Artifical Photosynthesis

[donnahardy2]

Hi Irregular,

I think your evaluation of the pH of the solutions is correct. The newspaper is too acidic and the sawdust is too basic. This is a critical parameter for your experiment because the pH of the samples will affect the growth of the yeast. You can verify that the pH strips you are using are working correctly by also using purple cabbage juice. The purple cabbage juice should be violet/purple if it is in the correct pH range. Here are the Science Buddies websites with the detailed information on doing this.

https://www.sciencebuddies.org/science- ... p041.shtml

https://www.sciencebuddies.org/science- ... p013.shtml

You probably do need to add some acid to the sawdust and base to the newspaper before you add the yeast. The orange peel is perfect.

Here’s how to calculate how much acid to add to the sawdust:

A pH of 12 is equivalent to 10 to the minus 12th moles of hydrogen ions per liter of solution and a pH of 6 is 10 to the minus 6th moles of hydrogen, so you need to increase the concentration of acid in the sample by 100,000 (10 to the 6th) times. If you start with a 1 M (moles/liter) solution of the HCl (concentrate diluted 1 part acid plus 7.5 parts water), you would need to add 1 milliliter of the HCl into a liter of water (0.001 M hydrogen or pH 3) and then add 1 ml of this dilution to a liter of your sample to achieve a pH of 6. A milliliter is about 20 drops, so you can use 2 drops to equal 0.1 mL. I don’t know what the total volume of your sample is at this point, so you will need to adjust the volume of acid to make is proportional to a liter (1000 mL).

However, there are complications because of your sample, which contains lots of organic molecules. Organic molecules tend to buffer a sample to maintain the pH either high or low. I think this is the reason that the pH of your newspaper and sawdust samples are so far off after the hydrolysis and neutralization steps, and why I suggested using the purple cabbage juice. I think you are going to have to titrate your samples with either acid or base to get to the right pH range, and that’s why I suggested the purple cabbage juice. You can even add this pH indicator directly to your sample. You can check the color range of the cabbage juice, and then acid to the sawdust and base to the newspaper, a drop or two at a time, until the color is in the blue/violet range. Swirl the sample to mix is very well before adding more acid otherwise the pH will shift too far all at once.

Here is some general information on what buffers are:

http://www.ehow.com/about_4609033_what- ... istry.html


I hope this helps you to get your samples into the right pH range. Let me know if you have any questions.

Donna Hardy

[irregular]

Hi Donna,

Could you please open the following papers for me?

http://www.springerlink.com/content/8673497r0j642660/
http://www.springerlink.com/content/b1l82w8573gn2p53/

http://www.springerlink.com/content/n8270406246112n5/
http://www.sciencedirect.com/science?_o ... archtype=a
http://www.sciencedirect.com/science?_o ... archtype=a

Since I will be focusing on newspaper, it will be helpful seeing what needs to be done in that biomass sector.

I will get back to you on the other matters.

Thanks.

[donnahardy2]

Hi Irregular,

You found some good references. Unfortunately I don't have access to SpringerLink, but here's what I could get:

Donna Hardy

Bioconversion or Waste Paper pt1.pdf

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[donnahardy2]

bioconversion of wastepaper pt II.pdf

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[donnahardy2]

Bioconversion of waste paper pt III.pdf

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[donnahardy2]

Hi Irregular,

I could not open the second Science Direct link for some reason, but I did find another pertinent article.

Donna Hardy

Saccharification & fermentation newspaper pt I.pdf

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[donnahardy2]

Saccharification & fermentation of newspaper pt II.pdf

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[donnahardy2]

Hi Irregular,

This reference is on removing lignin inhibition with the use of surfactants; this is definitely important for your topic because newspaper has a high lignin content.

Donna Hardy

Lignin inhibition removal pt I.pdf

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[donnahardy2]

lignin inhibition removal pt II.pdf

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[donnahardy2]

lignin inhibition removal pt III.pdf

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[donnahardy2]

Hi Irregular,

I will read these papers also. Let me know if you are missing any critical information and I'll look for more references.

Donna Hardy

[irregular]

Hi Donna,

Thank you very, very much for your help accessing the papers, I will finish reading them by tonight. Do let me know if you find anyhting interesting. I am almost positive that I will be using the organosolv process and newspaper for my independant variable, but I still must refine the details.

Yesterday, after much experimentation with the NaOH and HCI I managed to neutralized the 3 samples within the 4-7 range. Then, I added 20g yeast. Ever since, the sp gravity has been decreasing, which is good. Do note that after the very first neutralization I had done about a week ago, I had already added 20g yeast after that but it probably died due to the high pH. For the nextcouple of days I took measurements when the pH was high and the yeast was dead, I noticed that the sp gravity was increasing. Is this something which happens to bases left over time? Why was this so?

A concern of mine is the filtering. My newspaper and orange peel samples have become such that every time I take a reading I must filter them. I take readings 2-4 times a day, so I can accumulate as much data possible. The filtering is a little time consuming, so do you think there is something I can or should do?

Thanks!

[donnahardy2]

Hi Irregular,

Are your samples producing lots of bubbles? If the yeast is growing well, it will produce 2 carbon dioxide and 2 ethanol molecules from each glucose molecule, so you should see lots of bubbles if the fermentation is proceeding as expected.

http://en.wikipedia.org/wiki/Fermentati ... chemistry)

I don’t know why the specific gravity increased in the one sample. Did it look like something was growing in the sample? If not, them maybe the high pH was continuing to hydrolyze the sample, increasing the concentration of sugar molecules in the sample.

If you can’t measure the specific gravity without completely filtering the sample, then I would reduce the number of measurements to once, or at most twice a day. I would be concerned that continual filtration would remove some the yeast cells and affect the fermentation process. It’s the end result that you are interested in, although I can understand why you want to see all of the steps involved in getting to the final result.

I did not finish reading the articles last night either, but I will start earlier tonight.

Donna

[irregular]

Hi Donna,

I have been seeing bubbles in teh orange peel and sawdust samples. The top of the newspaper sample has turned white. However the specific gravity hasn't been decreasing very much ever since this morning, 24 h after fermentation.

No, at the time I don't think anything was growing inside. It was probably continuing hydrolysis like you suggested.

Also, my orange peel sample has become very small. I'm not sure how, but at first it was filling the whole bottle, but now barely a quarter! It is so little that I can't even take a measure with the hydrometer after filtering it. What should I do?

Thanks!

[donnahardy2]

Hi Irregular,

Excellent, the bubbles are a very good sign. Since we know that sodium chloride inhibits yeast growth, it's possible that the yeast cells are just growing more slowly than they would if the samples were very low salt. But the bubbles indicate that you do have fermentation in your samples.

For your orange peel sample, measure the volume and then add enough deionized water to allow enough volume for the hydrometer. You will be diluting the sample, so you will have to adjust the calculations based on the dilution factor. For example, if you dilute your sample 1 part sample plus 1 part water, then your sample will only have 1/2 of the original sugar concentration. It's not exactly a controlled situation because you will be diluting inhibitors also, but I think it's better to get some type of measurement in your preliminary experiment.

I'm still reading the articles. It's a lot of information to get through.

Donna Hardy

[irregular]

Hi,

1) I would like to get started on my next set of samples this weekend. Which kind of samples should I run and how many? I could have a sample of newspaper with no hydrolysis, only fermentation, a sample of newspaper with the HCI pretreatment/hydrolysis, and a sample of enwspaper with the organosolv pretreatment/hydrolysis. What do you suggest, and what kind of proportions will I need for the organosolv?

2) I will need to use a pressure cooker for the organosolv process, but one paper says that inorganic acid catalysts are unecessary at temperatures above 185 C. I will need to figure out an organic or queous organic solvent to use, the paper indicates methanol, ethanol, acetone, ethylene glycol, triethylene glyrcol and tetrahydrofufurl alcohol.

3) One paper I found really really helpful, about woody biomass pretreatment, says that organosolv results in high quality lignin but recovers hemicellulose poorly "because of sugar decomposition at high terperatures in the presence of acid. The hemicellulose sugars dissolved in the water-soluble steram are not fermentable without extensive detoxification".


I'm hoping I can contribute to the cellulosic ethanol field by doing some new work. What do you think about the three thigns I mentioned above?

4) I didn't completely understand the paper you sent me about the kinetic model for lignin elimination. Some of the terms are quite advanced.

5) I'm very confused with my readings at the moment. Why are my gravities higher than 1? Have I not made ethanol?
Orange Peel
Volume:106ml
Weight: 114.4g
Density (weight / vol): 1.079
Hydrometer reading: 1.072
(Note that I didn't add the extra water yet which you suggested in your last post)

Newspaper
Volume: 480ml
Weight: 484.3g
Density: 1.008
Hydrometer reading: 1.038

Saw Dust
Volume: 250ml
Weight: 258.1g
Density:1.048
Hydrometer reading: 1.048

I did these calculations so I could find out what gravity I should have (http://www.newton.dep.anl.gov/askasci/g ... n06645.htm)

Thanks!

[donnahardy2]

Hi Irregular,

I think your goal for setting up the next experiment next week-end is a good plan. Your questions are excellent; I'm so glad you did the pilot experiment.

1. How many containers fit into your water bath? 3? If you can run 3 samples at a time, I recommend testing acid hydrolyzed newspaper, organosolv-hydrolyzed newspaper, and then a positive control that uses sugar as a substrate instead of newspaper. The result on the positive control would represent the maximum concentration of ethanol that could be produced, and you could compare results of the two experimental samples to those results. For purposes of your experiment, you could assume that you would not get a significant quantity of ethanol without hydrolyzing the newspaper, but if can run 4 samples at a time, then you could run the negative control as well.

The Wayman paper from Process Biochemistry gives the best details for setting up the samples. They used 2-20% paper. Your first samples were apparently too viscous, so maybe you should use less paper this time, maybe in the 5-10% range; that would be 5 to 10 grams of newspaper per 100 ml of water. The authors also added YMP (0.3% yeast extract, 0.3 % malt extract, 0.5% peptone) or DAP (diammonium phosphate, 0.02% urea). Do you have anything in your kitchen that contains some pure soluble protein and a few B vitamins that would be comparable? Here is a website that describes how to make a protein extract from raw ground meat:

http://www.disknet.com/indiana_biolab/b029.htm

Please note that you do not want to add salt or any other carbohydrate (sugar) source to any of your samples. You could add purple cabbage extract to your growth medium to have a visual pH indicator in all of your samples.

The Wang paper studied the effect of adding polymers and nonionic surfactants on the enzymatic saccharification of cellulose, and found that adding polymers helped with the conversion of cellulose to glucose. You are not using enzymes to hydrolyze the cellulose in your samples, but this paper does give include some interesting ideas for future experiments.

2. For your organosolv process, I don’t think we have found a reference that would indicate an advantage of one solvent over another, so any of the solvents you mentioned would be suitable. Since you will be producing ethanol as an end-product, it would make sense to use ethanol if you can get this. Otherwise, use whatever you can get.

The temperature for your pressure cooker should be 121 degrees Centigrade
http://en.wikipedia.org/wiki/Pressure_cooking

3. The second page of the Saxena paper lists the composition of the newspaper they used in their experiments. They were 85% cellulose, 12% lignin, 2.7 % moisture, and 2.3 % ash. We know that the lignin can inhibit fermentation, but you don’t need to worry about hemicellulose, which is found in wood samples. You might consider contacting your newspaper supplier and ask if you could get the composition of the paper being used. However, if this is not possible, then you should assume that your newspaper has a similar composition as the sample used in this paper.

4. Don’t worry about the lignin elimination paper for now.

5. I am assuming that your values are correct. Do you have a weight standard to verify that your balance is giving you a correct reading? If not, then weigh something at home and take it to school tomorrow and measure it on another balance, preferably a balance that has been calibrated recently. Are you using a graduated cylinder for the volume measurements and are you measuring from the bottom of the meniscus?

Did you do the measurements at the beginning of the experiment, or are these reading the measurements after the fermentation? The specific gravity measurements for alcohol assume there is nothing else in the sample except for alcohol and water. The production of alcohol will decrease the density of a solution. Since your samples contained a high concentration of solids to begin with, maybe the density was much higher than 1.0 to begin with and has decreased. It’s possible that you won’t be able to use absolute values, but maybe you will need to rely on a change of values. For your next experiment, I recommend that you plan to measure the density of the freshly prepared sample, of the hydrolyzed sample, and of the sample again at the end of the fermentation process. Using the positive control of sugar should result in the production of ethanol, and you can verify that your density measurements correlate with your results. This will allow you to compare the density of the samples before and after fermentation.

The link you found for doing the density calculations is excellent. Thanks for sharing it. Did you understand the algebra part of the equation, which is used to convert the density measurements into % ethanol?

Donna Hardy

[donnahardy2]

Hi Irregular,

Here's information for how to read volumes in a graduated cylinder. You may already be doing this, but it's an important detail for your project:

http://www.youtube.com/watch?v=D_g0U3vxr18&NR=1

http://www.youtube.com/watch?v=D_g0U3vxr18&NR=1

This website describes how to calibrate a balance. Since you moved your balance from school to home, it may have lost calibration. You should be able compare the weights of 3 objects measured on your balance and with a reference balance (at school or at your Dad's work) to verify your balance is correct. If calibrated test weights are available, maybe you can borrow those for a day to check your balance.

http://education.ezinemark.com/how-to-c ... 20c9f.html

Donna Hardy

[irregular]

Hi Donna,

1) My water bath is quite large. In this coming experiment I will do 2 samples of acid hydrolysis and 2 samples of solvent (organosolv) hydrolysis(2 for repeatability), 1 with sugar as the substrate and no hydrolysis. I will need to cut down my newspaper to water and acid/solvent to 200ml this time, instead of 300ml. I will be using 5g newspaper/100ml water, but we might do 10g newspaper/100ml if that's too less. The experiment I just finished we used 100g newspaper with 1 270 ml water.

For acid hydrolysis, you said I would need a 300ml of 1:8.5 ( 7.5 part water and 1 part 31% HCI) for 1 M HCI. I will adjust this to 200ml.
For organosolv, what proportions should I use of the solvent to water? I will be using ethylene glycol as my solvent since I have some at home in the form of antifreeze coolant.For the sugar as a substrate, the sugar will represent the newspaper, right? So if I just add either 5g or 10g per 100ml water, whatever I decide upon.

2) I'm not sure if I will be able to addthe YMP or DAP protein, probably not, but I am considering adding the purple cabbage extract. Also, you said that I shouldn't add any salt/carbohydrate/sugar to my samples, however, I will need to add the NaOH to the samples when I neutralize them. What should I do?

3) How much time should I let the organosolv sample cook in the pressure cooker? The Garcia paper says 90mins at 160 C, the Pan paper says 30-60min at 185-198 C, the Zhu paper said that that a previous Pan paper cooked for 60mins at 180 C. Since I'm using the pressure cooker for the organosolv sample, should I do so with the acid sample as well? If yes, how much time should I cook that for?

4) I am using a graduated cylinder for the volume measurements. I will be extra conscious when taking my readings, that I am reading them properly. I will measure the density at the times you indicated for my next experiment as well. And yes, I did understand the algebra. I will try to calibrate my weighing scale as well.

5) I've reviewed my papers and searched online, and I haven't found any paper which deals with using the organosolv process to hydrolize newspaper. Could you please confirm this for me?

Thanks!

[irregular]

Hello,

I just purchased some acetone as well.

What would be the proportions if I was using that as a solvent?

Thanks!

[donnahardy2]

Hi Irregular,

Here is a reference using organosolv and newspapers, but I don’t have access to it. The abstract is intriguing, however:

https://springerlink3.metapress.com/con ... erlink.com

Yes, 5 to 10% newspaper would be your source of sugar for the experiment.

I am attaching another reference by Park et. al, from Bioresource Technology, that includes the details of the organosolv process. The authors describe using the organosolv process on pinewood with various catalysts. They used a 10% wood mixture; 20 grams of wood in 200 ml of ethanol: water, and used a 50% mixture of ethanol and water. They used temperatures ranging from 150 to 210 degrees C for 50 minutes and added either 1% sulfuric acid, 1% magnesium chloride, or 1% sodium hydroxide as a catalyst. The authors found that sulfuric acid worked best as a catalyst at lower temperatures. You can read the details.

50% organic solvent is a high concentration to work with if heated and would be an extreme safety hazard if used over an open flame or unventilated room.

http://www.pharmco-prod.com/pages/MSDS/ ... 50_MIX.pdf

So, please don’t plan to use the acetone yet until you can make sure it will be safe.

Ethylene glycol is less flammable, but heating it would release fumes.

http://www.jtbaker.com/msds/englishhtml/e5125.htm

Do you have access to an autoclave either at school or at your Dad’s work? We need to figure out a safe way for you to do this part of the experiment. I will do more research on the question also.

The organosolv process is a pretreatment method for breaking down plant cell walls to make the cellulose accessible to enzymes that are used to break down the cellulose to glucose. If you use just the organosolv process, you still need to hydrolyze the cellulose. For your experiment, you could use the acid hydrolysis since you don’t have any cellulase enzymes.

You need to add some additional nutrients to the samples. Saccharomyces cereviseae requires a nitrogen source and a few vitamins and minerals. Malt extract, Brewer’s yeast and some protein of some sort will allow the yeast to grow and utilize the sugars.

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

You will have to add the NaOH to the samples to neutralize the acid; what I meant was that you shouldn’t add any extra sodium chloride from other sources.

Running your experiment in duplicate is excellent.

Donna Hardy

organosolv ref pt 1.pdf

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organosolv ref pt 1.pdf

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[donnahardy2]

organosolv part II.pdf

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[donnahardy2]

organosolv part III.pdf

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[donnahardy2]

organosolv part IV.pdf

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[irregular]

Hi Donna,

Last night I created the samples - 4 samples with a 10g newspaper/200ml water solution. I cut up the newspaper into small pieces, but even after adding the water it was too viscous to test for sp gravity. Also, all my newspaper samples have a difference in density, conductivity, salinity and TDS. I added and weighed the water and newspaper and sugar carefully. I don't want this difference to affect my experiments, what should I do? I also created the 10ml sugar/200ml water solution. To help them decompose and mix, I left them in the water bath at a 26 C temperature.

For the acid hydrolysis samples, I will have to add approx 26.5ml HCI with the 200ml water&newspaper, to have the appropriate dilution, right?
For the organosolv samples, I would have to add 200ml acetone/ethlyene glycol with the 200ml water&newspaper, right?I would leave the sugar sample until the other two have been hydrolyzed, and then ferment at the same time.

It's very unfortunate that the process ends up being pretty dangerous, since I won't be able to gain access to an autoclave. What if I try the organosolv process at a lower temperature like with the acid, I can put my water bath upto 30 C or a bit more. If not possible, another option, what if I used oxidative delignification instead of organosolv, therefore adding hydrogen peroxide?

For the yeast, could I just buy yeast nutrient which was being sold beside the yeast at the grocery store?

I asked if there was a paper using organosolv and newspapers because I was hoping that it was the factor that would make my project original. It turns out that it doesn't happen to be so though. How can I make it end up being original in a certain way?

Thanks for all your help!

[irregular]

Hi,

Some of my water evaporated from my samples, so now I will be keeping everything anaerobic. I also added water to each of my samples until they reached 210ml. I will adjust my dilution and measurements of the acids/solvents. I also just realized that I forgot to mention the amount of acid for the organosolv sample, which is 1%. My samples are still too viscous, so I will be calculating all of my gravities my density. My gravitites continue to differ slightly, I will test the TDS/conductivity/salinity once again.

It's very unfortunate that the process ends up being pretty dangerous, since I won't be able to gain access to an autoclave. What if I try the organosolv process at a lower temperature like with the acid, I can put my water bath upto 30 C or a bit more. If not possible, another option, what if I used oxidative delignification instead of organosolv, therefore adding hydrogen peroxide?

For the yeast, could I just buy yeast nutrient which was being sold beside the yeast at the grocery store?

I asked if there was a paper using organosolv and newspapers because I was hoping that it was the factor that would make my project original. It turns out that it doesn't happen to be so though. How can I make it end up being original in a certain way?

Thanks!

[donnahardy2]

Hi Irregular,

If you set up 4 identical samples with 10 grams newspaper in 200 ml water, the results for density, conductivity, etc. should be identical. If you are getting different results, this must represent the variation in your test method. Can you post your actual results so I can see what your data looks like for these samples?

Yes, you will either add 26.5 ml of your acid to 200 ml of water to make a total of 226.5 ml , or you can add 23.5 ml of acid to 176.5 ml of water to make a total of 200 ml.

I apologize, but I had not considered the safety hazard of using the organic solvent in the pressure cooker when I originally suggested it. You could use the organosolv method by using the ethylene glycol, or perhaps some propylene glycol by heating a pan of water on the stove, turning the heat off, and then adding the sample in a flask and letting the water cool off. Perhaps two or three cycles of hot water would work for your sample. Propylene glycol would offer an advantage because it is less viscous and it would not be toxic if accidentally spilled and consumed by a pet. If you do use ethylene glycol, make sure to keep any pets out of the room when you do your experiment. The acetone has a low flash point, so you should not plan to heat it at all; it is so volatile that the vapors would ignite if exposed to a spark or open flame. We will look for another ambient temperature use for the acetone.

I will have to read about the hydrogen peroxide protocol again, but this might be a safer option for an experiment.

Is this the yeast nutrient available at your store? If so, it is exactly what you need to feed your yeast. Yeast actually prefer ammonium ions as their source of nitrogen, so this would be perfect. The directions call for using ½ to 1 tsp per gallon. You can weigh what you use.

http://www.eckraus.com/NUT110.html

Your project is original because you are using information from various references and trying to get to the next level on this topic. You will be able present data that will be unique and original. The fact that there is only one paper on this topic suggests that additional research is needed, so you may be able to make a significant contribution.

Donna Hardy

[irregular]

Hi Donna,

It's okay, I realize that safety is important, so hopefully next year I'll be able to gain access to a lab! I used the method you indicated for boiling for the organosolv method, and two days ago I started the hydrolysis. Yesterday I took the containers out of the water bath to neutralize them, but when I added the NaOH a bit at a time, it would suddenly become alkaline, so I had to add HCI, but then it would become suddenly basic. In the end, I used up a lot of NaOH, a lot of HCI, a LOT of time, a lot of pH papers but only the acid hydrolysis sample neutralized, I had to abandon the second acid hydrolysis sample because it wouldn't become neutral. So I left all of the containers out overnight. Today I will make the pH indicator from red cabbage and neutralize the rest of the samples. Then, I will add yeast.

I went to the grocery store, but I was sadly mistaken and the nutrient wasn't available. What kind of at-home things could I use for the nutrients/minerals that would work for the yeast? I will ask my parents at home if we have anything with nitrogen.

Thanks!

[donnahardy2]

Hi Irregular,

You experience of trying to neutralize the sample is typical. The cabbage juice will give you a visual indicator and you can add the base 1 drop at a time so the sample doesn't go suddenly too basic. You can dilute the base 1:10 or so when the pH gets close to the endpoint.

You can use anything with protein in it that will dissolve in water and give a relatively clear solution. The homemade broth I suggested in an earlier post used ground beef cooked in water to extract the soluble protein. You could boil any type of beef or other meat in plain water and let the water cool and skim off the fat. The beef broth would be perfect for the yeast and you could eat the meat for dinner. If you could find a commercial beef broth that didn't have added salt, that would work also.

Do you have any garden fertilizer that is in dry pellet form? If so, look at the label and let me know what the composition is. I'm sure you will have something available to use.


Donna Hardy

[irregular]

Hi Donna,

I will check if I have garden fertilizer.

As for protein, I don't think I have the time to leave the meat overnight as suggested in the procedure you sent. I have some One A Day Advanced Multi-Vitamin pills at home, which contain vitamins and minerlas, but they don't have protein in them. Will this work? However, I will follow the procedure you sent but skip that step, if the pills don't work.

I made the red cabbage pH indicator, and was able to successfully neutralize my samples. I then added 20g yeast. Do you think this is enough yeast?

Also, I wanted to confirm with you, does theyeast fermentation to ethanol have to be anaerobic?

Thanks.