Jack Foley1
1 Biology Department, Framingham State University, Framingham, MA 01710
Abstract
Acinetobacter venetianus and Alcanivorax borkumensis are bacterial strains capable of degrading hydrocarbons from oils or natural gases. Plastics are often derived from fossil fuels like crude oil and natural gas, with similar chemical structures and formulas to oil. Both products are commonly disposed of improperly and collected in the natural environment with few means for effective removal. Since A. venetianus and A. borkumensis are known to consume hydrocarbons, the ability to consume microplastic Polyethylene (PE), a type of hydrocarbon, will be explored. The bacteria are expected to consume the microplastic fastest when concentrations are highest. The premise of this experiment was to see if bacteria could consume PE and while there was bacterial growth, PE consumption was not being measured directly. Both strains were given high, medium and low concentrations of PE as the sole nutrient source and growth was measured with optical density. In the second trial, A. borkumensis demonstrated a significant difference between samples with PE and control samples, whereas there was no difference in the other trial. In one trial, through the growth phase and after the stationary phase in all concentrations of PE, A. venetianus had a significantly higher OD600 compared to the control. It appears both strains may be capable of consuming microplastics, however continued research is needed to explore whether microplastics are truly consumed or simply broken down further.
Introduction
Acinetobacter venetianus is a non-motile, aerobic bacterium found in marine environments that is capable of degrading hydrocarbons, an organic compound consisting of only carbons and hydrogens. A. venetianus thrives specifically with n-alkanes, a hydrocarbon with only single bonds (Fondi et al. 2016; Di Cello et al. 1997). The strain is unable to oxidize or utilize common sugars, such as D-glucose or DL-lactate, and amino acids, such as L-aspartate, for cellular functions; instead A .venetianus[Sigma Aldrich,(St. Louis, MO)] uses carbon nutrients (Vaneechoutte et al. 2009). The A. venetianus strain produces a biosurfactant that binds to the hydrocarbon, allowing the uptake into the cell. The biosurfactant is a glycoprotein that forms vesicles allowing emulsification of the hydrocarbon into the vesicle (Baldi et al. 2003). The biosurfactant causes the surface tension of a hydrocarbon to reduce and eventually break; as a result, microdroplets are formed, and the bacteria can surround the carbon source to transport it into the cell envelope (Baldi et al. 2003).
Alcanivorax borkumensis is another non-motile, aerobic bacterium found in marine environments and is capable of degrading hydrocarbons (Yakimov et al. 1998; Brooijmans et al. 2009). Alcanivorax borkumensis do not utilize common amino acids and sugars for cellular respiration, instead hydrocarbons are commonly used as the source of energy (Schneiker et al. 2006). When cultivated in the presence of hydrocarbons and alkanes, extra-cellular glycolipid biosurfactants are produced as a secondary metabolite which allows intake of the nutrients by breaking the carbon source (Yakimov et al. 1998). When degrading alkanes, A. borkumensis can modify fatty acids and incorporate them into the cell, increasing the bioavailability of the alkane within the bacterium, which allows degradation to occur (Naether et al. 2013).
Both strains of bacteria have been of interest in regard to bioremediation, the process of organisms removing environmental pollutants. Specific interest has been in cleaning up the ecosystem after an oil spill or contamination of oil (Brooijmans et al. 2009). Alcanivorax. borkumensis in the presence of oil can emulsify and degrade the oil in contaminated waters (Brooijman et al. 2009). Acinetobacter. venetianus then can adhere to degrade diesel fuel and n-hexadecane when present (Baldi et al. 1999).
Since most plastics, synthetic polymers, are derived from fossil hydrocarbons, they are not biodegradable and accumulate rather than decomposing (Geyer et al. 2017). The current estimates are that roughly 8300 million metric tons (Mt) of plastic have been produced to date, and of the 8300 Mt, 79% have accumulated in either landfills or in the natural environment (Geyer et al. 2017). If nothing changes, this number is projected to reach 12,000Mt by 2050 (Geyer et al. 2017). In 2019 alone, 7.8Mt of plastic leaked into aquatic and marine environments, with an estimated total 30Mt of plastic waste in the ocean and an estimated 109Mt of plastic accumulation in rivers (OECD 2022).
In the environment, the microplastics that can be found have been produced through two different means (Rillig 2012). A microplastic is defined as a plastic that has either been produced or degraded from a larger source and is between 1μm to 5mm (Gago et al. 2019). A primary microplastic is manufactured of the specific size from 1μm to 5mm, and secondary microplastics have been degraded from a larger plastic source already in the environment (Rillig 2012). Microplastics pose a unique problem as they can be ingested by other organisms, absorb organic nutrients from the surrounding environment, increase mortality rate or decrease fertility in organisms (Wagner et al. 2014).
Previous research demonstrates that A. borkumensis and A. venetianus can degrade fossil fuels, however no research to date has centered on microplastics (Brooijman et al. 2009; Baldi et al. 1999). With the majority of today’s plastics and microplastics deriving from fossil fuels (Geyer et al. 2017), this investigation explores if both strains may be capable of degrading microplastics since degradation of the predecessor is possible.
With issues concerning organismal health and continual increasing collection of microplastics in the natural environment, without a means of efficient bioremediation and removal of microplastics, the world faces a threat (Wagner et al. 2014; Geyer et al. 2017). With both A. borkumensis and A. venetianus having been demonstrated to consume oils and fossil fuels (Brooijmans et al. 2009; Baldi et al. 1999), this research will investigate the ability of the bacteria to consume Polyethylene, a derivative of fossil fuels, in the form of microplastic.
Methods
Materials
For this experiment, materials include: Marine Broth media, A. borkumensis, A. venetianus, 5mL test tubes, micropipettes, Eppendorf tubes, serological pipettes, powdered Polyethylene (PE), ethanol, an autoclave, a lab hood, an incubator, a rotator, a spectrophotometer and spectrophotometer cells.
The Marine Broth [Sigma Aldrich,(St. Louis, MO)] was used as the media, per recommendation of the American Type Culture Collection. The PE [Sigma Aldrich,(St. Louis, MO)] was used at 0.01g/mL, 0.02g/mL and 0.04g/mL. Ethanol [Sigma Aldrich,(St. Louis, MO)] was used in excess for sterilization of the PE in Eppendorf tubes in a lab hood. The excess ethanol was filtered out, and the PE was left to sit in a hood until the remaining ethanol completely evaporated. Once evaporated, the PE was added to the 5mL test tubes that contained 4mL of media. An autoclave [Consolidated Sterilizer Systems (Billerica, MA)] was used for sterilization of Marine Broth, and the test tubes before the PE was added. The bacteria were added to the test tubes with serological pipettes so that the starting Optical Density (OD600) was 0.01. Test tubes were then kept on the rotator within the incubator. The spectrophotometer and cells were used to measure the OD600 of the bacteria at roughly 24-hour intervals.
Culturing
Using a serological pipette, both strains of bacteria were added to sterile test tubes containing media to be cultured. Once in the media, the test tubes were capped and placed in the incubator set to 30°C with a rotator. This ensured the bacteria were kept at an optimal temperature with enough nutrients and constant oxygenation for growth. After approximately 72 hours, the bacteria were fully cultured and could be used for experimentation. Triplicates of each sample of bacteria at high, medium and low concentrations of PE were run at 30°C with aeration, with two trials for A. borkumensis and one trial for A. venetianus.
Optical Density and Spectrophotometer
A spectrophotometer was used to measure the OD600, and the culture was added so that the starting OD600 was 0.01. This low level of OD600 ensured growth to be accurately monitored. As accurate measurement for bacteria growth using OD600 is only between .2 and .8, data could only be collected when between these ranges. With measurements taken at roughly 24-hour intervals, measurements below .2 were discarded to ensure accuracy. Any data measuring above .8 was diluted until in the range of .2-.8 and then could be calculated to find the actual OD600 value. The spectrophotometer cells were not sterile, and once measurements were recorded, the bacteria were added to a waste beaker so that the rest of the sample would not be contaminated.
OD600 is a measurement of how much light is scattered in a solution, and as the bacteria grew in solution, the more the light scatters. This value can be used to find how much bacteria were in solution at the time of recording, to compare across PE concentrations and controls. To be able to compare the bacteria, the spectrophotometer needed to be “blanked” using a sample of only media, so that any changes in bacteria growth could be compared to the starting point.
Results
In the first trial of A. borkumensis, the only statistically significant increase in OD600 in test groups occurred at 198 hours in the low concentration of PE (Figure 1). From 0 hours through 168 hours, there was no significant increase in growth of any of the PE groups compared to the control with no PE (Figure 1).
Figure 1. The first trial using A. borkumensis. The average OD600 of three replicates with standard deviation plotted throughout 198 hours. The High, Medium, and Low concentrations of PE are colored in orange, grey, and yellow respectively. The A. borkumensis control is colored in blue. During the lag, exponential, and stationary phase there was no difference in growth between groups except at 198 hours when the Low concentration of PE group had significantly more than the control group.
In the second trial of A. borkumensis, the data show statistically significant increases in OD600 in test groups at 95 and 115 hours in the medium and low concentrations of PE (Figure 2). From 0 hours through 67 hours, there was no significant increase in OD600 in PE groups compared to the control with no PE (Figure 2).
Figure 2. The second trial using A. borkumensis. The average OD600 of three replicates with standard deviation plotted throughout 115 hours. The High, Medium, and Low concentrations of PE are colored in orange, grey, and yellow respectively. The A. borkumensis control is colored in blue. At 95 and 115 hours, the Medium and Low concentration groups had a higher OD600 than the control group.
For the one trial of A. venetianus, there was a statistically significant increase in OD600 across test groups at 18, 168, 186 and 211 hours in the high, medium and low concentration of PE groups compared to the control with no PE (Figure 3). The high concentration of PE had a significantly higher OD600 than the control at 18 hours and 186 hours (Figure 3). The medium concentration of PE had a significantly higher OD600 than the control at 211 hours (Figure 3). The low concentration of PE had a significantly higher OD600 than the control at 168 hours (Figure 3). Each of the high, medium and low concentration of PE groups is compared to the control at each time point using a two-sample t-test at a 95% confidence interval. Ideally a second trial of A. venetianus would have been done, however due to time constraints of the semester, only one trial was able to be run. Measurements would also have ideally been done at exactly 24-hour intervals, however due to access and availability to lab space and equipment, measurements were taken as close as possible to 24 hours apart.
Figure 3. The trial using A. venetianus. The average OD600 of three replicates with standard deviation plotted throughout 211 hours. The High, Medium, and Low concentrations of PE are colored in orange, grey, and yellow respectively. The A. venetianus control is colored in blue. At 18 and 186 hours, the High concentration of PE group was significantly higher than the control group. At 211 hours, the Medium concentration group of PE was significantly higher than the control group. At 168 hours, the Low concentration group of PE was significantly higher than the control group.
The results from the A. borkumensis have multiple time points with a statistically significant increase in OD600. The first trial has a gap from 101 to 168 hours with no data (Figure 1). This was over a weekend, and the laboratory was locked, so no data could be collected. The gap in time means that we do not know what happened during that time period, as the OD600 could have increased or decreased, however Figure 2 may better indicate the possibility.
Statistics
Each of the high, medium and low concentrations of PE groups are compared to the control at each time point using a two-sample t-test with a 95% confidence interval.
Discussion
The goal of this experiment was to determine whether or not the bacteria are able to consume microplastics, and it was found that both strains were able to display significantly more growth in the presence of PE than without PE.
The second trial of A. borkumensis better displays results (Figure 2), as data during the initial growth of the exponential phase were collected. This gives a better indication as to what the true growth curve may look like in comparison to results from the first trial (Figure 1).
In the two A. borkumensis trials, the first significant increase in growth may be due to the bacteria that were cultured being in different stages of the stationary phase when first added to the test environment. While it may cause a difference in when the first real growth is seen in the data, it does not change how much the bacteria grow overall, and it does not change anything within the individual trials, as all bacteria were taken from the same cultures. The higher amounts of growth support the idea that the bacteria can consume fossil fuels (Brooijman et al. 2009).
The trial using A. venetianus also has a gap in data collection between 49 and 138 hours (Figure 3), as it was over the weekend, and the laboratory was inaccessible. This means that we do not know for sure how the bacteria grew in the presence of the PE, so we are unable to draw conclusions from the times in between. After the 138-hour point, all groups declined in growth, however the high, medium and low concentration groups had at least one time point after this in which there were more bacteria in the sample than the control. This may be due to bacteria preferentially consuming all of the peptone first, the only other carbon source in the media, and having to consume the PE, supporting the idea that the bacteria are able to consume diesel fuel (Baldi et al. 1999).
For all trials of A. borkumensis and A. venetianus, taking a measurement at roughly 24- hour intervals is a good start, but to be able to more accurately report on the data, closer intervals should be measured. Continued research is needed to determine whether or not the PE was actually being consumed. While the question of this research is whether or not A. venetianus and A. borkumensis can consume microplastics, optical density is a measure of growth not consumption. Growth in this experiment is being used as a proxy of microplastic consumption. If the bacteria are able to grow more in the presence of the PE compared to no PE, it can be assumed that PE is being consumed. The result does not tell whether the PE is being consumed or broken down further, so a Carbon NMR spectral analysis could be used to make the determination. Had there been more time in the semester a Carbon NMR spectrum would have been obtained, but with limited time it was unable to be done.
The use of a minimal growth media instead of the Marine Broth in future studies may be used to determine if PE is being consumed. Since there will be no other carbon sources for the bacteria to consume, comparisons to environments with PE and without PE may better demonstrate the ability to consume microplastics. Marine Broth was chosen as the media because it is known that both bacteria are able to grow in it, whereas minimal growth media does not ensure the bacteria will be able to grow. Thus, allowing experimentation during the semester instead of trying to get a viable culture of bacteria.
Since the bacteria can be demonstrated to grow more in the presence of PE, continued studies may have interest in other types of plastic. PE is one of the major types produced and in use today, however it is not the only one. Just because A. borkumensis and A. venetianus may be able to consume PE does not mean the results will translate to the other kinds of plastics and microplastics as the structure varies in each type.
Due to having few replicates, 3 per group, the standard deviation is high relative to some of the data. Since there are few replicates and a higher standard deviation, the variation is more spread among the data and less reliable to conclude from than if there were more replicates per group.
This study indicates that A. borkumensis and A. venetianus may be able to consume the plastic due to the increase in amount of growth in the presence of PE compared to without PE. If it is determined through Carbon-13 NMR spectroscopy that these bacteria can consume PE, or any other type of plastic, the applications within bioremediation of plastics and microplastics in the environment are immense.
Acknowledgements
A sincere thank you goes to Dr. Cara Pina, Department of Biology at Framingham State University. I truly appreciate all of the provided guidance, instruction, patience and critiques to the experiment and paper. Another thank you goes to Kathryn Kaufman, Department of Biology at Framingham State University, for coordinating and ensuring proper supplies at all times throughout the experiment.
Without the assistance from Dr. Cara Pina and Kathryn Kaufman, this research would not have been possible.
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