Following the insertion of genetic constructs into vectors and transformation into yeast, the genetically engineered yeast cells were tested for starch degradation in known starch solutions. Three yeast strains were successfully engineered, each producing a different amylase enzyme. Once confirmed that the yeast were able to effectively degrade starch, the yeast was tested in industrial wastewater samples, using a prototype. Cell growth tests were used to calculate parameters for a mathematical model, and to determine if the genetically engineered yeast could survive in a glucose deficient media.
Testing in Known Starch solutions
To characterize starch degradation in a controlled environment, the yeast strains were tested in starch solutions of a known concentration. Four yeast strains were tested; 3 genetically modified (labeled Construct 1, Construct 2, and Construct 3 for simplicity) and 1 wild type strain, used as a control.
Short Term Testing
In the first test, 4 yeast strains were added to 0.5% starch solution in a 1:1 ratio. Undiluted samples were measured every hour for 6 hours, using iodine to cause a color change.The intensity of the color change correlates to the concentration of starch.
A spectrophotometer was used to quantify the intensity of color change. A spectrophotometer sends specific wavelengths of light through a sample and measures how much of the light was absorbed. If there is a high concentration of a substance in a solution, more light will be absorbed. Thus, higher spectrophotometric absorbance values correlate to higher quantities of starch. A higher starch concentration results in a darker blue color change, which causes more light to be absorbed, and thus a higher absorbance value.
Figure 1. Color change of starch solutions showing starch degradation at 5 and 6 hours. In the above images, a difference in color change is apparent. After 5 and 6 hours, the genetically modified yeast strains (C1, C2, C3) have largely degraded the starch; the low concentration of starch is indicated by the pale blue color. In contrast, the control, the wild type yeast (wt), has a darker blue color, indicating a higher concentration of starch.
Figure 2. Quantified starch degradation over 6 hours for three genetically modified strains and a control. The graph above shows the absorbance values for each yeast strain over time. The absorbance values correspond to the concentration of starch. An absorbance value of 1 corresponds to approximately 0.25% starch. All samples started with an absorbance value of ~1.
All three of the genetically modified strains degraded nearly all of the starch within 6 hours, indicated by the change in absorbance from a value of 1 to nearly 0. These results prove that the yeast are indeed producing and secreting functional amylase enzyme. In contrast, the level of starch for the control remained nearly the same throughout the 6 hours. The slight decrease seen in the control can be attributed to the very small amount of amylase enzyme that wild type yeast naturally produce.
Long Term Starch Degradation Testing
With confirmation that the yeast were producing amylase enzymes, the next test was to determine the rate of degradation over a longer period of time.
The 4 yeast strains were added in a 1:8 ratio of 3% starch, with a total volume of 250mL. By decreasing the yeast to starch ratio, and increasing the concentration of starch, it was ensured that there would be sufficient starch for the yeast to break down over a period of several days. Samples were diluted with water in a 1:8 ratio, iodine added to cause a color change, then absorbance was measured in the spectrophotometer every 24 hours, for 72 hours.
Figure 3. Color change of starch solutions 48 hours after yeast was added.In the above image, a difference in color change is apparent 48 hours after the addition of the yeast. The 3 genetically modified yeast strains (C1, C2, C3) have degraded a large portion of the starch. This is indicated by the pale, translucent blue seen in the three tubes on the right. The control, wildtype yeast, has a far darker, opaque blue color, indicating a much higher concentration of starch.
Figure 4. Starch degradation quantified over 72 hours using spectrophotometric absorbance values. Here, an absorbance value of ~1.1 corresponds to ~2% starch.
Within 72 hours all three of the genetically modified strains degraded a significant amount of the starch, indicated by the decrease in absorbance from 1.9 to nearly zero. Construct 3 appears to be the most efficient in degrading starch in the long term. The control, however, had almost no change in starch concentration. The slight decrease seen for the control can be attributed to the small amount of amylases that wildtype yeast produce.
For the genetically modified strains, the steep decline in absorbance after 24 hours, followed by a less steep decrease at 48 and 72 hours, is likely due to a high starting amount of amylase in the solution. Before the yeast were added to the starch solution, while the yeast were growing in liquid culture they were producing and secreting amylase into the liquid media. When this amylase rich yeast culture was added to the starch solution, the excess of amylase enzyme rapidly degraded starch. As this initial quantity of excess enzyme was used up after ~24 hours, the remaining starch began to be broken down by the amylase enzyme the yeast produced after being added to the starch solution. The rate at which the yeast produce the enzyme is lower than the rate at which the excess starting amount of amylase can break down starch, thus there is a lower rate of degradation from 24 to 72 hours than from 0 to 24 hours.
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.
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.
Cell Growth Testing
Two cell growth experiments were run; cell growth in standard yeast media (YPD media) and cell growth in starch media.
Cell Growth in YPD Media
This experiment was run to determine parameters for creating a mathematical model. It was also run to determine if the genetic modifications to the yeast and the increased metabolic strain of constitutively producing amylase enzymes would have an effect on cell growth rates and glucose consumption. YPD media (standard liquid yeast media) was used as a glucose rich substrate. Thus, at the first stages of cell growth, the amount of nutrients is not a limiting factor, the main factor limiting cell growth is the rate at which the cells reproduce. In addition to measuring the optical density of the cell cultures using a spectrophotometer, the level of glucose in the media was measured to determine the rate at which the cells consume glucose. To prevent settling of the cells, cell cultures were placed on stirrer plates with stirrer bars. Cell cultures were diluted 1:4 with water before measuring in the spectrophotometer for optical density. A standard blood glucose meter was used to measure glucose in the samples, after diluting 1:15. Due to logistical constraints (shortage of stirrer plates), only 2 cultures were run, wildtype yeast as a control, and Construct 2 yeast (simply referred to as ‘Genetically modified yeast’). All the constructs had roughly equal rates of starch degradation, thus it can be assumed there were roughly equal rates of amylase production and therefore the metabolic strain on each yeast strain, if any, would be similar.
Figures 10 and 11 display the growth characteristics of genetically modified yeast cells and wild type yeast cells grown in glucose rich YPD media.
Figure 10. Cell growth for wildtype and genetically modified yeast strains in glucose rich substrate.
The above graph shows the absorbance of cell cultures over a period of 192 hours (8 days), which correlates to cell density. Figure 10 displays a typical cell growth curve, characterized by an exponential growth phase, followed by a stationary phase, and a decline phase. For maximum specific growth rates, please see Data Analysis below. This kinetic behavior is modeled by a set of differential equations in the Model section of the wiki. Figure 11. Glucose consumption over time for the genetically modified yeast and wild type yeast grown in YPD yeast media. The above graph of glucose consumption for wildtype and genetically modified yeast exhibits an exponential decay phase. The level of glucose in the media is declining at an exponential rate, as the genetically modified and wildtype yeast consume glucose.
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.
Calibration Curves for Cell Concentration and Glucose Concentration
From the raw data of absorbance and glucose meter readings, the actual concentrations were determined using a calibration curve created by measuring absorbance and glucose levels in known dilutions. The experiments performed to generate the two calibration curves for cell concentration and glucose concentration is outlined in the Methods section. Using the two calibration curves, we converted the raw experimental measures to their actual values before using them to estimate model parameters. Figure 13 Calibration Curves for Determining Actual Cell and Glucose Concentrations from Experimental MeasurementsThe calibration curve for glucose shows the relationship between the reading of the blood glucose meter and the actual glucose concentration in a known dilution of YPD media. The glucose meter has a linear and non linear range, the linear range is approximately between 20-150 mg/dl (the typical range of glucose in blood), which is the range the meter was designed to be most accurate in. At higher ranges, the meter is non linear, thus it is not as accurate. The calibration curve for cell density relates the absorbance in known dilutions of cell culture to the cell count of each sample.
Data Analysis of Testing Results
A key parameter that enables comparison of exponential cell growth curves is the maximum specific growth rate, µ. Another parameter is the saturation constant Ks that captures the substrate’s effect on limiting growth; it is the concentration of substrate (glucose) when the specific growth rate is half its maximum value. Both these parameters are described in the Monod equation of cell growth in the Model [link] section of the wiki. Two graphical methods are used to estimate maximum specific growth rate, µs and the saturation constant Ks. These methods are based on literature readings, further explanations can be found on the Model page. Before making the graphs, the experimental data was first transformed using the calibration curves to give the actual cell concentration (X) and glucose concentration (S).Figure 14 Double-reciprocal Line-Weaver Burk plot for Cell Growth
The first graph is a double-reciprocal Line-Weaver Burk plot. From the slope and intercept, the maximum specific growth rate and the saturation constant are estimated. Figure 15. Log plot of exponential cell growth.
The second graphical method is a plot of the log ratios of cell concentrations against time. This method applies to the exponential phase of growth and is useful for getting an estimate of the maximum specific growth rate (mu) from the slope of the graph.The top two figures are for the genetically modified and wild type yeast in glucose rich YPD, and the bottom left graph is for the genetically modified yeast in starch. The Table summarizes the cell growth parameters estimated from the two graphical methods. Also included in the Table is the growth yield Yx/s which is the ratio of cell growth per glucose consumed.
Comparing Growth Characteristics
In addition to providing estimates of model parameters, the graphs in the preceding section enables comparison of growth characteristics. The maximum specific growth rates are 0.017 per hour and 0.020 per hour for the genetically modified and wild type yeast in glucose, respectively, indicating that the genetically modified yeast have slightly lower growth rates. Comparison of the genetically modified yeast performance in regards to substrate, show a lower growth rate of 0.007 per hour for the starch substrate compared to the 0.017 per hour for the glucose substrate.
The growth yield Yx/s is 5.07 and 5.91 million cells per mg of glucose consumed, for the genetically modified yeast and the wild type yeast, respectively. This tells us that during the exponetial growth phase for the same amount of glucose consumed as the wild type, the genetically modified yeast reproduce slower. The saturation constants relating to glucose limiting growth are 17.3 and 5.4 mg/dL glucose for the genetically modified yeast and the wild type yeast, respectively. The higher saturation constant value for the genetically modified yeast indicated that the glucose-limiting growth kinetics begins at higher glucose concentrations, and is consistent with the higher glucose demands of the genetically modified yeast.
