Here, we show how we have demonstrated the functionality of our solution in solving the problem faced by ceiling tile manufacturers. Following proof of concept
Testing In Industrial Water Samples
Having proved that in a controlled environment of known starch solutions, 1) the genetically modified yeast are producing amylase enzymes and 2) the amylase enzymes are able to effectively degrade starch in both the long and short term, the next phase of testing was with industrial water samples. Testing in actual industrial water samples enables a better understanding of how the solution will behave under real world conditions. After visiting the Armstrong ceiling tile manufacturing plant, 4 possible problematic areas were identified; aeration basin, secondary clarifier, primary clarifier, and thickener. 6 water samples were collected from these 4 locations;
1) aeration basin
2) secondary clarifier
3) primary clarifier to the equalization basin
4) thickener to primary clarifier
5) thickener to dry broke
6) primary clarifier to dry broke
Figure 5. Industrial water samples from a ceiling tile manufacturing plant. The six water samples were first tested to determine starch levels. The aeration basin, secondary clarifier, and primary clarifier to equalization basin samples did not contain a detectable level of starch. The thickener to primary clarifier and thickener to dry broke samples contained a small amount of starch, approximately 0.32% and 0.45%, respectively. The primary clarifier to dry broke contained a much higher percentage of starch, approximately 1.3%. Thus, the prototype testing was run with the following three samples; primary clarifier to dry broke, thickener to primary clarifier, and thickener to dry broke.
As construct 3 was found to be the most effective in degrading starch, this genetically modified yeast strain was used in prototype testing. Yeast cultures were mixed into the industrial water sample in a 1:8 ratio, with a total volume of 250ml. To account for starch degradation from other organisms in the water sample, a control without yeast cells was run. The control contained a 1:8 ratio of YPD media (without yeast cells) to industrial water sample.
Prototype
In order to simulate the physical conditions of the plant, a prototype was created. Dissolved oxygen levels were simulated by continuously aerating the samples, mimicking the large blowers used at the primary clarifier and throughout the ceiling tile plant’s board mill. In addition to aeration, wastewater and process water in the plant is mechanically agitated, usually with large rotating rakes in the clarifiers and thickeners. Figure 6. Setup of prototype with stirrer plates and air pump.The mechanical agitation was simulated by adding a magnetic stirrer bar and placing the beaker onto a stirrer plate, which kept cells, starch, and other organic compounds suspended in the water sample evenly mixed throughout, as in the ceiling tile plant. Samples were measured at 6, 24, 48, and 72 hours.
Figure 7. Starch degradation in Thickener to Primary Clarifier and Thickener to Dry Broke industrial water samples.
As seen in the graph, within 24 hours, the genetically modified yeast degraded all of the starch present in both of the thickener samples. The control without yeast, in contrast, had almost no change in absorbance, indicating only a negligible amount of starch was degraded.
Figure 8. Color Change of Primary Clarifier to Dry Broke water sample at 24, 48, and 72 hours.In Figure 8, a color change is apparent at 24 hours, 48 hours and 72 hours for the primary clarifier to dry broke industrial water sample. Differences in color intensity between the control (without yeast) and the sample with yeast at all three time points indicate starch degradation by the genetically modified yeast. The slight decrease in color intensity across the three time points can be attributed to the enzymatic activity of organisms that were already in the water sample. The purple color, compared to the blue color seen in previous tests, is likely due to differences in the composition of starch in the water, and other compounds in the water sample that are affecting the way our eyes see the sample. Figure 9. Starch degradation in Primary Clarifier to Dry Broke industrial water sample over time. Within 72 hours, the genetically modified yeast degraded nearly all of the starch in the primary clarifier to dry broke water sample. The control, without yeast, also saw a decrease in the amount of starch. This is due to the enzymatic activity of organisms already in the water, which results in a some starch degradation. However, the genetically modified yeast degraded the starch at a higher rate than the control. The steep decline in absorbance seen between time 0 and 24 hours is due to an excess of amylase enzymes in the YPD broth, produced before addition to the industrial water sample, when the yeast cultures were growing. Thus, when the culture was added to the industrial water sample, there was a high amount of amylase enzymes that had been built up over several days. This excess of enzyme rapidly degraded the starch, which explains why there is a faster rate of starch degradation in the first 24 hours.
h3>Cell Growth in Starch Media
To gain insight on the ability of the genetically modified yeast to grow in substrate that contains starch, but no glucose, cell growth testing was completed in a starch media. The media contained starch, the carbon source for the yeast, and boiled wildtype yeast, a nitrogen and amino acid source (specifics on the media preparation can be seen in Methods). It was hypothesized that because of the genetically modified yeast’s ability to create degrade starch quickly, the yeast would be able to degrade the starch into glucose, which could then be used as an energy source. In an industrial water treatment plant it would not be possible to pump yeast media into the water system, thus for implementation, the yeast would require the ability utilize nutrients and extract energy solely from dead yeast and starch. Figure 12. Cell growth over time for the genetically modified yeast grown in starch media that contains no glucose.The cell growth curve above indicates the genetically modified yeast are capable of surviving in a starch media, albeit at a far slower growth rate than in YPD media. This slowed growth rate is due to limitations on nutrient availability. Glucose is quickly and easily metabolized; however, starch must be broken down several times before the resultant glucose molecules can be metabolized by the yeast. These results indicate that the genetically modified yeast would be able to survive in an industrial plant: the yeast would be able to extract necessary nutrients from the dead yeast of previous generations and degrade starch into glucose for an energy source.
Implementation of Solution
The genetically modified yeast solution could be implemented inside the board mill or outside in the water treatment plant. Results indicated the primary clarifier or thickener could be places for implementation. While the thickener did not have a high content of starch, based upon the plant visit and technical reports, it is believed that the thickener had a higher starch content, which was broken down during transport. The same is also believed to have occurred to the aeration basin sample. More so because it is known that the aeration basin has a high percentage of bacteria needed for removal of solid and dissolved organic matter.The genetically engineered yeast could likely be implemented in the section of the primary clarifier as it goes to the dry broke, as that area appears to have problematic levels of starch and known butyric acid problems requiring biocides. Implementation in the thickener, to treat the wastewater before it enters the primary clarifier, could be an effective measure to prevent butyric acid buildup in the primary clarifier and aeration basin. The aeration basin has been identified as a problematic area; a bigger problem in the older plant, the new plant still has butyric acid fluctuations at the secondary clarifier despite higher aeration levels with powerful blowers. Furthermore, over aeration in the wastewater has caused other issues, such as bacterial filamentous ‘slime’. Implementation of our solution would prevent butyric acid production, and allow for aeration to decrease, which would eliminate the filamentous ‘slime’ issue and significantly reduce the energy consumed by the continuously running blowers. Lastly, the cell growth testing in starch media proves the ability of the yeast to survive in an industrial setting; the yeast are able to survive solely off of starch and the dead yeast of previous generations, eliminating the need for addition of nutrients or repeated applications of yeast cells.
