Science Buddies: "Ask an Expert"

Hey science buddies,

I'm doing a project where I measure how much crude oil is disappearing in my water samples (that may or may not have oil eating enzymes in them). Here's the link: viewtopic.php?f=28&t=18082

I can do this visually over a period of time, but I want to know if I can do this physically and with quantitive numbers. My samples are relatively small; I'm using water samples of 40mL, and dispersing 16ml, 10ml, and 4ml into three different sample containers. The research I've done online so far only shows large scale oil-water separation techniques, not small scale ones like mine. Any advice?

Marla

Hi Marla,

Bioremediatiion of oil is a great research topic and I hope your experiment works!

I wasn't sure from reading your post how much oil you were adding to the water but I was wondering if you could simply use a dropper or pipet to remove the oil (since it is separate from the water phase) and weigh it on a scale with a sensitivity of at least 0.1 g.

What bacteria did you finally use to degrade the oil or did you take CMS' idea and culture some samples of pond water in the presence of oil and use the organisms that survive? I really like that idea because it has the element of the unknown and also uses a technique that an environmental scientist might use to discover a new strain of oil-eating microbe.

I found a good article on enzymatic digestion of oil that you might not have seen so here it is: https://www.hindawi.com/journals/er/2011/475193/

Please post again and we'll try to help.

Sybee

Hi Sci B,

I terms of how much oil I am adding to the water, I am testing different concentrations of oil to water ratios to make sure that I am not adding to little or too much oil for the enzymes. I will collect water samples from several sources. In one of those water samples, I will take three different containers and fill them each with 40ml from that one water sample. In each of the three containers, I will disperse different amounts of oil. In one I will add 16ml(40% ratio), in the other I will add 10ml(25% ratio), and in the third I will add 4ml(10% ratio).

I definitely like the dropper/pipet idea. I am monitoring the oil over a month, so how often would you suggest that I pipet the oil and measure it? Weekly?

I am taking CMS's idea and culturing samples of ocean, lake, and pond water, but they are not oil contaminated (at least I think not). I want to see if these sources have possible oil degrading enzymes by directly adding oil into them, and measuring if any oil is disappearing. The element of the unknown, indeed!

Also, thank you so much for the article. I will most likely use it in my citations.

Marla

You're welcome, Marla. I'm glad my answer was helpful!

Can I get a few more details on your experiment? What kind of oil are you using? This may be important because some types may contain toxic hydrocarbons or metals that will kill any microbes in the water. On the other hand, you want to use an oil that would be authentic--one that a ship would be carrying when it spilled. I know many online science project sites use salad oil as a stand-in for crude but you want to test the real thing, right? Do you have samples of crude oil to test?

Another thing that is bothering me is the proportion of oil to water that you said you are going to use. Oil floats on water because it is less dense and does not mix with water. According to one site I looked at, three gallons of oil would be enough to create an oil slick one acre in area. Now that's big! OK. What I am trying to say is that what may be important here is the surface area of your water sample. I think you may need a lot more water than 40 ml--more like 3 or 4 liters.

Oil spreads itself on the surface and refuses to associate with water except a little bit because water is a polar molecule (has a charge) and oil is nonpolar (uncharged hydrocarbons). So, a little bit of oil is all it takes to make an oil slick. Adding more is not gonna do anything because the only point of contact with water is at the very surface and that may be only a couple of molecules thick. In order to get a large enough contact surface for your microbes to 'see' the oil, you need a fairly large surface area of water--how large I don't know.

Did that make sense? It's sometimes hard to communicate this way because I can't tell if you are following what I am saying and you can't stop me to ask a question.

This surface area problem is gonna make it harder to measure oil degradation. Let's say you fill a glass bowl 10 inches in diameter with water then put 1 ml of oil on the surface. The oil will float and spread out, maybe forming blobs or maybe an oil slick (https://response.restoration.noaa.gov/t ... water.html). So then you want to let the oil sit on the water so the microbes in the water, hopefully, can digest some of the oil. How long this will take I have no idea but the more water you can expose the oil to the better your chances of seeing some degradation.

So basically I think you need a fairly large amount of pond or ocean water relative to the amount of oil because I think the amount of digestion is going to be quite small over the relatively short time period you will be able to run the experiment. Maybe CMS will read this and can help with his comments.

Post again and let me know what you think about my comments and suggestions. The old saying "The devil is in the details" is always true when planning scientific experiments!

Sybee

Hi Marla,

I just came across an interesting news story about a fungus that is able to digest oil and thought you might be interested in reading about it: http://www.cbc.ca/news/canada/saskatoon ... -1.4369041

The article points out some advantages to using this fungus to clean up oil spills on land but it would not work in water. The way they found the fungus was interesting, however, and is sort of what you would like to happen with a microbe in the water sample being able to eat the oil and reproduce.

Please keep us posted on how you are doing so we can make suggestions to improve your experiments or avoid problems. That's what we are here for!

Sybee

Hey Sci B,

This is the crude oil I will be using. https://www.ebay.com/itm/Petroleum-Crud ... SwopRYkkfR

I will definitely acknowledge collecting larger samples of water for my experiment. I have decided to compare freshwater and saltwater sources, and I will try to collect 3-4 liter samples.

So I understand the problem with surface area. I will try to get a bowl with large diameter, but I'm debating whether or not I still want to experiment with different concentrations . According to CMS, "There are really two important factors to consider as you work this out. The first is, of course, that you don't want to add so much oil that you overwhelm and sicken even bacteria that potentially could survive in crude oil. Secondly, though, you want to add enough oil that if your water samples do contain oil-digesting bacteria, you would be able to reliably measure the difference in the amount of oil before and during/after the experiment duration. " You also stated that "the more water you can expose the oil to the better your chances of seeing some degradation."

I'm going to try to acknowledge both statements. What I think I will do is take 4 different source samples of both freshwater and salt water, collecting up to 4 liters of each. Then, each sample will get two bowls. Each bowl will be filled with at least 2 liters of water. Then, one bowl will get 5ml of crude oil, and the other will get 1ml (in order to make sure I am not adding too much or too little oil for the enzymes to feed off of). I will cover the bowls with saran wrap to prevent evaporation. My observations will be over the course of a month. Everyday, I will take pictures of the bowls and compare if any oil has disappeared. Then, once every two weeks, I will measure how much oil has disappeared through pipette or dropper. I know this will be tedious. I will try to be as accurate as possible, then measure the oil via gram scale.

If you don't think it's necessary to even compare different concentrations, as in the 5ml and 1ml, please explain. I'm not exactly up for the tediousness of the 5ml blobs... So if you don't think it's necessary I will definitely acknowledge that. I'm a little bit confused by "Adding more is not gonna do anything because the only point of contact with water is at the very surface and that may be only a couple of molecules thick". All I'm understanding is that CMS said I shouldn't overwhelm or underwhelm the possible enzymes in the water. So, should I compare different amounts of oil, just to make sure the enzymes have just enough to degrade the oil?

I plan to start my experiment in 3 weeks, and before that I have to buy materials. I'm also trying to consider as many factors as I can, like surface area. I would greatly appreciate it if you had any other pointers to give me.

Thanks,
Marla

Good job, Marla! You have really thought this one through and I hope I can help you to carry it off successfully.

In my mind, one of the main problems is going to be measuring the amount of oil accurately. As you said, this is going to be tedious if you do two amounts of oil. My previous point about the amount of oil to saturate the system is this: the oil will float on the surface of the water, so whatever amount of oil you put on will spread out until it covers the water. The microbes are in the water and the oil they are exposed to is right at the interface between the oil and water. Adding more oil to what is already there is not going to increase the amount that they are exposed to.

My concern is that if you add too much oil you are not going to be able to measure a change in the volume accurately because the change will be very small relative to the error involved in pipetting off the oil and measuring its weight or volume.

What I don't know, because I have not done an experiment with oil-digesting microbes is HOW MUCH oil you can expect them to eat in one month. If you were using a culture of bacteria that was concentrated--billions of bacteria--and a species that was known to digest oil, then you might reasonably expect to see a substantial amount of the oil disappear in a month. But since you are just using samples of water that may not contain very many oil-eating microbes, the amount of oil decomposed may be too small for you to measure this way.

But even if you are not able to accurately measure a change in the amount of oil, I predict that you will be able to see a change in its appearance on the surface of the water as the microbes work on it. So, we are back to the question of how much oil to add to coat the surface. My opinion is that it would be best to have an oil 'slick' rather than an actual layer of oil. As CMS said, you don't want to overwhelm and kill off the microbes. Crude oil and motor oil contain chemicals that are toxic to living organisms (including humans, so wear gloves and be careful with the oil) and it is possible to inhibit them with too much oil.

It would be helpful if other people would weigh in here with some suggestions. My feeling is that you should use a minimal amount of oil so as to preserve the integrity of the microbes and take daily photos of the oil slick to hopefully see a measurable change in its appearance. At the end of one month, remove the oil and weigh it (you want to weigh it at the beginning, of course). Hopefully there will be a correlation between the visual changes in the oil slick and the final amount of oil remaining.

Sorry I can't give you some really specific guidelines. In science often you have to do several experiments to determine the best conditions to do the actual experiments. Troubleshooting and modification of methods is an everyday event in a scientist's life.

Good luck!

Sybee

Hey SciB,

Thanks for weighing in on your suggestions and comments. As I am buying my materials online, I would appreciate your input on them.

I am choosing to buy bowls that are at least 10" in diameter, and can hold at least 2 liters. Would you suggest I go with stainless steel, (https://www.webstaurantstore.com/5-qt-h ... XBHD5.html) or glass (https://www.bedbathandbeyond.com/store/ ... 1040686578)?

The scale I will be using has to be sensitive enough to measure 1-2ml of the crude oil, and I found this digital spoon (https://www.homedepot.com/p/American-We ... gIC4fD_BwE). It has the readability of 0.1g/.01oz. According to this online conversion, 1ml is equal to .0338 fluid ounces, and 2ml is equal to 0.06 fluid ounces. Although it may be more tedious to add 2ml of oil, I don't want to risk the digital spoon not being sensitive enough to weigh 1ml. What are your suggestions?

I will also be using a mechanical pipette (10ul-100ul) to more accurately collect the oil. Something like this (https://www.socalbiomed.com/equipment/p ... ttors.html).

Here is the crude oil I will be using (https://www.ebay.com/itm/Crude-Oil-Samp ... dYyTzUOujg). Will possibly buy 2-4 to be safe in amounts.

My water samples will be from 6 different local sources, 3 being freshwater and 3 being salt water. I will collect 2 liters of each, and store them in 2 liter soda bottles at room temperature. Would you say 2 liters is alright?

I will also use saran wrap to cover the bowls to prevent evaporation. However, I am curious to how the oil will degrade, and if it is turned into a gas or simply mixed into the water. If it is turned into a gas, should I poke holes into the saran wrap? Or should I use saran wrap at all? I will be conducting this in my room.


I have to get pre-approvals from LA county, and they wanted me to further evaluate on my "protocol on water isolation procedure", and "how long the water samples will be kept before experimentation". I'm not exactly sure what they mean by water isolation procedure, but I will be simply collecting water with my plastic 2-liter bottles. Also, the length my samples will be kept before experimentation depends on when all my supplies ship… varying from a few days to two weeks. Does this sound alright?

The length of my experiment will last for 1-2 months. I will take qualitative measurements, and try for quantitative every 2 weeks or so.

I would appreciate further input, SciB. You're like a mentor to me, and your ideas and opinions really contribute to my experiment.

Marla

Hi Marla,

I am happy that my answers have been helpful for you. I hope you can be successful in getting some good data in this interesting project.

Your water samples are the key to the whole experiment and I would not get them until the day that you are ready to start the experiment. I realize this could be difficult because you might not be able to get all of them in one day. The concern I have is because your experiment requires living organisms capable of digesting oil. Bacteria and other single-celled organisms live in fresh water and sea water but remember that they live in an environment and not in a glass bowl. Taking them out of their normal home will have some effect on them but I don't know what it will be, but the less time they are in an artificial environment the better.

My feeling is that you should immediately add the crude oil to the water sample the same day you collect it. Don't store the waters until you have them all and then start the experiment because the storage may harm them. I don't know how they organisms will react to being put into your artificial conditions but minimizing the stress on them is what you are aiming for. This means that you need to keep the bowls in a location where the temperature is fairly constant and warm--probably 75-80F [24-27C]. Also, I would suggest putting the bowls under an LED light fixture that has an output spectrum similar to sunlight and keeping them in a cycle of 14 hours light, 10 hours dark to mimic a natural environment.

I don't know how important these independent variables are, but as a scientist you have to try and control everything except the variable that you are studying, which in your case is the type of water sample and its native population of organisms.

It would be useful and interesting to know what organisms are in each of your water samples. Do you have a microscope with a power of 100-400X or access to one at school? If you do you could put a drop of water on a slide and examine it under the microscope and note what you see. I am not saying that you have to identify all the critters in the water, just to try and characterize them as to bacteria, algae, diatoms, protozoans, phytoplankton, etc. You don't have to do this, but since your experiment depends on these little guys being there it would be nice to see them.

OK. Have I answered all your questions?
- Glass bowls, not steel.
- 2 liters? I don't know if that is enough. It depends on how many micro-organisms per ml there are and how hungry they are for oil.
- As to covering them with plastic wrap, that's ok but do make some small holes in it to allow air to enter and other gases to exit. Remember that most microbes living in water use dissolved oxygen or carbon dioxide so the water needs to be in constant contact with the atmosphere. I don't know what the breakdown products of crude oil are, but you could find this by doing a search of the literature.
- I don't know what the LA County means by asking about your isolation procedure. I would talk to them directly and tell them exactly what your experiment involves and ask them what they need to know and why. The water is basically harmless to people, right? You should have a way of safely disposing of the waste oil since this might be considered an environmental pollutant so don't just pour it down the drain.
- Weighing the oil accurately is critical to your experiment. This is especially difficult with a substance like oil that coats glass and plastic. Transferring oil quantitatively is not going to be easy. You want to create an oil slick on the water, or at least globules of oil that the microbes can work on. I don't know the best way to transfer the oil to the surface. You can use your spoon (I think the 1.5 Tb model would be best for 2 ml) to weigh the oil but I think 0.1 g is not accurate enough. There are digital scales that weigh with an accuracy of 0.01 g (10 mg) and these are what you need. Most are no more expensive than the spoon. There are many suppliers of these online. Here's one example: https://www.medical-and-lab-supplies.co ... plies.html?
- I don't know which volume would work best--1 or 2 ml. Remember, crude oil is less dense than water so 1 ml of oil would weigh less than 1 g, but I don't know how much less. You should measure the oil by weight rather than volume. You should try adding 1 g or 2 g of oil to 2 L of just tap water in one of your glass bowls and see how well it covers the surface. You are looking for maximum contact with the water because that's where the critters are that you hope will eat the oil.
- Collecting the oil from the water to weigh it is going to be a challenge and I don't have a good suggestion how to do it. If you end up adding 2 g of oil to 2 L of water, that means your oil volume will be a little more than 2 mL. Somehow, I think, you will need to use a pipet or dropper to collect ALL the oil, including some water, from the water's surface and then put it into a long cylindrical tube like a test tube so you can see where the oil/water interface is. The key point is to collect ALL the oil and I think the only way to do that is by pipetting up some of the water along with it and then separating the oil from the water. If you can get all the oil into a test tube then you have a better chance of transferring it to a weighing container without taking along any water or leaving behind any oil. Does that make sense?

OK. I think that's enough for one post!

I'm sure you'll have more questions, so just send them along and I will try to make good suggestions. This is a very challenging project! It may not work. There may not be enough oil-eating microbes in the water to digest a measurable amount of oil in the time allotted. That's why i suggested examining the water with a microscope. If you don't see any microorganisms then chances are there won't be enough to change the amount of oil. But, you don't KNOW that so let's hope for the best. Maybe they are just waiting for some tasty crude and will gobble it all down!

Sybee

Hey Sybee

Thank you, thank you, thank you for your help! I've got more questions, and your suggestions mean the world to me.

- In terms of keeping my bowls at a temperature of 75-80F, the only place I can think of is my garage, but I don't think I can necessarily modify its conditions or keep it at a constant temperature. Do you think I could use a heating blanket? (https://www.walmart.com/c/kp/electric-blankets) Would I need to keep the temperature at a constant 75-80F? I'm a little worried in terms of safety; I don't want to overuse the blanket to the point that it catches on fire.


- I found a nice LED light fixture (https://www.amazon.com/Sunlight-Spectru ... 6YVLQ?th=1). There's two different kinds and one's more expensive than the other. Which would you recommend I purchase, and how many? They're sort of expensive, and I've got 6 bowls worth. Also, would I simply turn the light switch off to simulate night time conditions?

- I was also reading a kit guide on the best conditions for oil eating microbes to survive in (http://www.edvotek.com/site/pdf/956.pdf). The kit covers temperature, salinity, pH, and growth nutrients. Other than temperature, do you think I should modify any other aspects of my water samples? I'm interested in pH, but I don't think modifying the salinity of my sources (some being freshwater) would make sense... and I'm not sure about growth nutrients either. Although I do want to simulate my water samples as if they were in their natural environment, I do want my microbes to thrive in the best conditions. What is your opinion?


- I was also very intrigued by the tetrazolium indicator dye in the kit, and how it "is used to detect active OEMs while culturing the bacteria in different growth condi- tions. This indicator is colorless in its native form, but it is rapidly converted into a red dye by metabolically active bacteria. Since only live and active bacteria can perform this reaction, tetrazolium allows for the quantification of live bacteria within a solution. Scientists have used tetrazolium to detect active bacteria in different environmental samples including soil, groundwater, and aquatic environments. In this experiment, the indicator provides a simple and fast way to detect the bacteria at work instead of waiting weeks for the oil to disappear." From my understanding, the dye becomes a darker red if the microbes in the sample are working efficiently. I like this because in case I'm not able to quantitatively measure my oil samples, I can see a visual difference in how well the microbes are working. What is your opinion? I'm not entirely sure if the tetrazolium indicator only works with supplementary OEMs and not naturally occurring ones... which is a little concerning.


- I also did a test run of the oil and tap water. I took 2.5ml of olive oil (I don't have any gram sensitive scales as of this moment, only a ml measuring cup) to a 12inch diameter bowl with 3 liters inside. The condensed blob turned into a "C" shaped slick after a few minutes, and I would say that it covered 40% of the water. In terms of surface area, I'm still a little confused by your suggestions. Do you want the oil slick to cover the entire surface of the water? Maybe if I gave the oil enough time, it would cover the entire surface.

- I also tried pipetting the olive oil, and it was incredibly tedious. Because the olive oil's color was so similar to the water's, I couldn't really differentiate the two in my pipette. The oil also kept coating the plastic pipette. Crude oil is a dark shade, meaning I could easily differentiate it from water, but I feel like I would still be struggling trying to measure it. I might attempt to measure it at the very end of my experiment, but we'll see how that goes. That's why I like the tetrazolium indicator dye.

- I'm also very interested by your suggestion in looking at the samples under a microscope. There's certain bacteria, including alcanivorax borkumensis (http://www.metamicrobe.com/petroleum-mi ... teria.html) , that are known oil eating bacteria species. I'll keep an eye out for those in particular, and I'll try my best to identify the other organisms. I've never looked at water samples under a microscope before, but I'll get some help from my teacher. I'd also love to take pictures of samples under the microscope. I'll read up on that on my own.

- I'm also taking an interest in swabbing the water samples on petri dishes before I put crude oil onto them, and then at the very end of my experiment after the enzymes have (possibly) removed some of the crude oil. Maybe I'll count colonies too. In one of my earlier posts (viewtopic.php?t=17962), CMS also said that with this I could "isolate bacteria that are able to survive in oil-contaminated water, and possibly have oil-degrading enzymes." ... but for now I think I have enough for this year's project... maybe the isolated enzymes could be next year's project...

Alright, sorry for this boatload. I appreciate everything, Sybee!

Hi Marla,

Thank you so much for taking the time to work out the details on your project. I really appreciate someone like you who replies to my suggestions!

OK. Let me see if I can answer all your queries. I’ll try to do it in the order of your post…

1. Temperature regulation of water bowls
An electric blanket should be able to keep the T in the 75-80 degree range with no risk of fire. There are heating pads with temp regulators but they are pretty expensive (https://www.amazon.com/dp/B013SGDRXY/re ... 6197798775).
A small electric blanket might work better because you can wrap it around the bowls to keep the T more constant.

2. LED light source
For illuminating your bowls evenly to mimic sunlight on water I recommend 40W LED shop-lights (https://www.walmart.com/ip/SUNCO-2-PACK ... 3=&veh=sem). I use these for growing plants indoors and they are really bright and look just like sunlight. The only drawback is that they have to be hung from the ceiling or mounted on the bottom of a shelf over your bowls. They come with chains and cables for hanging or slots for attaching to a board. Each fixture contains two lamps and would provide enough light for your water samples so maybe your dad could use the other one in the garage or workshop.

3. Potential alterations to the water samples
For your first set of experiments I would not add anything to the water. Just let the existing microbes do whatever they can. When you find some evidence of digestion of oil then you can get more samples of that water and try to enhance the microbial activity by altering pH or adding nutrients. Scientists doing research on biological oil removal (bioremediation) have identified specific species of bacteria capable of digesting oil and they are working on how to grow them in quantity for addition to oil spills.

4. Edvotek oil bioremediation kit with tetrazolium indicator dye
I read through the procedure and I’m sure you noticed that one thing they didn’t do was try to measure the amount of oil consumed. The tetrazolium chemical changes color because of the metabolically active bacteria and that just tells you that your culture is healthy. Your idea of using the dye to show that you have active bacteria is a good one except for one thing—you need a spectrophotometer to measure the color change. I don’t think you can do it by eye. If you have access to a research lab that has a spectrophotometer then you could try this test. You would also need to purchase a small amount of tetrazolium and that would have to be done through a school or university, not an individual—and it is expensive: (https://www.sigmaaldrich.com/catalog/pr ... &region=US)
I took a look at the Edvotek kit containing the oil-eating microbes and it would be useful if you had a culture of these OEMs to use as a positive control because you could be sure that they would digest some of the oil. But, I don’t know where you could get some oil-eating bacteria that would not be too costly.

5. Test of oil addition to water
Hmm. I wonder what would happen if you had used salt water (I assume you just used tap water). The bacteria are in the water, but the oil is ONLY on the surface so that means only a few of the bacteria would actually be in contact with the oil at any moment. So, if the oil covered the entire water surface it would maximize the contact with the bacteria. But, if the oil does not spread over the entire surface then you just have to do the experiment that way.

6. Pipetting the oil to measure the volume
Marla, I totally sympathize with you on this one! I knew from the beginning that this part of the procedure was going to be a really tough one to do and I struggled to come up with some way to make it easier and more accurate. That’s why I suggested pipetting the oil/water mixture into a test tube so you could more easily see where the oil was floating on the water and remove all of it without taking any water. The amount of oil digested by microbes in your water samples is probably going to be very small over the relatively short time they have to work on it and you need to be very accurate in measuring the weight of the oil. I wish I could tell you that there was some easier and better way to measure the oil but I don’t know of one.

7. Microscopic examination of bacteria in the water samples
I’m glad your teacher has a microscope that you can use to look for bacteria and other microorganisms in the water samples. Indirectly, an increase in bacteria in the presence of oil would suggest that they grew because they ate the oil, but there you have the problem of measuring the number of bacteria which is also difficult. There may only be a relatively small number of microbes in the water so it is necessary to take a portion of the water sample—say 100 mL—and either filter it through a 2 micron filter or centrifuge it for 10 minutes at 5000 rpm. I don’t know if your school lab has a centrifuge or ultrafiltration units. You may need to stain the bacteria with methylene blue or crystal violet in order to see them. Your teacher should know how to do this. These are common stains and safe to use.

8. Growing bacteria from water samples
This is a great idea, Marla! Be sure to take photos of the colonies in the Petri dishes but leave them sealed because there may be some bad bugs growing on the agar and you don’t want to risk infecting yourself with a nasty microbe. Also, I don’t know what kind of agar to grow marine microbes on. You’ll have to do a search for that. I would think that you could make some marine agar by dissolving agar in seawater, but you need to read up on this first.

I’m so excited by your progress and enthusiasm! Wish I was there to help. Don’t hesitate to continue asking questions—as many as necessary.

Good luck!

Sybee