Final Report for GNE13-052
The intent of this study was to determine if enzymatic removal of chicken feather lipids can enhance feather meal processing. With the majority of chicken feathers being the protein keratin, keratinolytic enzymes are often looked at as a method to improve feather meal digestibility. However, little attention has been given to the role of lipids, which protect the surface of the chicken feathers. Removal of these lipids should provide keratin degrading enzymes better access to their substrate. Work on this project has led to the assembly of over 50 cloning constructs looking at 40 different genes from a feather degrading bacterial strain and two of its taxonomic neighbors. Of all the genes, 14 were capable of hydrolyzing chemical bonds found in feather lipids.
Feather lipids were extracted from whole feathers using a Soxhlet apparatus with petroleum ether as the solvent. Feather lipids were used for the development of two novel wax ester hydrolase assays. Assay parameters will be disclosed after our formal publication has been accepted. However, despite the identified lipase enzymes being active against a synthetic substrate, we were unable to observe lipid hydrolysis above the detection threshold when challenged with chicken feather lipids. This may be due to the limited substrate specificity of the lipase enzymes.
The project has had two community outreach events with one more to occur early 2015. A manuscript containing data from this project is currently being drafted and will be submitted for publication in the near future.
Every year over 24 billion chickens are produced globally for human meat consumption. Improved global standards of living are expected to only increase this number. With scientific literature suggesting as much as 10% of a chicken’s weight as its feathers, one might imagine the problem of what to do with all the feathers. Most feathers end up in landfills. However, with feather composition largely being protein (in the form of keratin), it is believed that it is feasible to incorporate this byproduct into animal feed. Replacing corn and soy protein in an animal’s diet with feather protein could reduce competition between human foods and production animal feeds for these crops.
Feather meal has been used for years within the industry, but many research teams have hoped to replace the steam hydrolysis preparation method because the final product is low in digestibility. Groups have shown that enzymatic feather hydrolysis can increase protein digestibility of feathers. The purpose of this study is to enhance enzymatic feather hydrolysis by removing surface lipids from the feathers. Lipids are excreted by the birds and spread on the feathers in an act known as preening. The lipids form a protective, waterproof layer on feathers’ surfaces and a physical barrier for keratin-degrading enzymes to access their substrate. We investigated potential means for lipid removal with the intent of improving enzymatic feather hydrolysis. Learning the role lipids have on feather hydrolysis can help reevaluate necessary inputs for feather meal rendering and allow for a more energy efficient and sustainable process.
In the original plan for the research project, there were seven main objectives:
- Determine the chemical composition of feather lipids.
- Identify potential lipases to hydrolyze triacylglycerides from the feathers.
- Isolate, clone, and express a wax esterase from feather degrading bacteria.
- Determine if keratinase assisted by lipase enhances feather meal preparation.
- Determine if keratinase assisted by wax esterase enhances feather meal preparation.
- Analyze wax esterase for a synergistic effect when accompanied by lipase on the keratinase- mediated feather degradation.
- Contribute to the knowledgebase on sustainable feather meal production and share that knowledge with fellow researchers and farmers.
Objective one to identify the chemical composition of feather lipids was initially broken down into two steps. The first step required us to isolate the lipids, which we successfully did using a Soxhlet extraction method. The second half of the objective was to run the lipids on thin-layered chromatography plates and characterize the lipid profile. Acceptable separation of blots was never achieved and continued efforts to identify specific lipids were outweighed against accepting a general lipid profile already known from literature. With feather lipids in hand and a general idea of the lipid profile, we had enough material to move forward.
Objective two, identification of lipase for triglyceride hydrolysis, was largely understated in the initial proposal. Bioinformatics techniques were necessary in expanding the pool of potential enzymes to work with. Alternative gene cloning and protein expression strategies facilitated streamline analyses for enzyme evaluation. The initial proposal called for a pH-stat to measure enzyme activity along with the use of a synthetic substrate. The synthetic substrate method won over pH-stat due to its high throughput and effectiveness in lipase identification. We were successful in completing this objective with the identification of five lipase enzymes.
The third objective was focused on isolating, cloning, and expressing a wax esterase. We found objective two overlapped significantly with this objective, in that we were essentially looking for a lipase with broad substrate specificity. As a result, this early assumption led us to using the same enzyme identification strategies used for lipase. What was not stated in the initial proposal, however, was how to measure wax ester hydrolysis. To this end, we have developed two efficient platform assays to quantitatively detect feather lipid hydrolysis.
Objectives four, five, and six were mainly to look at whether or not removal of lipids would improve feather hydrolysis. This section of the project had challenges in developing consistent reaction conditions across treatments. Plans to measure amino acid content or released protein into solution were found impractical as a result of high background from using crude enzymes in our treatments. Other approaches were created, but this part of the project still remains incomplete.
The final objective was addressed through multiple presentations. First, a presentation was given to faculty and students illustrating the work being conducted. Also, early on in the project, we worked with local high school students and described work that was being conducted for the project. Work to publish our findings is still under way and a couple draft manuscripts have been constructed with the anticipation of publishing soon in peer-reviewed, research journals.
In this study we aimed to better understand feather hydrolysis and create a more systematic approach to the enzymatic feather rendering process. We aimed to look specifically at the role lipids played in feather hydrolysis. To begin, chicken feather lipids were collected using a Soxhlet extractor. To do this, feathers were removed from chicken carcasses and then packed into the thimble of the Soxhlet extractor. The Soxhlet was run overnight with petroleum ether as the solvent. Lipids were collected in the still pot and then transferred to a glass tube. Excess solvent was evaporated off and final tube weight was compared with initial weight to determine lipid yield. We then used the feather lipids for the development of novel assays.
A gram-positive bacterial strain in our lab previously showed feather digestion capability. This strain and two of its taxonomic neighbors were used as a basis for the identification of lipid hydrolyzing biocatalysts. In brief, bioinformatics techniques were used as a bottom-up approach in the discovery of enzymes to break down feather lipids. Novel wax esterase assays were also developed to measure specific activity against feather lipids. Details on biocatalyst identification, their cloning, and expression, as well as our assay conditions will be disclosed upon completion of the research in a formal publication.
There were several milestones achieved in this project. First, we were able to successfully remove lipids from the surface of feathers with the Soxhlet apparatus. The waxy lipids dissolved well in petroleum ether and the process was straightforward and efficient. Having lipids in hand paved the way for the project to move ahead and served as the foundation for what we would be working with.
Our project reached its next great milestone upon creating an assay to measure feather lipid hydrolysis. Further details on this subject will be elaborated in a formal publication. Briefly, we created not just one, but two platforms to look at feather lipid hydrolysis, each unique and capable of quantitatively measuring enzyme activity. Having a suitable assay in hand was critical for the work to move forward and to determine if any of the biocatalysts we have been working with were effective at removing feather lipids.
The third milestone was a rolling achievement. Throughout the course of the study new enzymes and constructs were being identified, cloned, and tested for activity. In the end we identified 14 enzymes capable of hydrolyzing chemical bonds found in feather lipids. Five of these enzymes worked well as lipases and were further characterized for ideal conditions and activity units. Experimenting with different expression techniques helped us find an appropriate condition for many of the enzymes.
During the duration of the project, we have been able to participate in two outreach events. The first of which displayed our research with high school students and allowed us to share with them alternative ways one might be participating in agricultural research. For some students it was their first time seeing a real research lab. The second event was within the Animal Science department at Cornell, where we presented our research to fellow graduate students and faculty. It was a great way to excite the department about some of the interesting issues we are addressing with the project. A follow up project presentation with the department is expected to take place early 2015.
Efforts from this project stand to benefit agricultural sustainability and will have an effect for years to come. The foundation of this project was to identify a current process in agriculture and design a systematic strategy to render the process more efficient. Our target for this project was feather meal processing where we projected the removal of lipids from the chicken feather surface would increase enzymatic hydrolysis efficiency.
Teams that intend to take this project further will build off the foundation that we created in addressing this issue. Our attempts at identifying enzymes capable of removing waxy feather lipids can be learned from and improved upon. The same holds true for the assays developed for this project, which can be used in continual study of the same problem, or adopted for the use in similar studies. Some of our strategies used for improving enzyme activity and expression can also be beneficial for groups working with a variety of proteins.
Education & Outreach Activities and Participation Summary
Outreach came as a natural extension of this exciting research. The first activity involved approximately 20 high school students visiting Cornell University for a Career X event. Our project was one of two selected from the animal science department to be highlighted in the program curriculum. Students had real, hands-on experience, first learning about chicken feather physiology and nutrition, then being introduced to the methodology of enzymatically hydrolyzing feathers. Students were given a tour of our research lab and were introduced to some of the molecular techniques employed in finding, cloning, and expressing biocatalysts. As a result, students got to see a glimpse of graduate school research and were exposed to the development of biotechnologies in animal production and global sustainability.
The second activity was a presentation about the project to fellow graduate students and faculty from the Cornell University Animal Science Department. The setting allowed for a more technical presentation, covering the problem at hand and strategies that would be used to address it. The presentation occurred during the early portion of the project’s timeline, which contained some of the earliest data. A follow up presentation for the department will occur in early 2015.
Results from the study are still being finalized, but a manuscript draft has been constructed. With the completion of a few additional trials we hope to publish in a relevant, peer-reviewed, research journal to share our findings with the scientific community.
Areas needing additional study
This project has laid the groundwork to push enzymatic feather hydrolysis further with a special emphasis on the impact lipids may have. Efforts going forward should focus on identifying a solid enzyme for feather ester hydrolysis. With a couple of assays to work off of, getting a project started should be simple and screening throughput should be high. With these components in place, wax ester hydrolase identification may also benefit from a top-down approach, specifically looking at sources likely to have such an enzyme.
Additionally, perhaps the most pressing issue with this study going forward is the need for a quantitative and repeatable feather hydrolysis assay. The effective design and construction of such an assay went beyond the scope of this project. Ideally, such an assay will be able to use natural substrates, while accounting for the variability of chemical compositions between each feather. The assay should provide the ability to test crude enzymes without affecting test results through non-specific hydrolysis. The assay will also ideally be scalable for testing different size reactions.