitin) : 60 g of Shrimp Shells (from 2 kilos of shrimps) 20 mL Muriatic Acid 30 mL of Lye 20 mL of Bleach Equipments: Stove Casserole Strainer Materials Used (for experimental setups) 4 Pots 4 potatos 12 kg of Loam soil 100 g of eggshells Equipments: Hammer Rake

Procedures: In the extraction of chitin, 2 kilos of shrimps were cooked. The shells were peeled off, gathering a total 60 g of shrimp shells. The shells were soaked in a dilute solution of lye in water at a boiling temperature for 15 minutes for deproteinezation. Then, the shells were soaked in muriatic acid and water for demineralization. After 1 day, the shells were then transferred into another bottle with dilute solution of bleach to decolorize the shells. For the experimental setups, 4 pots were prepared, and 3 kilos of soil was put in each pot. Each pot is mixed with 25 g of crushed egg shells. After a week, the researchers set up 4 treatments. In pot A, 15 g of chitin were mixed with the soil. In pot B, 20 g of chitin were mixed with the soil. In pot C, 25 g of chitin were mixed. And in pot D, no chitin were added. This is considered ad the control setup.

CHAPTER III RESULTS AND DISCUSSIONS Graphical Interpretation of Data This table shows the symptoms of Streptomyces Scaies found in the plant. S. scabies causes variable symptoms on the surface of potato tubers including erumpent, russet, and pitted lesions. Erumpent lesions are raised lesions, russet lesions are defined as superficial corky tissue that covers large areas of the tuber surface and pitted lesions are dark colored sunken areas up to in deep. Scab lesions can occur anywhere on the tuber surface and more than one type of lesion may be present on a single tuber. Scab affects young tubers with the lesions expanding as the tuber matures. Graph 1

Setup D

Setup C Pitted Lesions Russet Lesions Setup B Erum pent Lesions

Setup A

The researchers categorized the experiment into four set-ups. Set-up A has 15 g of chitin, Setup B has 20 g of chitin and Setup C has 25 g of chitin. The last setup is the independent setup, which contains no amount of chitin. From the graph above, Set up C got the least lesions and the most favorable appearance. While Setup A, B and D has the most lesions that makes its appearance not considerable. Graph 2

In the graph above the height of the plant in centimeters is shown. It shows that Setup C has the highest rate of growth followed by Setup B and A. It can be inferred that the plant in Setup C, which has the highest amount of chitin grow fastest.

Graph 3

In the graph above, the number of leaves in each setup is shown. Again, Setup C got the most number of leaves compared to Setups A, B and D.

CHAPTER IV SUMMARY OF FINDINGS, CONCLUSION AND RECOMMENDATION

Summary of Findings This study was conducted to see if chitin from shrimp shells can reduce Streptomyces Scabies in potato plants. Moreover, the study was conducted with the aim of finding a new way to recycle shrimp shells and use it as soil conditioner. The study started in extracting chitin from shrimp shells, undergoing three processes, demineralization, decolorization and the extraction. After getting the chitin, four setups of potato plants were prepared with each pot having 25 g of egg shells. After some months, the researchers gathered the results from the grown potato plant. Set-up C gives the most favorable results because it shows the least lesions among the four potatoes observed. It also contains the right amount of chitin in the soil and set-up C is found to be the tallest plant that has grown. Conclusions

Based on the researchers results and analysis of data, the following conclusions were formulated.
1. It is possible to extract chitin from shrimp shells. 2. It is possible to use chitin from shrimp shells as a soil conditioner. 3. The use of shrimp shells as soil conditioner prevent the potatoes from Streptomyces

scabies.

Recommendations From the investigations, the researchers would like to recommend a more systematized and accurate way of extracting chitin from shrimp shells and planting potatoes. They also recommend better and more effective techniques in growing potato plants. More time is needed for the researchers to observe the maximum growth of the plant because the bacteria, Streptomyces Scabies requires more time to develop.

The researchers must be patient and observant in these investigations. This is to ensure the validity and reliability of the information gathered from the experiments.

Documentation Materials

Addition of HCl

Boiling of Chitin

Gathering of Chitin Addition of NaOH