Progress 08/01/16 to 06/30/17
Outputs
Target Audience:DuringPhase I work on this project, several renderers, both independent and those associated with the packing industry, were contacted. In many cases they provided some key collaborations to help realize the successes experienced with the aims in Phase I. In addition, much communication in person and electronically was undertaken with the USDA lab with which an exclusive license is being sought. Those scientists conducted the original investigations that led to this effort to develop and commercialize this technology for the industry. Certain industry groups have been contacted and their annual meetings and trade shows have been targets of this effort to gain insight into the opportunities that exist as well as to begin the transfer of knowledge necessary to the successful commercialization of the products. Ultimately, society benefits the most as this project and product represents a natural alternative to the widely used synthetic products which are an environmental burden. Changes/Problems:We deviated from the original research plan after discovering that flocculant production needs greatly exceed the amount of hemoglobin available through our antioxidant production process. We developed a much cheaper manufacturing process using inexpensive chicken blood as a raw material. The final product has much lower zinc content (1% w/w ZnCl2) alleviating concerns about potential effect of elevated zinc content in wastewaters after application of our flocculant. Our product has similar viscosity to that of currently used synthetic flocculants and can be applied using the same equipment making it easy for our customers to switch to it. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Presenatations were made to the Fats and Protein Research Foundation which supported some of the original research. Also, a presentation was made to the National Renderers Association Annual Meeting where a large segment of our initial target audience was in attendance. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) Aim I: Development of a cost-efficient process for re-solubilization of precipitated hemoglobin A) Raw material. We were able to produce highly efficient flocculants from all four tested raw materials. Efficacy was roughly proportional to the concentration of hemoglobin in the sample. B) Zinc chloride and EDTA. We studied samples with 0%, 1%, and 5% wt. zinc chloride. None of the samples containing zinc chloride at any concentration exhibited any visible signs of microbiological growth, as assessed by lack of odor or change of appearance months after preparation. Therefore, use of zinc chloride appears to be necessary to achieve prolonged shelf life of the flocculant. Also, addition of EDTA did not change any visually observable properties of the samples. C) Characterization. Measured amount of zinc was always in good agreement with zinc content expected in the particular sample.Protein content in different samples varied from 10 to 15% and was generally slightly higher in the samples prepared from separated red blood cells versus samples prepared from the whole blood. Most of this protein (>95%) was hemoglobin. D) Conclusions for Aim I. We developed a much cheaper manufacturing process using inexpensive chicken blood as a raw material. The final product has much lower zinc content (1% w/w ZnCl2) alleviating concerns about potential effect of elevated zinc content in wastewaters after application of our flocculant. Our product has similar viscosity to that of currently used synthetic flocculants and can be applied using the same equipment making it easy for our customers to switch to it. 2) Aim II: Demonstration of the efficacy of re-solubilized hemoglobin as a flocculant A) Efficacy with model kaolin suspensions. Efficacy of the samples with different zinc and EDTA contents were tested with 3g/L kaolin suspensions with an ambient pH of 6.0. For the application rate of 200 ppm of our flocculant prepared from chicken blood by adding zinc chloride (1 wt. %) without addition of EDTA, precipitation of kaolin occurred within seconds. Interestingly, no precipitation was observed upon addition of anionic poly(acrylamide) (PAM) at 30 ppm, a concentration typically used for flocculation of wastewaters by rendering and animal processing industries. Similarly, no efficacy was observed for cationic PAM added at 30 ppm (not shown). According to our customers and an industrial partner, PAM-based synthetic flocculants have limited efficacy at pH below 7.0, often leading to the need for pH adjustment to more basic (7.0-8.0) to ensure efficient flocculation. Thus, use of our flocculant could be advantageous for these customers since it will eliminate the need for additional pH adjustment. We also determined the application rate of our flocculant with 3 g/L kaolin suspension (pH=6.0). The application rate that leads to immediate flocculation of this kaolin suspension was 100 ppm. Finally, we compared performance of the flocculants prepared from different species (chicken and porcine) using different zinc chloride (0%, 1%, 2% and 5% wt.) and EDTA (0%, 0.15%, and 1.5% wt.) concentrations. In summary, we found that efficacy of the samples is proportional to the total concentration of hemoglobin and does not depend on the species. Flocculants prepared from chicken blood generally contained less hemoglobin, but because of more than 10-fold cheaper price, chicken blood appears to be the most promising raw material. Zinc chloride is an essential component in our formulation, which function is protection from microbial contamination and improvement of performance. Therefore, based on these studies, samples with 1% w/w zinc chloride concentration showed the best price/performance ratio. Also, no improvement was found in performance of the samples containing EDTA. Overall, total hemoglobin concentration in the flocculant appears to be the most important factor that determines its efficacy. B) Pilot field-testing experiments. We visited several rendering plants to perform field-testing of our hemoglobin-based flocculant. Wastewaters in these facilities contain between 0.5 and 1.5 wt. % solids, mostly proteins and fat. Most of these facilities use both coagulants and flocculants (either cationic or anionic PAM, or both). Application rate of cationic or anionic poly(acrylamide) flocculants is 25-30 ppm based on the solid content; however, PAM must be dissolved in water prior to application and is used as 0.5 to 10% aqueous solution. Overall, our pilot testing experiments at these facilities resulted in the following findings: 1) Our flocculant was always efficient when used as a replacement of PAM flocculants currently used by the corresponding facility. If the facility used both coagulant and flocculant for their wastewater treatment, use of their coagulant in conjunction with our hemoglobin-based flocculant was also required. 2) Because of higher solid content, wastewaters at rendering and animal processing facilities generally required higher application rates of our flocculant than those we established for kaolin models. To treat wastewater samples from one cooperatingplant, we used four doses of our flocculant made from chicken blood using 1% ZnCl2, and no EDTA. Flocculation occurred at all four application rates but was faster at higher doses.Fat-rich solids accumulate at the top of the test tubes. C) Conclusions for Aim II. We demonstrated efficacy of our flocculant with model kaolin suspensions and found the application rate of ~100 ppm. We also performed field testing at several rendering and animal processing plants, and found that our flocculant was an efficient replacement of synthetic PAM based flocculants currently used by the industry. The application doses in these field tests varied between 500 and 1000 ppm. While these application rates are higher than application rates of currently used synthetic flocculants, the price of treatment by hemoglobin-based flocculant per ton treated wastewater will be lower. 3) Aim III: Testing susceptibility of re-solubilized hemoglobin to microbial contamination A) Microbiological contamination. We noticed that our flocculant prepared as proposed did not show any visible signs of microbiological contamination upon storage for up to three months. In this study, we seededsamples of our flocculant prepared from chicken blood using 1% ZnCl2 on agar plates at different dilutions to perform colony counting. As expected, no colonies were observed at any of the dilutions, indicating lack of live bacteria present in the sample. Furthermore, two batches of our flocculant manufactured from chicken blood with 1% ZnCl2 were sent for testing for the presence of Salmonella species to Iowa State University's Veterinary Diagnostic Lab. Testing was performedand results were negative for both batches. This result indicates lack of contamination by either live or dead bacteria. B) Microbiological challenge. We also studied resistance of our flocculant to microbial contamination by challenging it with three different bacteria: Enterobacter Cloacae, Salmonella Enterica, and Escherichia Coli. For these experiments, bacteria were grown to a log phase and plated to determine the number of colony forming units (CFUs) in the stock solution. Microbial suspension containing 105 CFUs was added to 1 mL of our flocculant (chicken blood, 1% ZnCl2) followed by incubation at 37°C for 24 h. No evidence of microbial growth was observed for any of the three studied strains added to our flocculant; at the same time all three untreated controls showed evidence of growth. Thus, our flocculant does not support microbial growth and is resistant to challenge by these three most common bacterial species C) Conclusions for Aim III. We did not observe any evidence of microbiological contamination of our flocculant. Also, we showed that it was resistant to challenge by 105 CFUs of three different species, Salmonella, E. Coli and Enterobacter.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
National Renderers Association, Floyd Bislinghoff Award, VRM Labs progress with antioxidants and flocculants, 2016 NRA Annual Meeting.
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Progress 08/01/16 to 06/30/17
Outputs
Target Audience:VRM Labs attended and presented at the annual meeting of the National Renderers Association Annual Meeting in Florida. During this conference, we visited with many packer renderers and independent renderers who are potential customers. We also attended the Internation Water Conference in Texas to speak to many in different fields of wastewater recovery. We made individual trips to many packer renderers such as JBS in Marshaltown, IA, Indiana Packers in Delphi, IN, Pine Ridge Packign in Des Moines, IA, and Fieldale Chicken Plant in Georgia. We also visited and ran tests for many independent renderers such as Wintzer and Sons in Ohio, Kaluzny Bros in Illinois, Sanimax in Wisconsin, Baker Commodities in California, and Darling in Des Moines. Changes/Problems:We initially thought we would manufacture our flocculant from the waste stream of our primary antioxidant product. However, during customer development interviews and after initial assessment of our application rates, we found that the target market for this product was much larger than we initially expected. This is true even for just the market entry strategy beginning with rendering and the livestock and poultry slaughtering industry, as the volume of hemoglobin needed would far exceed the amount of hemoglobin waste produced during the first five years of our antioxidant production. As an example, 25 tons of blood per day would yield the amount of antioxidant sufficient to cover 80% of the entire US antioxidant market but would supply only enough flocculant for 10-15 customers. Therefore, we decided to pursue this flocculant opportunity separately from our antioxidant production. Our challenge for Aim I was therefore to determine whether we could develop a commercially viable process for production of hemoglobin-based flocculant. Based on the market prices, chicken blood (priced at 1-2 cents per pound) appears to be much cheaper raw material than porcine or bovine blood (15-20 cents per pound) or separated red blood cells (~20-25 cents per pound). However, since flocculant efficacy could vary considerably depending on the species and overall hemoglobin concentration, we decided to test flocculant produced from chicken blood, chicken red blood cells, porcine blood, and porcine red blood cells. What opportunities for training and professional development has the project provided?VRM Labs was represented at the TechConnect Conference where a great amount of knowledge was gained about the agency and the process for grants. Also, our work with DeLaval, a commercial company with a lot of relevant equipment provided free consultation to our company on flocculants and wastewater recovery. How have the results been disseminated to communities of interest?We have made many presentations to our taget audience either individually at their places of business or in groups settings such as the National Renderers Association convention and the Fats and Protein Research Foundation semi-annual meetings. Also, we have conducted demonstrations of our product in tube tests at several packer renderers and at independent rendering plants. What do you plan to do during the next reporting period to accomplish the goals?Conclusions and future work. We did not observe any evidence of microbiological contamination of our flocculant. During the remainder of the project, we plant to challenge our flocculant with 105-107 colony forming units of several different strains of bacteria, including Salmonella, E. Coli and Enterobacter, to determine its tolerance to microbiological contamination. In the Phase II project, we will also study microbiological contamination of several scaled up batches and develop a screening quality control method to monitor potential viral contamination of the flocculant.
Impacts
What was accomplished under these goals?
The following results were obtained during the first six months of the project: 1) Aim I: Development of a cost-efficient process for re-solubilization of precipitated hemoglobin. We initially thought we would manufacture our flocculant from the waste stream of our primary antioxidant product. However, during customer development interviews and after initial assessment of our application rates, we found that the target market for this product was much larger than we initially expected. This is true even for just the market entry strategy beginning with rendering and the livestock and poultry slaughtering industry, as the volume of hemoglobin needed would far exceed the amount of hemoglobin waste produced during the first five years of our antioxidant production. As an example, 25 tons of blood per day would yield the amount of antioxidant sufficient to cover 80% of the entire US antioxidant market but would supply only enough flocculant for 10-15 customers. Therefore, we decided to pursue this flocculant opportunity separately from our antioxidant production. Our challenge for Aim I was therefore to determine whether we could develop a commercially viable process for production of hemoglobin-based flocculant. Based on the market prices, chicken blood (priced at 1-2 cents per pound) appears to be much cheaper raw material than porcine or bovine blood (15-20 cents per pound) or separated red blood cells (~20-25 cents per pound). However, since flocculant efficacy could vary considerably depending on the species and overall hemoglobin concentration, we decided to test flocculant produced from chicken blood, chicken red blood cells, porcine blood, and porcine red blood cells. We also needed to determine, whether or not adding zinc chloride and EDTA was necessary, and if yes, what was the optimal content of these compounds. Results for Aim I. We deviated from the original research plan after discovering that flocculant production needs greatly exceed the amount of hemoglobin available through our antioxidant production process. We developed a much cheaper and straightforward manufacturing process using inexpensive chicken blood as a raw material. The final product has much lower zinc content alleviating concerns about potential effect of elevated zinc content in wastewaters after application of this flocculant. The product we developed has similar viscosity to that of currently used synthetic flocculants and can be applied using the same equipment making it easy for our customers to switch to it. Overall, the goals of Aim I were achieved successfully. 2) Aim II: Demonstration of the efficacy of re-solubilized hemoglobin as a flocculant and comparison of its efficacy to that of currently available flocculants. Efficacy of hemoglobin-based flocculants was first tested with kaolin models in the lab, to determine application rates and compare difference samples with the same model. These experiments were followed by pilot field-testing with wastewaters at several rendering plants and one chicken processing plant to demonstrate robustness of the flocculant with different types of industrial wastewaters. Results for Aim 2. We demonstrated efficacy of our flocculant with model kaolin suspensions and found application rate of ~100 ppm. We also performed field testing at several rendering and animal processing plants, and found that our flocculant was an efficient replacement of synthetic poly(acrylamide) based flocculants currently used by the industry. The application doses in these field tests varied between 500 and 1000 ppm. Overall, we achieved the goal of Phase I proposal by manufacturing a working prototype and demonstrating the proof-of-concept both in the lab and in industrial settings. 3) Aim III: Testing susceptibility of re-solubilized hemoglobin to microbial contamination. Results for Aim 3. Potential microbiological contamination is a concern for any products manufactured from animal blood. Based on the findings of the first two Aims, we determined that our product should be manufactured from chicken blood by adding 1 wt. % of zinc chloride. We noticed that pour flocculant prepared in such did not show any visible signs of microbiological contamination (such as smell or visually observed evidence of microbial growth) upon storage for up to three months. In fact, Zn2+ has been shown to be a broad-spectrum antimicrobial agent in the literature. it could be expected that our flocculants would not support microbial growth. In our preliminary experiments we seeded samples of our flocculant prepared from chicken blood using 1% ZnCl2 on agar plates at different dilutions in order to perform colony counting. As expected, no colonies were observed at any of the dilutions, indicating lack of live bacteria present in the sample.
Publications
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Progress 08/01/16 to 03/31/17
Outputs
Target Audience:VRM Labs attended and presented at the annual meeting of the National Renderers Association Annual Meeting in Florida. During this conference, we visited with many packer renderers and independent renderers who are potential customers. We also attended the Internation Water Conference in Texas to speak to many in different fields of wastewater recovery. We made individual trips to many packer renderers such as JBS in Marshaltown, IA, Indiana Packers in Delphi, IN, Pine Ridge Packign in Des Moines, IA, and Fieldale Chicken Plant in Georgia. We also visited and ran tests for many independent renderers such as Wintzer and Sons in Ohio, Kaluzny Bros in Illinois, Sanimax in Wisconsin, Baker Commodities in California, and Darling in Des Moines. Changes/Problems:We initially thought we would manufacture our flocculant from the waste stream of our primary antioxidant product. However, during customer development interviews and after initial assessment of our application rates, we found that the target market for this product was much larger than we initially expected. This is true even for just the market entry strategy beginning with rendering and the livestock and poultry slaughtering industry, as the volume of hemoglobin needed would far exceed the amount of hemoglobin waste produced during the first five years of our antioxidant production. As an example, 25 tons of blood per day would yield the amount of antioxidant sufficient to cover 80% of the entire US antioxidant market but would supply only enough flocculant for 10-15 customers. Therefore, we decided to pursue this flocculant opportunity separately from our antioxidant production. Our challenge for Aim I was therefore to determine whether we could develop a commercially viable process for production of hemoglobin-based flocculant. Based on the market prices, chicken blood (priced at 1-2 cents per pound) appears to be much cheaper raw material than porcine or bovine blood (15-20 cents per pound) or separated red blood cells (~20-25 cents per pound). However, since flocculant efficacy could vary considerably depending on the species and overall hemoglobin concentration, we decided to test flocculant produced from chicken blood, chicken red blood cells, porcine blood, and porcine red blood cells. What opportunities for training and professional development has the project provided?VRM Labs was represented at the TechConnect Conference where a great amount of knowledge was gained about the agency and the process for grants. Also, our work with DeLaval, a commercial company with a lot of relevant equipment provided free consultation to our company on flocculants and wastewater recovery. How have the results been disseminated to communities of interest?We have made many presentations to our taget audience either individually at their places of business or in groups settings such as the National Renderers Association convention and the Fats and Protein Research Foundation semi-annual meetings. Also, we have conducted demonstrations of our product in tube tests at several packer renderers and at independent rendering plants. What do you plan to do during the next reporting period to accomplish the goals?Conclusions and future work. We did not observe any evidence of microbiological contamination of our flocculant. During the remainder of the project, we plant to challenge our flocculant with 105-107 colony forming units of several different strains of bacteria, including Salmonella, E. Coli and Enterobacter, to determine its tolerance to microbiological contamination. In the Phase II project, we will also study microbiological contamination of several scaled up batches and develop a screening quality control method to monitor potential viral contamination of the flocculant.
Impacts
What was accomplished under these goals?
The following results were obtained during the first six months of the project: 1) Aim I: Development of a cost-efficient process for re-solubilization of precipitated hemoglobin. We initially thought we would manufacture our flocculant from the waste stream of our primary antioxidant product. However, during customer development interviews and after initial assessment of our application rates, we found that the target market for this product was much larger than we initially expected. This is true even for just the market entry strategy beginning with rendering and the livestock and poultry slaughtering industry, as the volume of hemoglobin needed would far exceed the amount of hemoglobin waste produced during the first five years of our antioxidant production. As an example, 25 tons of blood per day would yield the amount of antioxidant sufficient to cover 80% of the entire US antioxidant market but would supply only enough flocculant for 10-15 customers. Therefore, we decided to pursue this flocculant opportunity separately from our antioxidant production. Our challenge for Aim I was therefore to determine whether we could develop a commercially viable process for production of hemoglobin-based flocculant. Based on the market prices, chicken blood (priced at 1-2 cents per pound) appears to be much cheaper raw material than porcine or bovine blood (15-20 cents per pound) or separated red blood cells (~20-25 cents per pound). However, since flocculant efficacy could vary considerably depending on the species and overall hemoglobin concentration, we decided to test flocculant produced from chicken blood, chicken red blood cells, porcine blood, and porcine red blood cells. We also needed to determine, whether or not adding zinc chloride and EDTA was necessary, and if yes, what was the optimal content of these compounds. Results for Aim I. We deviated from the original research plan after discovering that flocculant production needs greatly exceed the amount of hemoglobin available through our antioxidant production process. We developed a much cheaper and straightforward manufacturing process using inexpensive chicken blood as a raw material. The final product has much lower zinc content alleviating concerns about potential effect of elevated zinc content in wastewaters after application of this flocculant. The product we developed has similar viscosity to that of currently used synthetic flocculants and can be applied using the same equipment making it easy for our customers to switch to it. Overall, the goals of Aim I were achieved successfully. 2) Aim II: Demonstration of the efficacy of re-solubilized hemoglobin as a flocculant and comparison of its efficacy to that of currently available flocculants. Efficacy of hemoglobin-based flocculants was first tested with kaolin models in the lab, to determine application rates and compare difference samples with the same model. These experiments were followed by pilot field-testing with wastewaters at several rendering plants and one chicken processing plant to demonstrate robustness of the flocculant with different types of industrial wastewaters. Results for Aim 2. We demonstrated efficacy of our flocculant with model kaolin suspensions and found application rate of ~100 ppm. We also performed field testing at several rendering and animal processing plants, and found that our flocculant was an efficient replacement of synthetic poly(acrylamide) based flocculants currently used by the industry. The application doses in these field tests varied between 500 and 1000 ppm. Overall, we achieved the goal of Phase I proposal by manufacturing a working prototype and demonstrating the proof-of-concept both in the lab and in industrial settings. 3) Aim III: Testing susceptibility of re-solubilized hemoglobin to microbial contamination. Results for Aim 3. Potential microbiological contamination is a concern for any products manufactured from animal blood. Based on the findings of the first two Aims, we determined that our product should be manufactured from chicken blood by adding 1 wt. % of zinc chloride. We noticed that pour flocculant prepared in such did not show any visible signs of microbiological contamination (such as smell or visually observed evidence of microbial growth) upon storage for up to three months. In fact, Zn2+ has been shown to be a broad-spectrum antimicrobial agent in the literature. it could be expected that our flocculants would not support microbial growth. In our preliminary experiments we seeded samples of our flocculant prepared from chicken blood using 1% ZnCl2 on agar plates at different dilutions in order to perform colony counting. As expected, no colonies were observed at any of the dilutions, indicating lack of live bacteria present in the sample.
Publications
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