Our PBL Project

Project Title: Agricultural Use of Chicken Feather by Novel Bacillus Strain Isolated from the Soils of Houston TX

Introduction:

Problem Statement:

Feathers contain a protein called keratin which is insoluble due to its chemical bonds and is near impossible to decompose. Instead of trying to dispose of the feathers, we have found a way to reuse them to benefit the ecosystem in multiple ways. By using bacillus and its keratinase we can efficiently break down the protein to use it agriculturally.

Purpose:

The purpose for our project is to potentially improve the environment by reducing the amount of feather by-products being wasted. Feathers contain keratin that is insoluble and is not being decomposed in a successful manner. Instead of trying to dispose of the feathers, we have found a way to reuse it to benefit the ecosystem in a myriad of ways. By using bacillus, we can efficiently break down the protein and use it agriculturally.

Research Question:

Which bacillus strain is the best at digesting the keratin in Bacillus.

Hypothesis:

If we use bacillus to degrade feathers into a digestible form of protein, we can use it as animal feed, probiotics, fertilizer and to stop the drains from clogging in slaughterhouses. Our main goal is to reduce the heavy pollution of keratin in feathers, nails, hair, etc which is mindlessly placed in landfills to even the bottom of your bathtub which can cause diseases by their misplacement, the toxins they create and discomfort by their presence.

Our Methods:

Variables:

Our independent variable is the type of bacillus strain being used while our dependent variable is the are of digested rate and it's effectiveness. Our control group was the keratin agar because all petri dishes had the same concentration of feathers.

Materials:

For this experiment we collected some chicken feathers from specific pillows, as well as Bacillus from a library we created from Houston soil. We also used petri dishes in order to place the keratin feather agar (nutribullet was used to grind feather powder) (0.5g NH4Cl, 0.5g NaCl, 0.3g K2HPO4, 0.4g KH2PO4, 0.1g MgCl2 6H2O, 0.1g Yeast Extract, 10g Feather, 15g Agar), turning them into agar plates. LB broth was used. A heating bath was used to isolate bacillus. An incubator was used to to allow the bacteria to grow. A refrigerator was used to help keep them at a workable state. Spreaders or loop were used to spread on the plate. We cleaned the feathers using detergent wash. After we used chloroform and methanol to deep clean them further to sterilize. Afterwards a nutribullet was used to grind up the feathers. DMSO was used to to help break down protein. During the process we used ethanol to sanitize certain equipment and our hands. The vortex was used to shake mix and combine. We used a fume hood to accomplish certain procedures. While doing doing all these procedures we had to use safety equipment such as goggles, gloves, and an apron.

Procedure:

First we obtained feathers and washed them by using detergent then drying them at 65 degrees overnight and washed them again with methanol and chloroform intensely and leaving it overnight. We then dried it at 65 degrees Celsius overnight, then DMSO at 100 degrees for 2 hours and allow it to cool. We then added ½ cold acetone for precipitation, filter, wash and dry it at 65 degrees overnight. We chop it up by homogenizer and combined it with agar. We then test the agar with different bacillus.

Safety:

Some safety precautions we must take are the ones involving chemicals. Working on this experiment requires gloves, goggles, and aprons to protect ourselves against some of the harsh products. All things were dispensed into their appropriate disposal containers. The fume hood was sanitized with alcohol after experiments and made sure everything was in order. We had to take extra precautions to prevent contamination of the petri dishes and the bacteria itself.

Results:

Pictures

Graphs:

Analysis:

The result we collected were mostly positive. Out of many strains of bacillus we tested most had keratinase activity, and we identified the ones with highest activity. The strains that were the most effective were Bacillus siamensis, Bacillus methylotrophicus, and Bacillus subtilis which showed a greater quantitative value of 1.5, 1.5 and 1.25, respectively. One of the main limitation we encountered was time. Because we had to spend most of our day in our classes, we had to take several hiatuses from working in the lab. Many times our bacteria samples needed to be placed in a fridge because we couldn’t continuously work on it, and it took much longer to get something done because of our time restraint.

Conclusion:

We obtained very positive results and we were successful at finding the most useful bacillus that can digest keratin and turn it into amino acids or polypeptide which can later used to create a consumable product. The most effective bacillus were methylotrophicus and siamensis based on our data. We can conclude that these species of bacillus were the best at digesting Keratin and creating the most amount of usable protein. We can use this information to create household and agricultural products such as fertilizers, animal feed and probiotics which can benefit anything from plants to our own selves.

Application and Further Research:

We can apply our knowledge from this project to create a dietary supplement for both humans and animals or even as a way to provide proteins to plants who are lacking in important nutrients. And the specific bacilli we found can be used to drain slaughterhouse blockage because these are very effective at digesting and breaking down some of the animal and feather remains.

References:

Agrahari, S., & Wadhwa, N. (2010). Degradation of Chicken Feather a Poultry Waste Product by Keratinolytic Bacteria Isolated from Dumping Site at Ghazipur Poultry

Processing Plant. International Journal of Poultry Science, 9(5), 482-489.

Bihari, Z., Vidéki, D., Mihalik, E., Szvetnik, A., Szabó, Z., Balázs, M., . . . Kiss, I. (2010). Degradation of Native Feathers by a Novel Keratinase-Producing, Thermophilic

Isolate, Brevibacillus thermoruber T1E. Zeitschrift Für Naturforschung C, 65(1-2).

Cai, C., Lou, B., & Zheng, X. (2008). Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis . Journal of Zhejiang University. Science. B, 9(1),

60–67. http://doi.org/10.1631/jzus.B061620

Friedrich, A. B., & Antranikian, G. (1996). Keratin Degradation by Fervidobacteriumpennavorans, a Novel Thermophilic Anaerobic Species of the Order

Thermotogales. Applied and Environmental Microbiology, 62(8), 2875–2882.

., M. M., ., K. A., ., H. K., & ., T. S. (2005). Keratinolytic Activity of Some Newly Isolated Bacillus Species. J. of Biological Sciences Journal of Biological Sciences, 5(2), 193- 200.

P. Jeevana Lakshmi, Ch. M. Kumari Chitturi, and V. V. Lakshmi, “Efficient Degradation of Feather by Keratinase Producing Bacillus sp.,” International Journal of Microbiology,

vol. 2013, Article ID 608321, 7 pages, 2013. doi:10.1155/2013/608321

Park, G., & Son, H. (2009). Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiological Research, 164(4),

478-485.

Sangali, S., & Brandelli, A. (2000). Isolation and Characterization of a Novel Feather-Degrading Bacterial Strain. Applied Biochemistry and Biotechnology

ABAB, 87(1), 17-24.

T. Sivakumar, V. Ramasubramanian, V.T. Arasu, T. Shankar and D. Prabhu , 2012. Screening of Keratinolytic Bacteria from the Feather Dumping Site of Sivakasi.

Insight Bacteriology, 1: 1-6