Introduction

“Modern technology owes ecology an apology.” States Alan M. Eddison, director of Green Earth’s Affairs. Technology has improved the way of life for people in the 21^st century yet it creates more conflict everywhere else. A great example of this claim would be the Oil rigs that are established worldwide. These rigs are a not an amenity but rather a need. In fact, statistics show that in 1996 the world consumes 71.7 million barrels of oil per day. By 2020 the total amount is supposed to be a groundbreaking 100 million barrels per day. Oil drives both our economy and our way of life. However, ecology does not agree. 0.25% of the oil taken out is released back into the oceans. Out of this total amount 50% comes from oil spills! Though the amounts do not seem to be extraneous one must take into account the effects that oil has on the ecosystem and ecology in general. Land, sea, and air dwelling organisms can be affected by an oil spill. An example would be a bird, when an oil spill occurs the oil covers their feathers and the bird attempts to clean themselves up. However the oil enters their inner body and acts as a poison and furthermore kills this vital part of nature. Another effect of oil spills is on physical features such as beaches and other shorelines. Oil deposits and often ruins a beach making the beach useless. One cannot stop an oil spill from occurring but one can help keep future oil spills clean for the environment and useful for the people. Crude oil is a substance made of carbon, hydrogen, oxygen and sulfur. Crude oil can be found in ocean floor and in the floor of freshwater ecosystems. In both these ecosystems bacteria thrive and feed on the crude oil released in oil spills. However, one can question whether salt water compatible bacteria are able to digest crude oil at a greater pace than those bacteria indigenous to freshwater. Due to the abundance of salt water rigs in the oil industry one can hypothesize that the salt water bacteria have a greater efficiency rate in the digestion of crude compared to the freshwater bacteria. The independent variable is the saltwater and freshwater environments whereas the dependent variable is the rate of carbon degradation due to the crude oil loss in the water.

The bibliography for the project is located under References in the Blog Archive.

Problem and Hypothesis

Problem:
Do salt water compatible bacteria have the ability to digest crude oil at a greater pace than those bacteria indigenous to freshwater.


Hypothesis:
Salt water bacteria have a greater efficiency rate in the digestion of crude compared to the freshwater bacteria.

Safety

            The beginning of the experimentation starts with the collection of sea water. This will be collected from Galveston, Texas. This will be done with the aid of gloves so that the experimenter does not contaminate the water and also so that experimenter is safe from any potential danger. Also the experimenter will wash their hands and refrain from eating on the site of collection. Another safety risk is the growing of the bacteria with the use of mixed liquor (sewage slush), which is basically human waste, in the freshwater environment. However, the experimenter will wear gloves, mask and  apron and the experimenter will cover the cultures after inserting the bacteria into the environment thus that the air will not get contaminated. Also this experimental step will be done outside to prevent any air borne bacteria from harming humans. When removing the bacteria the experimenter will be in a level two lab thus all safety measures such as lab coats/aprons, and gloves will also be taken. When using the dechlorinator the experimenter will have gloves and an apron on.  Upon the TOC analyses the experimenter will get the lab technician (or the qualified scientists) help in order to avoid damage to both self and the machine. Last but not least the cleanup will be done with the help of a certified environmental scientist who is the mentor for this project.

Materials

·         9 40 mL glass vials (supplied by the lab)

·         12 pH strips

·         1 liter Glass containers with Screw on top

·         latex gloves

·         mask

·         apron

·         sharpie

·         1 liter of sea water

·         2.5 liter of freshwater

·         3 ml of crude oil

·         TOC analyzer

·         Incubator

·         petri dish and cover

·         2 clean 2% milk carton

·         Start Right Liquid (Dechlorinator)

·         2 rolls of scotch tape

·         5 grams of household fertilizer

·         teaspoon

·          pipette and more than 11 disposable tips

·         ¼ liter of Mixed liquor

Procedures

1.      In the first leg of the experiment, one will first start by collecting the necessary materials. First, grab an empty 2% milk carton and its corresponding cap. Both the carton and its cap must be clean and dry. Along with the milk carton and cap one must get a roll of scotch tape. One the three materials are gathered that are needed for this part of the experiment one must go down to Galveston, Texas where they will fill up the milk carton with only water from one of the numerous beaches.  After collecting a carton full of water, close the carton with its cap and using the scotch tape go around the cap twice. Then take the seawater carton and store it in the BSL-II lab. At the lab one must remember to feed the bacteria already existing in the sample with approximately 5 grams of fertilizer. Then reseal the carton and keep the water in an incubator with the temperature set at about 76°F.

2.      For the next part of the experiment, gather another empty and clean milk carton, its corresponding cap, and a roll of scotch tape. Take the beaker and measure out 2 liters of fresh water. Next, take the empty 2% milk carton and fill it with fresh water. To the freshwater add 2 drops of the Dechlorinator. At this point, if the project is to be continued later, one must put the corresponding cap on the milk carton and seal the bottle shut with the scotch tape just as one did with the bottle of sea water. Then one must put the fresh water in the same area as the salt water, however the two containers must be placed at least a foot apart from each other.

3.      The next leg of the experiment requires the Petri dish and its cap as well as the tape, the dechlorinated water, a pipette, the crude oil and the mixed liquor. From this point on the experiment will not leave the BSL-2 laboratory unless it is part of the samples that are being sent out for the TOC analyzing. Now take the container filled with mixed liquor and open it. After that take the pipette and take out 10 mL of mixed liquor.  Place those 10 mL into the Petri dish. Change the tip of the pipette and then draw out 5 mL of crude oil and place that into the Petri dish as well. Last but not least fill the rest of the dish with the dechlorinated water and then place the top of the Petri dish on top and seal with tape. Keep the Petri dish in the incubator that the Sea Water should currently be residing in.

4.      After a day take the Petri dish out of its incubator. Then gather the following materials: 1 of the glass jars along with its top, a pipette along with a tip, the milk carton filled with fresh water, a beaker, and the sample of crude oil. Now take the beaker and from the milk carton with fresh water measure out 1 Liter of water. Pour this water into the glass jar. Then take the Petri dish and pour its contents into the jar. Next take the pipette and from the crude oil sample measure out approximately 1 mL of crude oil and dispense it in the glass jar. Close the jar tightly and shake well for 30 seconds. Then taking a Sharpie© label the container as Fresh water. Then go ahead and place the container in the incubator.

5.      Next is the control sample which requires the pipette with a clean tip, a crude oil sample, the milk carton filled with fresh water, a beaker, and a new glass container and its lid. Take the beaker and once more measure out 1 Liter of dechlorinated water/fresh water.  Pour that water into the glass jar. Then take the pipette and once again measure out 1 mL of crude oil and dispense that into the glass container as well. Then close the container with its lid and shake well for 30 seconds. Finally, take the Sharpie© and label the glass container control. Then go ahead and place the container in the incubator.  

6.      The last sample that needs to be created is the sea water sample which requires the pipette and a clean tip along with a crude oil sample, the milk carton with sea water in it, a beaker and a new glass container with its appropriate lid. Taking the beaker measure out 1 Liter of sea water from the milk carton and pour it into the glass container. Then using the pipette measure out 1 mL of crude oil and dispense the 1 mL into the glass container. Close the container with its corresponding lid and shake well for 30 seconds. Then take the Sharpie© and label the container sea water.

7.      After one day, one must revisit the samples and bring with them the 3 vials supplied by the lab along with the pipette and three disposable tips. First, one must open a sample by unscrewing the lid. Then one must take one of the pH papers and hold it in the sample for 10 minutes, after 10 minutes one must compare the colors visible on the pH to the key that analyzes what color(s) belongs to what pH level. Note the pH level of the sample. Then using the pipette they must measure and transfer 40 mL of the sample into one of the vials. Then close both the vial and the container and then put that sample back into the incubator while the vial goes inside the bubble wrap bag supplied by the lab. Once one sample is done the same thing must be done for the other two samples thus that at the end, one has three different vials each filled with a different sample as well as a pH level for each sample. All three of the vials will go inside the bubble wrap bag and the bag will go into the icebox which should always contain ice.  This procedure of drawing samples and putting it into vials must be done thrice for each sample. The first time is the day after mixing the sample and then two days later another three samples must be taken and two days after that the last three samples must be taken. The pH test must be done the 2^nd, 3^rd, 4^th, and 5^th day of the experiment.

8.      For the testing carbon part of the experiment one needs to take the glass vials to the Total Organic Carbon Analyzer to find the carbon content in each of the samples. A certified professional will do the testing. The machine will calculate the total amount of carbon in the sample and the experimenter must note this. After the testing is over the samples need to be disposed of as a biological agent, and the disposal must occur in the lab containing the TOC Analyzer.

Results

Upon adding the mixed liquor, which had a dark drown color, to the dechlorinated fresh water one can notice that the newly created sample had a light brown tinge and small pieces of human waste. The sample after a day had more solid pieces that were attached throughout the petri dish. Upon creating the control, sea water, and freshwater samples one can observe the visual differences between the samples. The fresh water (before the addition of the mixed liquor) and saltwater were both clear and after the addition of the cultures grown in the Petri dish the fresh water sample started to acquire a brown tinge. The pieces of human waste that were from the petri dish sample tended to rest at the bottom of the sample. The sea water upon acquiring it from the sea had a lighter brown shade than the mixed liquor when it was mixed with water but it was darker than the color the fresh water had when it was mixed with the bacteria from the Petri dish. The fertilizer used to preserve the bacteria dispersed inside the sample. Upon mixing the samples with crude oil, many more observations were drawn. The crude oil floated to the top of all the samples. Thus the chemical remained suspended in the mixture. The crude oil started to disperse within the water quicker in the control sample as well as the sample with Fresh water. The salt water sample had crude oil that took longer to disperse. As the experiment proceeded there was plenty of qualitative data that was taken. As the days progressed the samples turned a darker color, this was true for all the samples. Another observation that is worth noting is the fact that the human waste/bacteria in the fresh water increased in size. Overall, these were all the visible changes that could be noted.

The first test that was conducted on the samples would be the TOC analyzer. However, due to some lag in testing the results will not be in the experimenter’s possession till January 26^th, 2011. Upon receiving the information from the TOC analyzer as well as from the TPH analyzer the experimenter will include those results as well as the appropriate graphs and other illustration in a revised results and discussion.

 The results of the PH test are illustrated in figure 1 below. The pH value for sea water was lower and more acidic than that of the control and fresh water. As 7 is neither basic nor acidic one the control and sea water samples both were neither basic nor acidic if one disregarded the average. The mode and the median was the same for the control and sea water and the two samples had the same standard deviation of 0.5 as well. The control and sea water samples also contained outliers. The outlier would be 8 for the control and 6 for the freshwater sample.  Thus only the sea water was actually acidic.

Figure 1: pH Test

	Monday, January 17, 2011	Tuesday January 18, 2011	Wednesday January 19, 2011	Thursday January 20, 2011	Average	mode	median
Control	8	7	7	7	7.25	7	7
Sea Water	6	6	6	6	6	6	6
Freshwater	7	6	7	7	6.75	7	7

           

Upon conducting the t-test on the samples and comparing them to the data one would have gotten had they taken the pH of just water. The pH of water is seven, and when it is comparatively analyzed against the control and fresh water sample the p value for both was p=0.391 at six degrees of freedom. When referring to the table one can see that the null hypothesis should be rejected.

            Figure 2 below showcases a histogram that shows how the averages and standard deviations from the 3 different samples compare. One can see that the sea water had the lowest average pH level as well no standard deviation. The other two samples had the standard deviations and the fresh water’s +1 standard deviation corresponded with the control’s average. As far as acidic to basic goes when deriving from this graph one can say that the sea water was the most acidic followed by the fresh water which is less acidic and increasingly basic and then the control is the most basic of all the samples.

Discussion

            The major findings in this section are that the sea water was more acidic than any of the other samples. Another major finding is that the crude oil took longer to disperse in the sea water sample then any of the other samples. Another major finding is that the sea water bacteria was more efficient overall in the digestion of the crude. The last major finding is that the solids in the mixed liquor grew in size over the course

of the experiment.

            Now going back to the first finding the fact that the sea water is more acidic is not due to the fact that the sea water contains salt. This is because the sea water/salt water places as an 7- 8 on the pH scale. In fact, this mixture is considered a weak alkali. Thus the only difference between this sample and the two others are not the reason for the surprising difference in the acidic levels. However, as this water came from Galveston, a utopia for oil rigs, there is already a great abundance of crude oil in the water. Then upon adding more crude oil to the water the water became more acidic.  Now the reason that this water was more acidic than the rest was due to the fact that the crude oil added to the other samples floated on the top as it did in the sea water sample but as already stated the sea water had crude oil dispersed within the sample. This makes a difference as to take the pH level one must put the strip within the water and as the other two samples only had crude oil on the top and not dispersed evenly within the crude oil did not factor into the overall pH level of the sample, while the sea water already had the existing crude oil mixed within.

            However, the finding of the sea water being more acidic than basic is contradictory to one site which states that “In the ocean…a pH of 8 is found…” (Report of Royal Society 2005). Now from the introduction the introduction one knows that crude oil escapes from sea bed through natural occurrences as well as human facilitated ones. Thus crude oil would be present in all samples of sea water. Crude oil is definitely acidic as it facilitates corrosion in many forms. However, in the report the crude oils acidity does not seem to factor in the overall acidic level of the ocean water. Thus the results of this experiment are not in correlation with the facts reported by a government database.
            The sea water bacteria was more efficient in the digestion of crude oil due to the fact that the bacteria in the water had previously an environment that had a source of crude oil in the water already. On the other hand, the bacteria in the fresh water had to be acclimated to the environment and food source. On thing to note however is that there was a lag period before the bacteria in the sea water started to digest bacteria. To figure out the reason for the lag period one would have to conduct further and more in depth research on the anatomy and habits of the sea water bacteria.

            The crude oils lag in dispersing in the sea water environment showcases why most companies need to use dispersant when an oil spill occurs. Unlike the fresh water environment the salt water environment restricts the dispersing of oil within the medium. The last major finding was the enlarging of the solids in the fresh water samples, the reason for that enlarging would be the abundance of nutrient the bacteria on the solids would receive. One must remember in the Petri dish the bacteria did not get as much crude oil to digest as they would in the sample. Thus this sudden food source caused an overall growth in the bacteria which are presumably the solids as mixed liquor is a liquefied version of human waste. 

            There are three sources of major errors. The first source directly effects the result of the pH test. When testing with the strips the colors for the different pH levels tend to be very similar to a point. Humans cannot catch the color difference very well and thus have to call it to the very best of their ability. Also as the strips only give a whole number as the pH level, one cannot be sure which way the solution actually leans. This can mess up the results as the difference between a 6.0 and 7.8 can be the difference between an acid and a base. The next major mistake would be the shaking of the container in order to get the crude oil dispersed within the container before the taking of the sample. As one cannot keep exact consistency of shaking there is a chance that one was shaken more than another. This could lead to more crude oil in one sample as the experimenter takes the sample from within the container not from the top. The last major source of error is in essence systematic, this is due to the fact that the crude oil started to stick to the sides of the glass container and a lot of crude oil was lost in that way.

            Once again the problem behind which this experiment was born was whether crude oil was digested better with salt water organisms rather than fresh water organisms. The hypothesis was proved accurate as the sea water did digest a greater range of crude oil over the six days that this experiment was conducted.  The results show that oil companies should concentrate in drilling for oil in sea water environment on the basis that in case an oil spill occurs clean up would be more effective in this environment rather than the other environment.

            In the future scientists would want to concentrate on whether dispersant would improve performance in ocean bacteria compared to fresh water bacteria. Another project that can aide the future of oil research would be for scientists to see whether a large amount of added crude oil from oil spills would affect the acidity level of the water. As the project showcased there is a discrepancy between the acidity level of water with a natural amount of crude oil compared to when a human introduces large level of crude oil.