Artifical Photosynthesis

[donnahardy2]

Hi Irregular,

Vitamin pills usually don’t contain nitrogen, but they do contain other essential nutrients such as zinc and B vitamins that are required for yeast growth and probably not present in the newspaper that will be your main carbon source. Maybe you could use ¼ to ½ of a vitamin pill plus 50-100 ml of low sodium beef broth from the grocery store to each flask to support the growth of the yeast. Check out the list from this website and compare it with the label of the vitamins you are using:

http://www.beer-brewing.com/beer-brewin ... ements.htm

20 grams of yeast is plenty. We have seen this quantity of yeast used in many of the references we have read. Be sure to record the brand name and the lot of yeast in your lab notebook.

Yeast fermentation is an anaerobic process; however, unlike the bacteria you grew in the microbial fuel cell last yeast, they will not be killed by the presence of oxygen. As the yeast grow in the medium, they will use up the oxygen and the sample will contain reduced oxygen levels. Since the oxygen levels can affect the growth of the yeast and the production of ethanol, you should try to handle all of your samples consistently so that oxygen is a controlled parameter in the experiment. You will have to mix your samples to take a hydrometer reading and you should mix gently before taking other measurements, but you should do this consistently for each sample.


Donna Hardy

[irregular]

Hi Donna,

I will probably add in 1/4 to 1/2 of the vitamin pill to each sample 24 hours after the yeast was added. I am considering making the beef broth.

For my containers to store my samples, I have flasks and graduated cylinders. To make them anaerobic, I must cover the flask with a special flask lid, and for the graduated cylinder I would use duct tape to seal the top. I am using the graduated cylinder procedure because it is much more convenient - if I use the flask then, to measure the volume and density every time I have to pour all the contents in the flask to the cylinder and make sure there are no remains, and pour it back when I'm done. I am scared that in the duct tape there might be some small opening which allows the air/oxygen to seep in. Do you think I should switch over to the flasks?

Also, after I added the yeast to the sugar sample last night, there were A LOT of bubbles, it was overflowing so I couldn't make it anaerobic. However today there were no more bubbles so I made the mixture anaerobic. There's sa bit yeast and water liquid left in the baggie where I put my container in. I hope that there's still some yeast in the sample. I hope that all this didn't affect the yeast..

Thanks!

[donnahardy2]

Hi Irregular,

The vitamin pill and beef broth, if you use it, will help with the growth of the yeast.

I’m sorry, I don’t think my explanation was clear. Fermentation in yeast is an anaerobic process, but it’s OK if the samples are exposed to oxygen. Duct tape will be difficult to work with and the presence of oxygen won’t hurt your culture; as the yeast grow, they will use up the oxygen in the medium and will naturally keep the culture anaerobic.

The yeast culture produced lots of carbon dioxide bubbles since it was a fresh culture, but it should have continued producing bubbles if it was growing. Hopefully the cells are still alive and will start growing again when you add some more nutrients. Production of bubbles is a good sign. Check and notice if the culture starts growing again. Have you tried your test strips to see if you can detect any ethanol?

Do you have a microscope available? If so, do you have cover slips and slides? It might be helpful to check on the yeast culture if you don’t observe continued bubble formation.

Donna Hardy

[irregular]

Hi Donna,

Ah, now I understand. Thanks for the explanation, by the way.

I have used my pH strips, and there has been a slight change one of my organosolv samples and sugar samples since the pH has gone done a little. So right now I'm calculating pH, salinity, conductivity, TDS, weight, volume, density and now the samples have become less viscous; I can use the hydrometer as well. I have three though, two which measure sp gravity (one of them measuring sugar and po. alcohol as well), one which measures Brix. Each sample takes quite a long time, so I'll be doing 1-2 samples per day.

There still are bubbles forming but not as overwhelmingly many than in the beginning, sorry I used the wrong word.

Thanks!

[donnahardy2]

Hi Irregular,

Thanks for the explanation. The decrease in viscosity and the lower rate of bubble formation are good news; the yeast are apparently fermenting the sample and growing.
Testing a sample once every other day might be frequently enough with the rate of growth that you are seeing.

Donna Hardy

[irregular]

Hi

Here are the ingredients in the fertilizers that I have:

1:
Total Nitrogen: 2%
Available Phosphoric acid 11%
Total Phosphoric acid 22%
Water soluble potash 0%

2: Total Nitrogen 30%
Available Phosphate 10%
Soluble Potash 10%
Boron Actual 0.025%
Copper Actual 0.100%
Iron Actual 0.338%
Manganese CHelated ACtual 0.055%
Molybdenum Actual 0.00079%
Zinc Chelated Actual 0.110%
EDTA Chelating Agent 3%

3: Total Nitrogen 5%
Available Phosphoric Acid 9%
Soluble Potash 8%
Calcium 6%
Magnesium 2.5%
Sulphur 3%
Iron actual 1%
Organic Matter 18%

Which one should I use, and how much?

Also, if I'm adding the fertilizer, do you think I should still add the multivitamin? Fertilizers 2 and 3 contain some vitamins/minerals present in the multivitamin.

Thanks!

[donnahardy2]

Hi Irregular,

I like the second fertilizer the best because of the high nitrogen content. The trace metals will also be helpful. The vitamin pill contains preformed vitamins like biotin and thiamine that the yeast cells can use directly. Use 0.5% fertilizer in your sample, or 1 gram per 200 ml. Use both the vitamin pill and the fertilizer and I think the yeast will be able to grow well.

Donna

[irregular]

Hello Donna,

I think that it is time to conclude my experiment (not take any more readings), but will not until you confirm so. I have attached an Excel spreadsheet of my data, since I will be analyzing my data now and would like you point out anything you see.

My density/sp gravities seem to be quite different and no close to 0.789.. I'm wondering if I even MADE any ethanol, even though I did see bubbles and smell some alcoholic scent.

Also, my volume and weight seem to be decreasing with every reading I take. Is this due to evaporation?

All of my temperatures aren't exactly equal. Will I have to adjust my data to fit on temperature so they are comparable? For example, I know that when you're testing a solution which isn't at room temperature but more, you must add a certain number depending on the temperature of the solution.

Thanks!

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[donnahardy2]

Hi Irregular,

Sorry for the delay in responding. This was a lot of work. You should definitely conclude your experiment; I don't think anything else will happen in the fermentation of these samples. I'm not sure why your hydrometer readings are not as expected, especially since you have made other observations that indicate that ethanol was produced. Perhaps the newspaper, acid, and base affected the readings. I will review the data completely and send more additional comments tomorrow.

Donna Hardy

[irregular]

Hi Donna,

Don't worry, I understand.

I'm surprised especially with the reuslts of the sugar sample - the specific gravity decreased, yes, but not very much. I will continue to tackle my own questions as well.

My density/sp gravities seem to be quite different and no close to 0.789.. I'm wondering if I even MADE any ethanol, even though I did see bubbles and smell some alcoholic scent.

Also, my volume and weight seem to be decreasing with every reading I take. Is this due to evaporation?

All of my temperatures aren't exactly equal. Will I have to adjust my data to fit on temperature so they are comparable? For example, I know that when you're testing a solution which isn't at room temperature but more, you must add a certain number depending on the temperature of the solution.

I took some readings last night. The volume has decreased a lot, the sawdust sample is only 50ml.
I added 10g yeast to all except the sawdust sample to see if anything changes.

Do you think I might even have to redo my experiments..?

Thank you!

[irregular]

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I have added more data to the spreadsheet:

[donnahardy2]

Hi Irregular,

What a lot of work! Thanks for posting all of the data; you have an amazing amount of data and you will need to plot it using Excel graphs eventually. To help make sense of it, I have abstracted just a few values to start with and I would like to review these with you to confirm the results and to make sure I understand exactly what you have done.

The important readings are the density, specific gravity, and the brix. If ethanol is produced during fermentation, all of these values should decrease over time. Here are the changes in your results from February 16 to February 24, from the time that you added the yeast to the end of the fermentation:

Acid1: Density 1.105 to 1.131 (increase)
Specific gravity 1.11 to 1.122 (increase)
Brix: 25 to 28 (increase)

Acid 2: Density 1.0472 to 1.1286 (increase)
Specific gravity: 1.085 to 1.138 (increase)
Brix: 20 to 25 (increase)

Organo 1: Density: 1.0482 5 to 1.0769 ( increase)
Specific gravity: 1.078 to 1.106 (increase)
Brix: 18 to 25 (increase)

Organo 2: Density: 1.0482 to 1.060 (increase)
Specific gravity: 1.07 to 1.092 (increase)
Brix: 16 to 21 (increase)

Sugar: Density: 0.0976 to .0997 (very slight increase)
Specific gravity: 1.012 to 1.014 (?)
Brix 1 to 3(increase)

Sawdust: Density 1.227 to 1.019 (decrease)
Specific gravity: 1.09 to 1.138 (increase)
Brix 21 to 28 (increase)


Please confirm that I have interpreted your tables correctly.

Did you use the alcohol test strips with any of the samples? If so, what were the results?
Did you see bubbles in all of the samples? What are the exact names of the hydrometers you used for each table of results? What are the values listed in the Proten Al and Corunna columns?

Thanks!


At this point, I would say that your results are unexpected and it is not apparent that you were successful in converting the cellulose in the newspaper to ethanol. However, further evaluation of the data may help clarify the results.

I noticed that the density of the two acid samples were different at the point that you added the yeast, however the relative changes in the two samples were consistent. I assume this is because this is one of the samples that you had to add extra acid and base to achieve a netral pH. The values for the two organosolv samples were remarkably consistent.

While you continue analysis of the data, are you going to have time to repeat the experiment? If you have not discarded the samples yet, I recommend that you freeze at least a portion of the sample for further analysis, if necessary.


Donna Hardy

[irregular]

Hi Donna,

Yes, your interpretations were quite accurate.
Acid 2: Density: 1.105 to 1.1286

Sugar: Density: 0.0976 to 0.967 (very slight decrease)
Specific Gravity: 1.012 to 1.016 (very slight increase)

Sawdust: Density: 1.227 to 2.354 (the most recent sample, if you are counting those additional readings without the hydrmeter)

Please note that I did not add an extra 10g yeast to the sawdust sample, and that at this point there is no water in the sample, it is solid.

I used the pH strips with the samples. I will add a category for pH on my spreadsheet and send it to you. It has not changed very much over fermentation.

Yes, I saw bubbles in all of the samples, but I am not sure about the sawdust sample. The Lakeshore and School categories represent my hydrometers specific gravity readings (They are named after where I got them from). Corunna represents my third hydrometer, but it reads sugar, in Brix. Poten Al and Sug represent the potential alcohol (in %) and sugar readings (g/L) using the Lakeshore hydrometer. My hydrometers don't have a 'brand' - the Lakeshore box simply says "Wine & Beer hydrometer, Triple Scale, Made in France", the Corunna box says "Brew Tester with instructions, W.-Germany" and the school one doesn't say anything.

I may be mistaken, but do you mean that the acid samples were different at the point of neutralization? I then added yeast, and then the next day the acid densities were both 1.105. I agree with your assumption.

I think that I will have time to repeat my experiment. I will collect data from two readings today and update my chart and send it to you with the pH column.

How do you recommend I run my repeated experiment? One sample of sugar, one sample of acid, and one sample of organosolv? I do not think that I should do a sample with sawdust.

I am confused about what my acid sample represents; 'no pretreatment, acid (chemical) hydrolysis', right? I am confused because when discussing acids, papers seem to use the acid pretreatment and acid hydrolysis interchangeably. When I referred to the wikipedia article, it seems like adding acid is both pretreatment and hydrolysis, too. I only added acid once to the sample, does this make the sample both pretreated and hydrolyzed like texts suggest? It basically seems like I'm comparing acid and organosolv methods, which seems like not very much. What if I do an oxidative delignification sample, where I use hydrogen peroxide as pretreatment?

I have also been working on my research report.

Also, do I need to adjust any data because my readings have slight temperature variations of a couple degrees?

Also, when the water in my sample is evaporating over time, and suppose there is ethanol made in there too, wouldn't the ethanol evaporate with the water since their boiling points are close (ethanol 78C, water 100C)

Thanks!

[donnahardy2]

Hi Irregular,

Thanks for the clarification. Your hydrometers are all standardized for brewing applications, so you will need to plot the change in density over time for your experiments. You obviously won’t be able to use the standard values use for other applications on these samples. The pH results will be useful and will help confirm if fermentation did occur, the pH should decrease as fermentation progresses. What about the alcohol strips?

You need to explain the reason for the increase in density. Did all of your samples evaporate during the experiment? Is that the reason for the increase in density? Or, is the decrease in weigh of the samples over time due to unavoidable loss while taking measurements. If you are losing just the water and retaining all of the solids, then the samples may be getting too concentrated. Yes, you have made an excellent point; the ethanol would evaporate more quickly than the water.

For the repeat experiment, I think one sample of each will be sufficient, and you can exclude the sawdust sample. A solid sample is not going to ferment. If you want to add one more sample using the hydrogen peroxide to delignify the sample, you will still need to hydrolyze the cellulose. Maybe you could cover the samples to prevent evaporation of water and ethanol in the next experiment. Also, I think you could plan to do measurements just once a day. The results don’t vary that much during a day, and I think reducing the sample handling will help reduce the loss of liquid.

Adding the acid hydrolyzes the cellulose in the newspaper to glucose, which can then be used by the yeast. And the acid hydrolysis could be considered a pretreatment step prior to fermentation. You only need to add the acid to the sample one time to hydrolyze the cellulose to glucose.

The temperature was a controlled parameter because all of your samples were kept at the same temperature. You can mention that there was a variation in temperature, but a couple of degrees in variation did not significantly affect the outcome.

It will be good to have your research report written, because the data analysis is going to be a major project for this experiment.


Donna Hardy

[irregular]

Hi Donna,

By alcohol strips, do you mean coolant strips? If so, then I didn't use them in this experiment because I had the pH strips available. I also didn't use them because they don't have a full colour reference chart, only for 5, 6.5, 7.5, 9 and 11 (since it is meant for coolant testing). The coolant strips measure FP/BP (freeze and boiling point), RA (reserve alkalinity) and pH. Sorry, I didn't realize that I should've tested for the FP/BP and RA.

I noticed that the sample sizes were becoming smaller, so I assumed that it was because of evaporation. When I transferred the samples into a small cylinder for hydrometer testing (the only time I had to transfer samples), a very, very small amount of sample was lost.

So in my repeated experiment, I will have one sugar sample (representing fermentation), acid sample (representing muriatic acid pretreatment and hydrolysis), organosolv (ethylene glycol pretreatment, muriatic acid hydrolysis) and oxidative delignification (hydrogen peroxide pretreatment, muriatic acid hydrolysis).

1) Since hydrogen peroxide is sold in various forms, how much of concentration of hydrogen peroxide should be present in the product I buy/find? This will affect my proportions as well.

2) When I add the chemicals for pretreatment (in organosolv and oxidative delignification) can I put in the acid in right after, or should I wait for a day and then add it in?

I am planning on returning the conductivity meter. With that, I will lose the capability to measure the temperature accurately. I will have the choice to a) assume all of my samples are at 32C b) stick the digital aquarium thermometer on the outside of the samples (it has a range of 20-30C, in whole numbers) b) put in a mercury thermometer. I will probably end up using the digital thermometer.

If I will be covering the cylinders, I will need to figure out a way to have a good seal. I have flasks with their appropriate "cap seals", but if I use flasks I will need to do a lot of transferring (once to a big cylinder for vol and weight, again to a small cylinder for a hydrometer reading). I don't think I will do this. The cylinders don't have a "lid", but I can ask for any ideas at school. I will see what solutions I can come up with. EDIT: I talked to the teacher at school, and she recommended that I use this plastic wrap, but it's more opaque and "sticky". She couldn't couldn't remember the brand, but I think she was talking about this: http://www.biteofthebest.com/glad-wrap-pressn-seal/ and http://www.hippocketaeronautics.com/hpa ... pic=4714.0

Attached is the Excel spreadsheet. I have added newer readings and a pH category.

Thanks!

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[donnahardy2]

Hi Irregular,

Yes, do use the coolant strips on the next experiment. The problem is that since the density did not decrease as expected, you really don’t have any results that document the production of ethanol. The freezing and boiling point information will be a helpful addition.

In your repeat experiment, do you have space to include one more control? A newspaper sample with acid hydrolysis and no yeast. That will allow you to compare results with and without yeast; the difference in the results should be the ethanol produced.

I will have to review the information on delignification again. I have forgotten the details. I take home papers tonight to read on the topic. The standard hydrogen peroxide available is usually 3%.

The organosolv procedure involves heating the sample, you are using very gentle heating because of safety considerations, but you can add the acid immediately after that. I’m pretty sure you can do the same for the hydrogen peroxide sample, but let me verify this for you.

I’m sorry you are losing the conductivity monitor; the results would be helpful in analyzing the results. The 20 to 30 degree thermometer should be good enough for monitoring the temperature, however. Can you keep the conductivity monitor until you do an initial measurement on the next experiment?

The Glad brand press and seal wrap should work well to cover the containers. You will probably have to use a fresh piece every time you take a measurement to avoid cross contamination. Your teacher had a good idea, and I can’t think of anything better.

Thanks for the additional results with pH readings. I have printed these out and will look at everything again.


Donna Hardy

[donnahardy2]

Hi Irregular,

I found a good reference from Industrial Crops and Products comparing different conditions for delignifying rye straw and I will attach it to this message. The optimum conditions were 2% hydrogen peroxide pH 11.5 for 12 hours at 25 degrees Centigrade. There is a flow chart on page 73 that includes all of the steps. I recall that the natural pH of your sample was high, so a blended sample might have the optimum pH for the hydrogen peroxide step.

If you can get 3% hydrogen peroxide, you will dilute it one part water to 2 parts hydrogen peroxide, blend your sample and then incubate it for 12 hours. Be careful when you work with the hydrogen peroxide as it will bleach everything it touches, so wear old clothes and safety glasses.

After the delignification step, then add the acid to hydrolyze the bleached newspaper sample to glucose.

You had mentioned that you are working on your research paper, and the introduction section is very important to explain the purpose and the science behind your project. This paper has an exceptionally good introduction second, so I recommend that you read it for inspiration. The authors explain the problem they are researching, they review the results that previous authors have reported, and they provide excellent details about the chemistry of the project. You are working on a different problem, but you will want to use the same approach in your introduction.

Donna Hardy

delignification of rye hydrogen peroxide pt I.pdf

(167.24 KiB) Downloaded 408 times

[donnahardy2]

delignification of rye straw hydrogen peroxide ptII.pdf

(165.16 KiB) Downloaded 380 times

[donnahardy2]

delignification of rye straw hydrogen peroxide pt III.pdf

(150 KiB) Downloaded 396 times

[donnahardy2]

delignification of rye straw pt IV.pdf

(182.32 KiB) Downloaded 389 times

[irregular]

Hi Donna,

All of the coolant strips I bought before are finished, I only have the bottle and instructions. Is there another way to figure out the boiling and freezing point? (http://answers.yahoo.com/question/index ... 246AAupmSP)

I think that I will be able to do another sample. However, wouldn't my acid hydrolysis sample be the same as this sample except that I don't add yeast? I could take readings (like I did in my last experiment) after the sample is hydrolyzed and before yeast is added. If you would still like me to do this sample, I will do one of each sample we have talked about including this. If not, I will do one sugar sample, 2 acid hydrolysis samples, 2 organosolv samples, and 2 hydrogen peroxide samples. I would like to do these in duplicates to make sure my data is accurate.

I will see if I am able to use the conductivity meter.
My dad got 3% hydrogen peroxide from work, and some more pH strips as I had run out.
Thank you for the reference and your advice for my research paper!
Tonight I will take final readings for my current samples and then store them in a bottle each.



I would like to finalize and confirm the procedure and proportions with you. I will write as if I am doing one sugar sample, 2 acid hydrolysis samples, 2 organosolv samples, and 2 hydrogen peroxide samples:

For my newspaper samples, I will cut and mash 10g of newspaper. I will measure 200ml distilled water, and add the 10g newspaper to that (5g/100ml). The volume would exceed 200ml a little bit due to the newspaper, right? I will take volume, weight, density, and hydrometer readings and if possible, conductivity readings. I will refer to all of these as "measurements". Should I incubate the newspaper-water samples until they reach 30C (sealed with plastic wrap) and then take measurements again, and then do the pretreatment, or skip this and continue on with my samples being at room temperature? Then for pretreatment, I will:

1) add 26.5ml of 31.45% HCI to each of the acid samples
2) heat the two organosolv samples until right before they boil. Then, 200ml of ethylene glycol (coolant/antifreeze) will be added.
3) add 400ml of 3% hydrogen peroxide to each sample. If I do two samples, I would need a total of 800ml of hydrogen peroxide, which is quite a lot. My bottle only contains 500ml, I might be able to get more from dad's work, I'm not guaranteed though.

I will incubate the samples for 24 hours (it will be easier doing all ofthe experiment in the evenings, 12 hours later would be in the morning, but if you recommend I incubate the samples for only 12 hours and then proceed with hydrolysis, then I will do so). I will proceed to take measurements. Then, I will add:

1) nothing to the acid hydrolysis samples. (The acid hydrolysis samples get 24 hours of hydrolysis this way as opposed to 12 hours for the other two)
2) _____ml of 31.45% HCI to each of the organosolv samples each
3) _____ml of 31.45% HCI to each of the oxidative delignification samples each
(I'm not sure how much HCI I would add in the organosolv and oxidative delignification samples, what do you think? Would it still follow the 1:8.5 dilution (1 part HCI and 7.5 parts water) rule?)

I will incubate the samples again. After 24 hours, I will have to do neutralization. At this point I would have made the red cabbage pH indicator with red cabbage and water. To confirm; would you recommend I use distilled water instead of tap water when making the pH indicator? Also, since I will be adding this cabbage-water mixture to my samples, I would need to add an equal amount of pH indicator to each sample, right? Also, I don't think that the cabbage would do any bad do the samples.. Once I start adding the NaOH to neutralize each sample, I am concerned that if I add to much and need to add HCI, and need to experiment this way with some samples, my samples will not remain identical and the content of HCI and NaOH won't be the same. Do you have any comments about this?

Once everything is neutralized, I will take measurements. Then, I will add 20g yeast to each sample, along with 1/2 a multivitamin pill and 1g of "soil acidifier plant food" (contains 30% nitrogen) to each sample. Do you agree with the amount of yeast, multivitamin and plant food?After that, I will take measurements everyday for about 5 days.

For my sugar sample last time, I made a 5g/200ml distilled water sample. However, what if I made a mixture of how much sugar I have in my acid hydrolysis sample after all hydrolysis is complete (right before neutralization) with the 200ml distilled water? I would know how much sugar to add by measuring the sugar (g/L) using the hydrometer. But what if the sugar levels in both of my acid hydrolysis samples differ? Also, I would be adding the amount of sugar to 200ml distilled water, but my acid hydrolysis sample volumes would be higher, so would I have to add the sugar to the volume of the acid hydrolysis sample or just 200ml?

After creating my sugar mixture I'd follow the end (2 paragraphs above this) of the procedure for the newspaper samples.

Sorry if I confused you. I will ponder over my questions and concerns (in bold) myself, as well.

Thank you so, so, so much, I'm extremely grateful!

[donnahardy2]

Hi Irregular,

If you are out of coolant strips, then you can do an actual freezing point test on the final sample at the end of the experiment. It sounds like you have saved the first samples, so you can also do the freezing point test on these samples. You have found an explanation for the freezing point test for antifreeze, but we need to find the information for ethanol. I will see what I can find. This information should verify the quantity of ethanol in each sample.

It’s probably better to do duplicate samples rather than to do just one of a sample, so you should skip the no yeast sample. I had suggested the newspaper acid hydrolyzed sample with no yeast to see what the effect of the yeast on the samples is. The reason for suggesting the no-yeast control was that since you are using non-sterile technique, it might be possible for other microorganisms to contaminate the sample. I really would expect the 20 grams of yeast to provide an overwhelming number of live organisms at the beginning of the experiment, and the sugar and pH levels should be ideal for yeast growth, the acid treatment steps should kill off all the contaminants, so this is not a major concern. Did you say that you have a microscope to use? Maybe you could use the microscope to verify the samples are populated primarily by yeast.

Your samples list is good: one sugar, 2 no pretreatment, 2 organosolv, and 2 hydrogen peroxide and acid hydrolysis for all newspaper samples.

I would take the measurements of all of your samples after you have added the newspaper to the water, and then again after the no treatment/organosolv/ or hydrogen peroxide, plus the acid hydrolysis step on each of the samples. On the sugar water, you need one measurement since this sample does not contain newspaper. Please let me know if I have misunderstood any of your samples.

The organosolv and hydrogen peroxide steps are pretreatment steps that remove inhibitors of fermentation and do not break the newspaper down to cellulose. Only the acid hydrolysis step will do this. So you should not add anything to the acid hydrolysis sample until after you have completed the organosolv and hydrogen peroxide treatments on the samples. It’s fine to incubate the organosolv and hydrogen peroxide samples for 24 hours instead of 12 hours. The organosolv procedure sounds good, for the hydrogen peroxide, the authors used 2% so you can dilute the 500 ml bottle to 800 ml with deionized water (to make a 1.8% solution of hydrogen peroxide). After the pretreatment, you can try to separate the newspaper from the ethylene glycol and hydrogen peroxide, and then do the acid hydrolysis on all of the samples at one time.

Unfortunately, I am out of time, so I will complete the rest of the response later today or early tomorrow morning. Let me know if you have any questions so far.

Donna Hardy

[irregular]

Hello Donna,

I will do a freezing test on the samples from my last experiment which I have stored. I found out the boiling point (78.5° C) and freezing point (-115) of ethanol. I found that the range of FP possible to be measured using the strips is 0C to -51C and range of BP possible to be measured is 121C to 132C, so ethanol would not be detected using the strips. Research showed me that FP and BP can also be detected using a hydrometer and refractometer. The hydrometer needed is a special antifreeze hydrometer, but it only measures the same range of the test strips. So, I will only be able to do the tests at the end of the experiment.

Yes, I will do one sugar sample, two no pretreatment/acid, two ethylene glycol/acid, and two hydrogen peroxide/acid experiments.

Here is the revised version of my procedure with the questions:

For my newspaper samples, I will cut and mash 10g of newspaper. I will measure 200ml distilled water, and add the 10g newspaper to that (5g/100ml). will take volume, weight, density, pH, temperature and hydrometer readings and if possible, conductivity readings. I will refer to all of these as "measurements". Then for pretreatment, I will:

1) add nothing to the acid hydrolysis samples.
2) heat the two organosolv samples until right before they boil. Then, 200ml of ethylene glycol (coolant/antifreeze) will be added to each sample.
3) dilute 500ml hydrogen peroxide with 300ml deionized water, to create a 1.8% hydrogen peroxide sample.
***** My dad and I did the math, since the hydrogen peroxide is only 3% in concentration, that makes it a dilution of 15ml hydrogen peroxide and 485ml water. If I add 300ml of distilled water to the 500ml of 3% hydrogen peroxide dilution, I will get a total of 800ml of 1.8% hydrogen peroxide, 15ml being hydrogen peroxide and 785ml being water. How much of the 1.8% hydrogen peroxide mixture would I add to each sample? *****

I will incubate the samples for 24 hours. I will proceed to take measurements. Then, I will add:

1) add 26.5ml of 31.45% HCI to each of the acid samples
2) _____ml of 31.45% HCI to each of the organosolv samples each
3) _____ml of 31.45% HCI to each of the oxidative delignification samples each
***** (How much HCI I would add in the organosolv and oxidative delignification samples? Would it still follow the 1:8.5 dilution (1 part HCI and 7.5 parts water) rule?) *****

I will incubate the samples again. After 24 hours, I will take out the samples. I will attempt to remove all solid pieces, i.e. newspaper which isn't broken down. Then, I will have to do neutralization. At this point I would have made the red cabbage pH indicator with red cabbage and water. To confirm; would you recommend I use distilled water instead of tap water when making the pH indicator? Also, since I will be adding this cabbage-water mixture to my samples, I would need to add an equal amount of pH indicator to each sample to keep it a controlled variable, right? I don't think that the cabbage would do any bad do the samples.. Once I start adding the NaOH to neutralize each sample, I am concerned that if I add to much and need to add HCI, and need to experiment this way with some samples, my samples will not have the same volume; they would not remain identical and the content of HCI and NaOH won't be the same. Do you have any comments about this?

Once everything is neutralized, I will take measurements. Then, I will add 20g yeast to each sample, along with 1/2 a multivitamin pill and 1g of "soil acidifier plant food" (contains 30% nitrogen) to each sample. Do you agree with the amount of yeast, multivitamin and plant food? I am sensing maybe this is too much yeast. After that, I will take measurements everyday for about 5 days.

For my sugar sample last time, I made a 5g/200ml distilled water sample. However, what if I made a mixture of how much sugar I have in my acid hydrolysis sample after all hydrolysis is complete (right before neutralization) with the 200ml distilled water? I would know how much sugar to add by measuring the sugar (g/L) using the hydrometer. But what if the sugar levels in both of my acid hydrolysis samples differ? Also, I would be adding the amount of sugar to 200ml distilled water, but my acid hydrolysis sample volumes would be higher, so would I have to add the sugar to the volume of the acid hydrolysis sample or just 200ml?

After creating my sugar mixture I'd follow the end (2 paragraphs above this) of the procedure for the newspaper samples.

I am hoping to start the pretreatment tomorrow, after you reply back.

Thanks!

[donnahardy2]

Hi Irregular,

Excellent questions. Here are some answers:

Here is a table that shows the freezing temperature of different concentrations of ethanol, so you will be able to estimate the concentration of ethanol based on the decrease in the freezing point. Doing the test at the end of the experiment is perfect.

http://www.engineeringtoolbox.com/ethan ... d_989.html

Your Dad and you did the math correctly on the concentration of the hydrogen peroxide, but I made a mistake on the total volume of hydrogen peroxide you will need. For some reason, yesterday I was thinking that you would need 400 ml for each sample, but you only need 200 ml, or a total of 400 ml so you can use 133 ml of deionized water plus 267 ml of the 3% hydrogen peroxide to make 400 ml of 2% hydrogen peroxide (have your Dad check the math again). Each sample should contain 10 grams of newspaper and 200 ml of liquid.

After you incubate 1,2 and 3 for 24 hours using the conditions, which you have described, , if you add the acid directly to the sample, you will carry over some hydrogen peroxide and ethylene glycol into the fermentation sample, and this could affect the growth of the yeast. So after the hydrogen peroxide and organosolv samples are pretreated, it would be best to separate the newspaper fragments from the liquid and resuspend the newspaper in deionized water before you add the acid to all samples for the hydrolysis step. Do you have some way to filter out the hydrogen peroxide and ethylene glycol and recover the newspaper? Do your best with this suggestion depending on what you can do.

Next, you should add the 26.5 ml of 31.45% HCl to each sample. Since all of the samples should have a 200 ml volume, this should be a 1: 8.5 dilution for each sample and the 24 hour incubation time is good. What temperature will you be using?

If you have to remove pieces of newspaper that are still whole, you should save them and dry them and then weigh the quantity, so you can estimate the potential glucose in the sample. For example, if you use 5 grams of newspaper and have to remove 1 gram, then you will know that you had only a total potential of 4 grams.

You should make the cabbage indicator in deionized water and add an equal volume to each sample so that the color will be visible. Maybe 20 ml? You will have to look at the color to make sure it will be visible. I think the cabbage indicator will really help avoid adding too much NaOH during the neutralization step, and it should not affect the growth of the yeast.

The amount of plant food and vitamin is good. If the 20 grams of yeast seems like it’s too much (you made the observations last time), then reduce the quantity, but of course, add the same quantity to each sample.

For your sugar sample, use 5 grams sugar per 200 ml, and add the cabbage juice, fertilizer, vitamin, and yeast so your volume should match the other samples. . I recommend that you start all of the samples with the same volume and seal them this time to protect against evaporation.

Donna Hardy

[irregular]

Hi Donna,

Thank you for confirming, I used that same table as a reference for freezing point yesteday as well!

I started the pretreatment and took my first measurements, and think that everything went pretty smoothly!
By the way, the temperature of my water bath will be 32C, the highest setting that my 200 watt aquarium heater runs at. I do have another 50 watt aquarium heater but I haven't been using it simultaneously, it does not have a temperature setting like the 200watt. Do you suggest that I use a pressure cooker for a specific duration of time after I add the acid (for hydrolysis), and then add the samples in the water bath? I have sealed, and will keep the samples sealed throughout the experiment.

Revised Procedure:

Already done:
For my newspaper samples, I will cut and mash 10g of newspaper and add it to each sample, except in the sugar sample I will substitute 10g of newspaper with 5g of table sugar. Then for pretreatment, I will:

1) Acid samples: add 200ml of distilled water. add nothing to the acid hydrolysis samples.
2) Organosolv Samples: add 100ml of distilled water. heat the two organosolv samples until right before they boil. Then, 100ml of ethylene glycol (coolant/antifreeze) will be added to each sample.
3) Hydrogen Peroxide Samples: dilute 267ml of 3% hydrogen peroxide with 133ml of distilled water, to create a 2% hydrogen peroxide sample, and add this to the sample.
4) Sugar Sample: Add nothing.

Then, I will take volume, weight, density, pH, temperature, conductivity, salinity and TDS measurements. I will refer to all of these as "measurements". I will incubate the samples for 24 hours.

To Do:
After 24 hours, I will remove them from the water bath and proceed to take measurements. Next, I will remove the leftover newspaper in each of the newspaper samples and leave them in separate containers of distilled water to let them soak. Next, the newspaper will be filtered out from the distilled water and dried. Once dry, the amount of newspaper removed from each sample will be weighed to calculate potential glucose. Newspaper will not be re-added to any of the samples again. (This is how I interpret your suggestion of newspaper removal. However, even if I remove the newspaper from the samples, won't there be ethylene glycol or hydrogen peroxide still in the samples? Or will the newspaper soak it all up? What do you mean by "resuspend the newspaper in deionized water", and what is the purpose of this? I have found a link on ethylene glycol filtering, http://www.epa.gov/osw/conserve/materials/antifree.htm, but I don't think filtration, distillation, reverse osmosis, or ion exchange will be possible. What do you mean by "recover the newspaper"?) Then, I will add:

1) Acid Samples: add 26.5ml of 31.45% HCI to each
2) Organosolv Samples: add 26.5ml of 31.45% HCI to each
3) Hydrogen Peroxide Samples: add 26.5ml of 31.45% HCI to each
4) Sugar Sample: Add nothing.

I will incubate the samples again. Then, I will have to do neutralization. At this point I would have made the red cabbage pH indicator with red cabbage and distilled water. I will pour approximately 20ml into ALL samples, or any amount as long as they are equal in each sample. Then, I will start adding NaOH to neutralize each newspaper sample, and note down how many grams I had to add to neutralize each sample.

Once everything is neutralized, I will take measurements. Then, I will add 20g yeast (I will have to review different sources and choose an amount of yeast to use, one source, http://www.practicalchemistry.org/exper ... 09,EX.html, recommends 1g yeast per 5g glucose.. my potential glucose should be less than 10, but 1-2g yeast is a very low quantity..) to ALL samples, along with 1/2 a multivitamin pill and 1g of "soil acidifier plant food" (contains 30% nitrogen) to each sample. After that, I will take measurements everyday for about 5 days, plus hydrometer measurements. At the end of my experiment, I will do the freezing and boiling point test.

Thanks!

[donnahardy2]

Hi Irregular,

The 32 degrees Centigrade temperature is fine, although it’s probably not good to run the heater at maximum heat all the time. You could turn it down slightly and use 30 degrees C if you want to. It will be OK if the temperature fluctuates a degree or two during the experiment like it did last time.

Do not put the acid samples in the pressure cooker. The low pH will kill any microorganisms that are present in the other components and it would not be safe to heat the acid under pressure. It might be a good idea to boil the sugar only sample for a few minutes since it will not be acid treated before you add the yeast. The purpose would be to kill any microorganisms that are present so they won’t be able to compete with the yeast.

You have done a lot of work on this experiment so far. Everything sounds good.

Don’t discard newspaper: after the pretreatment step. I need to clarify what you are planning to do with the newspaper because the newspaper has to stay in the sample, otherwise there will be no source of glucose available for the yeast. The organosolv and hydrogen peroxide steps remove inhibitors of fermentation, so it would be best if you can separate the newspaper from the pretreatment solution and rinse it with water, and so the composition matches the acid hydrolysis samples. So keep as much of the newspaper as you can and eliminate as much of the pretreatment solution as possible . . . I’m sorry I was not clear yesterday; I hope today’s explanation is better. Don’t discard the newspaper even if you can’t completely eliminate the pretreatment solutions.

The acid steps look correct: if you have 200 ml of each sample.

The cabbage step looks good.

Your information on the amount of yeast looks good; do add plenty of yeast and add the same amount to each sample; 2to 5 grams might be enough, but you can decide based on your background reading.

Your hydrogen peroxide step should be bleached white by now. Can you confirm this? Let me know how everything else is going.

Donna Hardy

[irregular]

Hi Donna,

I lowered the temperature to 30C. You're right, it's probably the best turning it down so I can avoid it possibly burning out.

Thank you for the newspaper-removal clarification.
I checked my hydrogen peroxide samples, and yes, it looks like the newspaper is bleached white.

To Do:
After 24 hours, I will remove them from the water bath and proceed to take measurements. Next, I will remove as much leftover newspaper in the organosolv and hydrogen peroxide samples and rinse them, so their composition will match the newspaper in the acid hydrolysis samples. If possible, I will filter out as much pretreatment solution possible. Next, I will take out the rinsed newspaper, wring it to remove excess water and put it back in it’s original sample. (1. In your previous post, you mentioned drying the newspaper and weighing them to estimate the potential glucose. I love this idea, but leaving the newspaper samples to dry would take a while; how will I do this if I have to straightaway add the newspaper back to their original samples? If I only removed the newspaper to dry [how long would it take? 2-3 hours?], that will mean the samples just sitting in the water bath with no purpose. 2. I am still not sure how I will do the filteration, but I will keep researching.) Then, I will add:

1) Acid Samples: add 26.5ml of 31.45% HCI to each
2) Organosolv Samples: add 26.5ml of 31.45% HCI to each
3) Hydrogen Peroxide Samples: add 26.5ml of 31.45% HCI to each
4) Sugar Sample: Add nothing.

I will incubate the samples again. Then, I will have to do neutralization. At this point I would have made the red cabbage pH indicator with red cabbage and distilled water. I will pour approximately 20ml into ALL samples, or any amount as long as they are equal in each sample. Then, I will start adding NaOH to neutralize each newspaper sample, and note down how many grams I had to add to neutralize each sample.

Once all of the newspaper samples are neutralized, I will take measurements. Then, I will put the sugar sample in a pressure cooker and boil for about 5 minutes. This will kill other competing microorganisms. Next, I will add (5,10 or 15, still undecided, will decide after reviewing all references)g yeast to ALL samples, along with 1/2 a multivitamin pill and 1g of "soil acidifier plant food" (contains 30% nitrogen) to each sample. After that, I will take measurements everyday for about 5 days, plus hydrometer measurements. At the end of my experiment, I will do the freezing and boiling point test.

Thanks!

[donnahardy2]

Hi Irregular,

Good, it sounds like you received my message before you got to the next step. I think you can skip the measurements after the organosolv/hydrogen peroxide step; there is not any reason they should be different than before you started, and it will save time.

To do:

After the 24 hour organosolv/ hydrogen peroxide step, you should wring out the newspaper and put it into new distilled water. You don’t want the contaminants you removed from the newspaper and the hydrogen peroxide/ethylene glycol to be in the sample during the fermentation step.

I had mentioned a drying step because this would be a quantitative way to measure the actual amount of newspaper that you are using in your sample. If you weigh the 5 grams of newspaper at the beginning of the experiment, and then dry and reweigh the sample after the organosolv/hydrogen peroxide step, then you would know what weight of the newspaper would actually be in the sample at the beginning of the fermentation step. However, I realize that this will take too long and will delay your experiment, so I recommend not doing it.

The rest of your procedure still sounds perfect.

Donna Hardy

[irregular]

Hi Donna,

Already done:For my newspaper samples, I will cut and mash 10g of newspaper and add it to each sample, except in the sugar sample I will substitute 10g of newspaper with 5g of table sugar. Then for pretreatment, I will:

1) Acid samples: add 200ml of distilled water. add nothing to the acid hydrolysis samples.
2) Organosolv Samples: add 100ml of distilled water. heat the two organosolv samples until right before they boil. Then, 100ml of ethylene glycol (coolant/antifreeze) will be added to each sample.
3) Hydrogen Peroxide Samples: dilute 267ml of 3% hydrogen peroxide with 133ml of distilled water, to create a 2% hydrogen peroxide sample, and add this to the sample.
4) Sugar Sample: Add nothing.

Then, I will take volume, weight, density, pH, temperature, conductivity, salinity and TDS measurements. I will refer to all of these as "measurements". I will incubate the samples for 24 hours.

After 24 hours, I will remove them from the water bath and proceed to take measurements. Next, I will filter out the pretreatment solution from the newspaper and store it separately. The newspaper will be wringed out and placed in a container. Distilled water will be added to this container, and the newspaper will be rinsed out. Then, the distilled water will be filtered out from the newspaper and kept in another container. 200ml of fresh distilled water will be added to the original cylinder, and the wringed and rinsed newspaper will be added back to the sample cylinder. (Is it okay if there are some traces of ethylene glycol in the cylinder? I didn’t wash the cylinders out or wipe them because I would lose even more pieces of newspaper attached to the inner walls of the cylinder; a small quantity was lost in each sample due to the filtering, etc.) Then, I will add:

1) Acid Samples: add 26.5ml of 31.45% HCI to each
2) Organosolv Samples: add 26.5ml of 31.45% HCI to each
3) Hydrogen Peroxide Samples: add 26.5ml of 31.45% HCI to each
4) Sugar Sample: Add nothing.

I will incubate the samples again for 24 hours.

To Do:After 24 hours, I will have to do neutralization. At this point I would have made the red cabbage pH indicator with red cabbage and distilled water. I will pour approximately 20ml into ALL samples, or any amount as long as they are equal in each sample. Then, I will start adding NaOH to neutralize each newspaper sample, and note down how many grams I had to add to neutralize each sample.

Once all of the newspaper samples are neutralized, I will take measurements. Then, I will put the sugar sample in a pressure cooker and boil for about 5 minutes. This will kill other competing microorganisms. Next, I will add (5,10 or 15, still undecided, will decide after reviewing all references)g yeast to ALL samples, along with 1/2 a multivitamin pill and 1g of "soil acidifier plant food" (contains 30% nitrogen) to each sample. After that, I will take measurements everyday for about 5 days, plus hydrometer measurements. At the end of my experiment, I will do the freezing and boiling point test.

Thanks!

[donnahardy2]

Hi Irregular,

Did you stay up all night?

Your experimental protocol is excellent and I think you are on track for a successful experiment. After you add the cabbage juice, make sure the color is visible. To help with your data analysis and conclusions next week, it would be helpful to save a small volume, maybe 20 ml, from each sample to compare the freezing/boiling point results to. This will give you a before and after result for each sample. The freezing point test is going to be your best check for the presence of ethanol, so it is a very important analysis for your project.

Good luck! Let me know about your progress.

Donna Hardy