Science Buddies: "Ask an Expert"

Hello, I am doing a project to find out if bacteria such as Pseudomonas putida will degrade polyethylene microparticles in water. I plan to weigh the microparticles before bacterial exposure, and then after exposure for a certain amount of time to obtain the mass degraded. However, in order to get and accurate ending weight, I would have to get the bacteria off of the surface of the microparticle. With microparticles being less than a millimeter in diameter, is there any way to get the bacteria off of the microparticles without harming the particles itself? It was suggested to use sodium hydroxide to dissolve the bacteria off of the surface, bit I am unsure if it will affect the microparticles.

Thank you very much.

Hi Matthew,

That's a great idea. It relates to a very important practical issue of how to degrade plastic refuse, and it seems fairly straight-forward to do with minimal equipment. I did find one research article on this very topic, here: http://omicsonline.org/2155-6199/2155-6 ... p?aid=5697

As far as chemically removing the bacteria from the polyethylene beads is concerned, I would recommend staying away from corrosive chemicals like NaOH if you can avoid them... because of safety concerns. If you DO use NaOH, I would urge you to do so in a chem lab at school, if possible, with supervision and eye & skin protection. Concentrated NaOH and KOH solutions (the active ingredient in Liquid Plumber) can cause extremely nasty burns. NaOH could probably be effective in killing and removing the bacteria at low concentrations, however. I don't think it would have any effect on the polyethylene beads, especially at low concentrations, since solid and concentrated NaOH is often stored in polyethylene containers. See here: https://www.sigmaaldrich.com/content/da ... 045pis.pdf

That said, I think there are other, less nasty chemicals that could be used to kill and remove bacteria from the polyethylene beads, such as commercially available detergents/soaps. You might also be able to use rubbing alcohol (isopropanol), although alcohols might dissolve some plastics. If you use alcohol, you'd need to confirm that it does not dissolve (reduce the mass) of your plastic beads in a control experiment.

Those are some thoughts. Good luck. Post additional questions if you have any.

Scott Currie

Thank you very much.

Hello, I am doing a project to see whether or not bacteria such as Pseudomonas putida are capable of degrading polyethylene microbeads in water. I have already set up the experiment by mixing water, bacteria, and microbeads into containers. In each container, I put 2 grams of microbeads, and I plan to weigh them after the experiment is over in order to find the change in mass. Each of the tests have around 16 milligrams of bacteria in them, which is a pretty significant amount, considering that I am planning to weigh the microbeads to the nearest milligram. As the bacteria degrades the plastic, they will attach to the microbeads, and will have to be weighed along with the beads. After I finish the experiment, I will take the micobeads out of the water and dry them to get accurate weight measurements. However, I cannot find any way to remove the bacteria from the microbead, being that each microbead is less than one millimeter in diameter. After being dried, the bacteria would have lost much of its body weight, but will the bacteria’s weight still be heavy enough to affect the data of the experiment? If so, are there any ways to remove the bacteria?

Hi Matthew,

I agree with what Scott said about using a detergent to remove any bacteria stuck to the PE beads. I would use a laundry detergent rather than a dish detergent because i think the former would be better at removing bacteria. I would not use alcohol as it might denature the bacterial proteins and cause them to stick to the plastic.

You did not say how you plan to separate the beads from the bacterial culture. Are you going to filter them out or are they large enough that they settle out on their own? If you filter them i would rinse the beads a couple of times with dilute detergent to remove as much bacteria as possible.

I took a look at some of the references for using Pseudomonas bacteria to digest PE and it is possible, but my question would be how long does it take? How long did you plan to let the bacteria digest the plastic? Also, how are you growing the P putida? In order for bacteria to use plastic as a carbon source there can't be another carbon source such as glucose in the culture medium or they will use that preferentially. Bacteria normally go through a growth phase that is called logarithmic during which they grow and divide rapidly but as the numbers of bacteria increase and they use up the nutrients in the culture medium, they slow down and eventually stop dividing. I don't know how long this would be for P putida but it is something that you need to consider.

How did you determine that each test solution contained 16 mg of bacteria? Normally bacterial cultures are defined according to the number of bacteria per mL which can be determined by making serial dilutions and plating on nutrient agar. Where did you get the P putida and how did you decide how many bacteria to add to a certain amount of beads? If you made an experiment in which you varied the number of bacteria while keeping the weight of PE the same you should be able to show that PE digestion increases with increasing numbers of P putida. Likewise if you did a time course where you weighed the beads at several times over a period of weeks you could show that digestion increases with time of incubation with the bacteria. As Scott also suggested, you should include a control with just beads--no bacteria.

Let us know more about the bacteria and the details of your procedure and we may be able to help in planning the experiments and avoiding potential problems.

Sybee

Hello. Thank you for your advice to use the detergent to remove the bacteria. To answer your questions, I am planning to filter out the microbeads out of the solution after letting them degrade for two months. Hopefully, that will be a long enough time to make a significant difference. I had ordered the bacteria from the Carolina institute, and it arrived live in an agar slant. I swabbed the bacteria and cultured them on nutrient agar plates. After about a week and a half, the bacteria had grown a lot and I used a sterile scalpel to scrape off as much of the bacteria from the agar as possible. I weighed the amount of bacteria produced, and got 50 milligrams of P. putida. I then divided that amount equally among the three testing containers, so they each received about 16 milligrams. I do have controls without any bacteria. Also, if there are any bacteria left on the microbeads after rinsing them with laundry detergent, would that be a significant weight to disrupt the data? I know bacteria has a large percentage of water, so if I dried them along with the microbeads, would the dried bacteria make much of a difference either?

Thank you very much.

Hi,

I would not be concerned about the weight of the bacteria. The detergent treatment and rinsing the beads after filtering will remove most of them.

You said you are going to allow the bacteria to degrade the PE for 2 months. How are you going to do this? As I said in my previous post, bacteria will quickly use up all the nutrients in a culture medium and die or at least stop growing. What 'solution' were you planning on using to incubate the bacteria with the beads? Bacteria need nutrients to grow and divide and you want them to use polyethylene as one of the nutrients, so you have to keep them alive and growing for 2 months. How did you plan to do that? The cultures also need to be kept at a temperature of about 24C for the P putida to metabolize at a reasonable rate.

Let us know the details about your experiment and we can better advise you on how to avoid problems and get good data.

Sybee

Hello,

I was hoping that the bacteria would be able to use the plastic as a nutrient and energy source for the span of two months. This is one of the factors that I am experimenting for, that the bacteria will degrade the plastic and use it to survive in the water.

Thank you for your advice.

I understand that, but bacteria need more to grow than just a carbon source, which is what polyethylene will provide. They will die otherwise.

A commonly used minimal salts medium is M9 (http://openwetware.org/wiki/M9_medium/minimal) which contains M9 salts (http://openwetware.org/wiki/M9_salts). I checked with Carolina Bio but they don't have M9 salts listed. I would call them and ask if they can sell you something comparable. You can buy M9 salts from Sigma Chem Co. (http://www.sigmaaldrich.com/catalog/pro ... &region=US) but the quantities are much more than you need and quite expensive.

Do you have access to a chemistry or biology lab at your school? The recipe for M9 and the minimal medium is fairly simple (http://www.teknova.com/M9-Minimal-Salts-Broths-s/39.htm):

Formula for 1X M9 (per liter)
0.3% (22mM) Potassium Phosphate (KH2PO4) (3.0g/L)
0.6% (22mM) Sodium Phosphate (Na2HPO4) (6.0g/L)
0.5% (85mM) Sodium chloride (NaCl) (5.0g/L)
0.1% Ammonium Chloride (NH4Cl)(1.0g/L)
2mM Magnesium Sulfate (MgSO4)
0.1mM Calcium Chloride (CaCl2) (15.0mg/L) (optional)
0.4% Glucose (or Glycerol) (4.0g/L) (optional) [OMIT THIS BECAUSE THE BEADS PROVIDE THE CARBON SOURCE]

If you have a friend in college who has access to a microbiology lab, you could ask them if you could have a small amount of sterile minimal medium and some sterile disposable culture tubes. This would be the easiest way to grow your bacteria. The culture medium needs to be sterile because if it is not other bacteria or yeasts will grow in it and ruin your experiment.

If you haven't done so, read the Scibuddies guides on working with microorganisms:
https://www.sciencebuddies.org/science- ... fety.shtml
https://www.sciencebuddies.org/science- ... ents.shtml
https://www.sciencebuddies.org/science- ... ains.shtml

Let us know if you have more questions.

Sybee

Thank you very much. You have just saved my project.

Hello,

I actually have one last question. It has already been about three weeks since I put the bacteria in the water with he microbeads, but without the minimal salts medium, and I am wondering if there are enough bacteria alive to continue on with this project. When I scraped the bacteria off of the agar plates, I had scraped a small amount of the agar as well, so the bacteria probably had some food for a time. Would it be ok to put in the salt medium even this far into the project? It would be very difficult if I had to redo everything.

Thank you.

I can't say whether your bacteria are still viable since I have never worked with that species. Some species can survive with a minimum of nutrients but their rate of metabolism slows way down and many of them die.

Do you have a microscope or is there one that you can use at school? If so, put a drop of your culture on a slide, add a cover slip and take a look at them at a magnification of 400X. If you see a lot of nice intact little rods that would be a good sign but if there's a lot of debris then they may be mostly dead.

The scientific way to tell viability under the microscope is to use a special dye called trypan blue that is excluded from live cells. When you mix a little of the dye with your culture and then look at it under the scope the dead cells appear blue. If you don't have trypan there are some other stains that might work: http://durack.moodlesites.com/pluginfil ... cteria.htm

I'm hoping that your starving the P putida made them especially hungry so they devoured the plastic but you won't know till you weigh the beads.

Let us know what the cultures' viability looks like when you examine them under the scope.

Good luck!

Sybee

Thank you!

Hello,

I have another question. after the experiment is done, I will have to somehow filter out the microbeads from the water. I have considered the idea to use filters, but I need one that has holes less than .3 millimeters. Many of the metal mesh ones are pretty expensive, and some of the nylon meshes are too big. Also, many microbeads could be lost during the process. I have also thought about evaporating the liquid inside of the container to be left with the microbeads, but i had also added salt in there and it would form crystals and not evaporate out. Are there any other better options than these? If not, I will just have to try and be as accurate as possible with the nylon meshes.

Thank you.

Hi there,

You might want to consider filter paper, often used to separate fine solids from liquids or air. Filter paper often can filter out at the micron level (smaller than a mm), and I believe this would apply to you. Here is just one of many potential products you can order from Amazon: http://www.amazon.com/Ahlstrom-Qualitat ... B007FXL7X4

A Google search would yield even more results!

I hope you find something that helps you! Please post back with more questions.

Thanks,
scibuddyAK

Thank you.

Hello,

I have one more question. I have finished collecting all of the data for my microbeads samples, but the data doesn't make sense. The data shows that for control one, the microbeads gained 0.017 grams, and for control 2, the microbeads increased by 0.019 grams. For the experimental groups, group 1 gained 0.003 grams, while group 3 gained 0.004 grams. Unfortunately, group 2 was lost. Why is it that the microbeads gained weight, instead of decreasing as the bacteria ate them? When I inspected the microbeads afterwards, there seemed to be some white foreign particles mixed in. However, there is no reason for the particles to be there, because I kept kept the microbeads in a sealed place throughout the experiment. Is there any idea what those white particles could be? Finally, I do see a faint correlation between microbeads with bacteria and without. The groups of microbeads with bacteria seemed to have gained less weight than its control counterparts. However, the experimental microbeads had unidentified flakes, which I am thinking are the dried skins of bacteria that couldn't wash off when rinsed. Is this relation significant enough, and would the flakes make too much of a difference?

Thank you very much.

Hi,

I don't know why your particles gained weight. Did you rinse them really well after you filtered them? What did the cultures look like before you filtered them?

In my previous post I had explained that bacteria will die if you don't feed them and they cannot live on just plastic. Did you add the nutrient medium to the cultures? If the bacteria were mostly dead at the end of two months then there could have been a lot of bacterial debris in the liquid with the beads. Did you see anything like this? The debris would have been filtered out along with the beads but should have been eliminated in the rinse step.

The harder question is why the control beads which had no bacteria showed an increase in weight. Did you rinse the control beads the same way as the ones with bacteria? If any salts remained on the beads that would add to their weight. Is it possible that the beads absorbed water that was not lost when you dried them? I don't know the chemistry of polypropylene but you could do some searching and see if there's any reference to this.

It is unfortunate that you lost one of the experimental groups. When scientists run an experiment they usually include at least three samples for each group and then take the average. A statistical comparison can then be done between control and experimental groups to test whether there really is a difference. Without this test you cannot say with certainty that the bacteria digested the beads. I would express the weight change as a percent of the starting weight so people looking at your results can tell how large the change was.

How could you have improved your experiment? Run more samples--at least 3 and preferably 5 each of controls and experimentals. Another thing that strengthens your data is to run what is know as a time course. You would have used double the number of beads and double the volume of your culture. After one month you would have removed half of each culture, being careful to shake the tubes before you pipetted so you got a uniform sample. You would then have measured the weight of the beads to see how much change there was in 1 month. At the end of 2 months you would have done the same thing to the remaining beads. In this way you would have two sets of points to plot--one at 1 month and one at 2 months. If the bacteria were digesting the beads efficiently, you would have seen the weight of the beads decrease at 1 month and even more at 2 months. These data would make your argument much stronger.

I hope this helps. If you have more questions, let us know.

Sybee

Hello,

Thank you for your reply. I did add the nutrient medium that you recommended, but I am not sure how the microbeads gained weight. I filtered all of the samples multiple times, with distilled water, isopropyl alcohol to kill the bacteria, and even with hot water. As I poured the experimental groups through a filter, I did notice some bacterial clumps at the bottom of the glass jar, but I did swirl around the water to evenly distribute the bacteria before filtering it. However, after drying the microbeads, I observed white foreign particles mixed in with the mocrobeads. Maybe those contributed to the extra weight, though I have no idea where they could have come from. I guess I will conclude in my research paper that there is a possible slight correlation, but further testing over a longer period of time would be required to obtain more accurate results.

Again, thank you very much for answering many of my questions about my project.

Hi,

Yes, i think you can conclude that the beads with the bacteria gained less weight, but since you don't know where the weight gain came from this doesn't prove that the bacteria digested the plastic. Also, in your follow-up statement you need to say that next time you will make sure that you have live bacteria over the whole digestion period by spreading a drop of your bead suspension on a nutrient agar plate and counting the bacterial colonies. Dead bacteria are not going to digest anything.

You said in an earlier post that you weighed out 2000 mg of beads for each tube. What was the sensitivity of your electronic balance? An increase in weight of 17 mg would be less than 1% and an increase of 3 mg would be even smaller. Weighing always involves some error and you need to say what this error was in your results.

The lack of a larger weight loss from the beads treated with bacteria could have been a result of the bacteria having all died in the three weeks they were in water only. I should have suggested that you add more bacteria along with the salts medium. By the way, a simpler and more accurate way to have added equal amounts of the bacteria to the beads would have been to scrape them off the agar plate and add them to a tube with 5 mL or so of salts medium, swirl the tube well to resuspend the bacteria in the liquid then pipet equal amounts into each of your experimental tubes containing beads. It is almost always more accurate [and easier] to pipet a specific volume of liquid than to have to weigh out a small amount of a solid each time.

One other thing that I can’t emphasize enough and that is to run multiple samples for each test--a minimum of 3 and preferably 5. Your results cannot prove anything unless you can do statistical tests on your data, and in order to get a meaningful average and standard deviation you need to have a large enough sample number. You will learn more about this as you go on to study more math and science.

Good luck on your next science project! We’ll be here to help you decide on a great hypothesis and plan the most efficient and accurate experiments.

Best wishes,

Sybee