Artifical Photosynthesis

[donnahardy2]

Hi Irregular,

You are really making good progress, and you are very wise to have a pilot experiment planned while you are finalizing your independent variable.

Cellulose is a polymer or long chain of glucose monomers. The yeast cannot break the cellulose bonds (neither can humans), so the plant material must be broken down so there will be glucose to feed the yeast cells.

http://en.wikipedia.org/wiki/Cellulose

http://en.wikipedia.org/wiki/Glucose

Cellulose can be hydrolyzed to glucose either by acid hydrolysis in strong sulfuric acid or by cellulase enzymes. Even with these methods, the lignins and other plant molecules must be removed. If acid hydrolysis is used, then the sample has to be neutralized before the yeast can grow in the sample. Some methods of hydrolysis leave yeast growth inhibitors that must be removed before the yeast can grow.

http://www.eng.umd.edu/~nsw/ench485/lab4.htm

The article you found is a good one and gives details of the hydrolysis method. Were you thinking about doing this method and a variation that would improve results? Did you get a chance to read the article that I attached last week? It includes a review and brief description of several hydrolysis methods, so you should look through all of the methods before you make a final decision. There are problems inherent in all of the methods, so research in this topic would be a really good idea. What idea do you have that would improve any of these methods.

What size are the containers that you are using? Here are a couple of typical experiments to give you an idea of the proportion of glucose, water, and yeast:

http://www.practicalchemistry.org/exper ... 09,EX.html
http://spot.colorado.edu/~kompala/lab2.html

You need to plan to have enough volume to remove samples for analysis periodically.

Donna Hardy

[irregular]

Hi Donna,

I just started my test to make sure that the water bath works - I have my aquarium thermometer, aquarium heater and mercury thermometer in. Hopefully the temperature increases soon.

Okay, so that means that the acid hydrolysis can substitute for the enzymes as well, but the pentoses are left unfermented (hemicellulose to pentoses to ethanol). I noticed in the Wikipedia article on Cellulose: "Processes do exist however for the breakdown of cellulose such as the Lyocell process [21] which uses a combination of heated water and acetone to break down the cellulose strands." Acetone can be bought at hardware stores.

The article you attached previously was actually the article that gave me the idea of organolv and alkaline hydrolysis (I had seen that article previously and actually wanted you to check it out as well, it's in a website PDF format right before the post where you sent me the link). I will review all the articles I have on hydrolysis before making a final decision and reply back in my next post.

My water bath container is 12 by 21 by 16 inches. However my beakers, where the fermentation will occur, are 500 ml.

Thanks!

EDIT: At what intervals do you suggest I take measurements? Different student experiments I've read have different intervals. So, I will be conducting 1 experiment for 5 days. For the first 3 hours of each experiment, I'll measure and test every 15 minutes. There on, I'm not sure every "so many" hours I should take measurements. Maybe 2-3 hours? I must also consider time lost while at school or sleeping. Will I have to take out a sample periodically or can I just measure inside the beaker?

[donnahardy2]

Hi Irregular,

Your aquarium water bath incubator sounds perfect. What temperature are you getting now?

I read the details on the organosolv hydrolysis process last night, and I understand why you chose this method. Thanks for confirming that you had reviewed all of the other methods. Whenever I see multiple methods for a protocol I know there are problems and room for improvement if the technical problems can be resolved, so I’m glad you are working on this problem. The organosolv method sounds like it has several advantages.

It will take the yeast a while to start growing after you inoculate the growth medium. I would recommend testing every 6 to 12 hours for the first trial. Depending on the temperature, the lag time will be a few hours and then the yeast will grow in log phase for about a day and then the metabolism will change when the yeast gets to stationary phase. A 5-day trial sounds perfect.

Since the alcohol test strips are qualitative, you can try making a dilution of the sample, maybe a 1:10 and a 1:100 dilution after the samples turn positive with the undiluted sample. This might give you a ballpark idea of the ethanol concentration in your sample. Were you able to find a hydrometer to use to measure the specific gravity?
Is there a refractometer available that you could use? This would be helpful to measure the decrease in sugar concentration in your samples. Since you are using test strips, you can test directly in the sample, but it would be best to use a sterile pipette or other device to transfer the sample to the test strip. This will help prevent contamination of your sample.

What type of plant material are you going to use?

Donna Hardy

[donnahardy2]

Hi Irregular,

Here is a growth curve for Saccharomyces cerevisiae that shows a lag time of about 8 hours, a log phase of about 24 hours, and a stationary phase continuing past 80 hours. Eventually the culture will reach death phase as the food supply is depleted and the ethanol and other waste molecules accumulate in the culture. Of course this growth curve could vary depending on the initial concentration, temperature, and type of growth medium:

http://www.ggause.com/gfg04.htm

But it appears that you could do a sample every 6 hours for the first day and a half, and then test every 12 to 24 hours after that.

Donna Hardy

[irregular]

Hello Donna,

At 8am this morning I got 13 C, at 4pm 15 C, and at 7pm about 16 C.

I think I wrote back to you using the wrong words last time, I meant that I would review all of the methods after that post and before this post. As I was most interested in organosolv, I was reading the paper I sent previously to you about the organosolv process, and I realized that required temperatures for reaction are very high - 130C. I'm not sure how this will be possible. I went back to the original paper with all the hydrolysis methods, and noticed that two other possibly do-able methods would be alkaline hydrolysis and oxidative delignification. For alkaline hydrolysis, would it be possible to open a battery which has stopped using, instead of throwing it out, extract the alkaline and using it for the hydrolysis? For oxidative delignification, wouldn't it be possible to buy a cleaning agent which contains hydrogen peroxide?

Thank you for indicating how often I should take my samples and giving me that reference. If I take some of the ethanol out of breaker every time for a sample, the amoung inside the beaker will decrease, won't this affect my data since the volume doesn't stay the same?

The alcohol test strips, I haven't bought yet, but we got pH strips from my dad's work. It will work the same purpose, however my dad says it will be better to buy coolant strips since they are cheap and we can give most of the pH strips back. I will look into that soon. I bought a hydrometer, and it measures starting at 0.89. However, ethanol is supposed to be 0.789 in gravity. Will the refractometer work the same purpose as the glucose strips, which I have?
My cellulose source will be recycled newspaper.

Thanks!

EDIT: I couldn't obtain the refractometer at school, and don't want to buy it since it's about $150. Will the glucose strips work instead?
EDIT 2: Are there any other parameters you suggest which I should measure, other than glucose content and ethanol measurement? I will read my papers and try to find out more.

[donnahardy2]

Hi,

Your water bath incubator is lower than the optimum 30 to 32 degrees Centigrade needed for yeast growth. If the temperature doesn’t go above 16 degrees, then your yeast will grow, but much more slowly than I had predicted yesterday. It might take 48 hours to get through log phase growth, so maybe you should do a sample every 24 hours or so.

Do you have a pressure cooker available? You can cook your cellulose in a pressure cooker and will reach at least 120 degrees Centigrade. Several of the methods used this technique to help break down the plant cells and extract the cellulose. The researchers probably used an autoclave, and they released the pressure on the autoclave quickly to create a pressure /vacuum shock, but you could do something similar with a pressure cooker. Hopefully, newspaper won’t smell too bad when it is cooked.

For the base needed for alkaline hydrolysis, I’ve never taken a batter apart. Have you tried this? How much alkaline solution does one battery contain? I am not finding the exact conditions for alkaline hydrolysis right now, but I will look and see if there are details in any of the articles we have.

The pH strips will be useful to monitor acid produced from the fermentation. It might be useful to try a few and see how quickly the sample becomes acidic.

The refractometer will measure total dissolved solids, including the glucose. The glucose strips will give you the specific information you need.

In addition to the ethanol and glucose, you could include the pH measurements and record the temperature, since that will be a critical parameter. I’ll see if I can think of anything else.

Donna Hardy

[irregular]

Hello Donna,

We bought another aquarium heater, the mercury thermometer indicates 18 C and the aquarium strip thermometer indicates 21 C. We are waiting for it to reach 25 C, since that will be the constant temperature (I decided that by http://www.sre.urv.es/web/amb/Webgrup/Y ... ja2002.pdf)

Yes, I will be able to use a pressure cooker. I've never tooken a battery apart either, I've tried finding some information about alkaline batteries either. I'm trying to find information on organolsolv and oxidative delignification, but I'm not very successful.

Some questions:
1) Papers say that acid hydrolysis should take 2 hours, and then hydrolysis should be stopped by neutralizing the acid. How do I know exactly when the hydrolysis IS complete (by taking samples, but measuring what?), and how do I neutralize it (by adding what?)?
2) Will it be necessary that I preform fractional distillation? Since my variable will be the pretreatment/hydrolysis, all fractional distillation will do totally extract the ethanol, when I'll have a rough idea already.
3) My total mixture for fermentation will be 450 ml - 20 g for yeast, 430 for water+acid+newspaper. I have found two sources which indicate proportions, and have done the math to make the total approx 430 for the hydrolysis.

source 1 (http://www.odec.ca/projects/2007/cheu7j2/materials.html - materials and procedure section)
90ml acid
180ml water
155g (2/3 cup) lignocellulosic biomass

source 2 (http://www.eng.umd.edu/~nsw/ench485/lab4.htm)
126ml acid
252ml water
52ml lignocellulosic biomass

Both proportions differ greatly, which should I use?
Thanks!

[donnahardy2]

Hi Irregular,

An additional aquarium heater will help speed up growth of the yeast. However, the temperature you have will work, so go ahead and start the experiment even if the temperature is quite warm enough. 25 degrees Centigrade is an excellent temperature for yeast if you can do this.

I found a review paper on alkaline hydrolysis of cellulose and I’m reading it. I will post it tomorrow if it looks like it will be helpful .

It’s hard to find the information you need about alkaline batteries. Potassium hydroxide (KOH) is used, but I can’t find the volume or concentration.

http://www.ehow.com/facts_7261064_alkal ... work_.html

You might have to take one apart (carefully using gloves and safety glasses) and pour out the liquid and measure the volume and the pH.

Here’s an MSDS for an alkaline battery, which includes the composition, 35% KOH. This would be 35 grams of potassium hydroxide per 100 ml volume (water) KOH has a molecular weight of 56 grams per mole. Have you had any chemistry this year? Do you know how to do this type of calculation? If not, let me know and I’ll explain more.

This is equivalent to 6 M KOH, which is a very concentrated solution. Be very, very careful with KOH at this concentration. If you get a drop on you, rinse with lots of water immediately. Read all of the safety information in this document.

http://www.alliedelec.com/Images/Produc ... 370520.pdf

Answers to your questions.

1. You will neutralize the sample using potassium hydroxide. KOH. This is usually sold in dry pellets, and you would make a fairly concentration solution to add to your sample. Can you obtain KOH? If not, do you have access to any other basic materials? Drain cleaners contain sodium hydroxide, but you would have to find a brand that did not have any other additives that you inhibit yeast growth. Knowing the concentration of KOH would be critical for your experiment.

2. I do not think you need to do the distillation step. You will have the test strips, and I think you will be able to make dilutions so you can get quantitative results. The distillation step would be a good addition to your project, but you should concentrate on all of the other details for now. There’s a lot for you to do.

2. You have excellent questions. The first source uses muriatic acid, which is about 17% HCl (17 grams HCl/ 100 mL); the second source uses 5% HCl or 5% sulfuric acid, so the concentration of acid is similar. I believe that sulfuric acid is usually used for cellulose hydrolysis, and I don’t know if you can obtain that. Sulfuric acid is sold as battery acid. I would use 5-6% sulfuric acid, if you can obtain this acid.

Donna Hardy

[irregular]

Hi Donna,

We bought a 200-watt aquarium heater, and hopefully it heats up buy tomorrow.

Thank you for the information on alkaline hydrolsysis. Unfortunately, I haven't learned any chemistry in school this year and am unfamiliar with the calculation, please do explain the chemistry. I will continue finding information myself.

I will see how I can find KOH, or sodium hydroxide. I found out that it's also known as lye or potash, used in farming.

I had bought muriatic acid previously from the hardware store, since it is slightly milder than sulfuric acid and available in stores. Will I still want to use 5 grams of muriatic acid for every 100 ml, or 17 grams of muriatic acid for every 100 ml?

How will I know when my hydrolysis reaction has finished, is there something I have to measure at intervals? Papers say that it should take at least 2 hours, but my reactions will be done at 25 C, which is a much lower temperature compared to the temperature used in 'dilute acid hydrolysis'.

Thanks!

EDIT: I've called many, many places and asked for sodium or potassium hydroxide in the form of caustic soda, lye or potash. No one carries some, but my dad's plant has some, although we were hoping to start the experiment this weekend..

My muriatic acid is 20 Baume commerical, 31.45%/

If the pH of HCI is 2, then to make it neutral I need a base of 9. Potassium and sodium hydroxide are in pH from 12-14, so can I use a base like borax? (http://www.engineeringtoolbox.com/bases-ph-d_402.html)

[donnahardy2]

Hi Irregular,

If you visit a farm supply or garden supply store, you should be able to buy lime, which is calcium carbonate. This will work to neutralize your acid hydrolyzed samples. When you add carbonate to acid, carbon dioxide will be formed and a lot of bubbles will be formed. So you will need to add it slowly so your sample doesn't bubble over. If you add the lime to the sample just to the point where no more bubbles are produced, this should give you a neutral pH solution, although you should check this with pH paper.

http://en.wikipedia.org/wiki/Agricultural_lime

I did not comment on your proposed volumes yesterday; the proportion is good, but I think you should reduce the total volume to 300 ml in a 500 ml flask. You need room for bubbles.

Your muriatic acid is 31 grams of HCL per 100 ml. Hydrochloric acid is composed of one atom of hydrogen and one atom of the element chlorine. Hydrogen has an atomic mass of one and chlorine has a mass of 35.5, so the total atomic weight, or molar mass is 36.5 grams. Chemists prefer to describe the concentration of chemicals in terms of moles per liter because this describes the number of atoms of an atom or molecule in solution. One mole of a compound contains 6.23 x 10 to the 23rd atoms or molecules. Your HCl contains 310 grams HCL per liter, or 310/36.5 or 8.5 moles per liter.

http://en.wikipedia.org/wiki/Avogadro_constant

http://en.wikipedia.org/wiki/Hydrochloric_acid

Sulfuric acid has a chemical formula of H2SO4, so has twice as many hydrogen ions compared to HCl. The concentration of H+ or hydrogen ions determines the strength of an acid. So a 5% sulfuric acid will be twice as strong as 5% HCl.

http://en.wikipedia.org/wiki/Sulfuric_acid

Most of the references I have seen use a 5% or 0.5 M concentration of sulfuric acid, although I have seen 6 M sulfuric acid protocols also. Cellulose is hydrolyzed more quickly at a higher temperature and with a higher concentration of acid. To obtain the same concentration of hydrogen ions using HCl, you would need a 1 M solution, so you would dilute your HCl 1 part HCl plus 7.5 parts water, or 1:8.5.

The rate of hydrolysis is approximately doubled for every 10 degrees increase in temperature. The protocols you have found range from 40 degrees to 160 degrees C for the hydrolysis step. Since it will be safer to use a lower temperature, I recommend that you use 1 M HCl and incubate it overnight and plan to observe the integrity of the cellulose at intervals every two hours or so to observe how quickly the cellulose dissolves. You will want to start with very small pieces of newspaper, shredded as finely as possible.

I am little concerned about using the HCl instead of sulfuric acid. Every commercial process I have seen described dating back to the early 1900’s uses sulfuric acid to hydrolyze cellulose. The Cl will remain in the glucose sample and a high concentration of Cl will inhibit yeast growth, so maybe that’s the reason that sulfuric acid is used. HCl is also more corrosive, which means that it reacts with metals and turns iron to rust very quickly. But I really don’t know why the commercial sites don’t use HCl. Maybe you will find out.

Donna Hardy

[donnahardy2]

Hi,

I forgot to answer the question about borax. Your research skill are excellent in identify possible solutions to the problem of neutralizing the acid! Yes, borax would work to neutralize the sample. However, boric acid is a classic antimicrobial solution used to treat infections, so I think it might also be toxic to yeast. You can't add anything to your sample that will inhibit yeast growth, and that is also one of my concerns about using HCl; it will add Cl to the sample.

Donna Hardy

[irregular]

Hi Donna,

Thank you for the information. I'll see what I can do in terms of calcium carbonate. So the borax and chlorine in HCI will inhibit yeast growth? The HCI could end up being a problem then.. What are other things inhibit yeast growth? Is it the pH?

How exactly should I "observe the integrity of the cellulose"? I am not exactly sure what you mean.. Do you mean that I stay up overnight and every 2 hours I should use the pH strips? I was thinking about shredding the newspaper, and then blending it so it becomes liquidy.

Thanks.

EDIT: I obtained the sodium hydroxide as 100% lye Roebic drain cleaner. I took pH tests with the coolant and pH strips of alcohol, glucose, water, sodium hydroxide and muriatic acid. I will be testing the glucose strips tonight. The water bath is at perfect temperature right now, I will monitor it so it maintains the temperature.

Do you have the paper on alkaline hydrolysis you were talking about? I would like to finalize my variables in the next few days.

[donnahardy2]

Hi Irregular,

Your idea to use the blender to break the newspaper down to very small particles is a good idea. You can blend your sample in water and then dilute it with acid to the correct concentration. If the cellulose is then hydrolyzed, the cellulose fibers will be broken down to individual glucose units, so the solution should become clear. I’ve never done this, so you will have to check your sample and observe the visual differences between an unhydrolyzed and hydrolyzed sample. Don’t get up every two hours; just check your sample before you go to bed and first thing in the morning. You may need to do an overnight hydrolysis instead of the standard two hour hydrolysis since you will be using a lower temperature.

If you use sulfuric acid to hydrolyze the newspaper and calcium carbonate to neutralize the sample, you will get a precipitate of calcium sulfate, which has limited solubility in water: If you put the sample outside in the snow for a little while, even more would precipitate. You could then filter the sample through a coffee filter to remove the calcium sulfate and you would then obtain a glucose solution with a little calcium sulfate in it. Calcium sulfate should not be particularly toxic to yeast growth, but you could verify this by growing yeast with and without calcium sulfate to determine the exact effect.

http://en.wikipedia.org/wiki/Calcium_sulfate

Here’s a bakery website that contains useful information about yeast and ingredients that inhibit yeast growth. Here’s the information on calcium sulfate:

Calcium Sulfate search for term
A compound of sulfate and calcium that is used to fortify breads and other baked products with calcium and also as yeast food. Calcium sulfate has minimal impact on yeast activity and is thus a preferential source of calcium in yeast raised products.


http://bread.com/glossary/9/letterc

Calcium chloride is very soluble in water and chloride levels above 1% will inhibit yeast growth.

http://en.wikipedia.org/wiki/Calcium_chloride

How are you going to measure your acid? Here is critical safety information for mixing acid and water. You will want to put the water or water-newspaper in the container first, and then pour the acid into the water. Mixing acid and water is an exothermic reaction, so the solution will get warm.

http://chestofbooks.com/crafts/popular- ... -Acid.html

'The 100% lye sounds very good; this will work well to neutralize the acid.

The alkaline hydrolysis paper is attached, or at least the first few pages. This is a review paper and is helpful for understanding the chemistry of the process. Read the first few paragraphs and then skip to the page that describes hydrolysis of cellulose. I would not expect you to understand everything since you haven't had chemistry, but let me know if you have any questions. This is not a how-to paper, so we still need another reference.

Donna Hardy

alkaline hydrolysis of cellulose part I.pdf

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[donnahardy2]

alkaline hydrolysis of cellulose part II.pdf

(181.75 KiB) Downloaded 433 times

[donnahardy2]

alkaline hydrolysis of cellulose part III.pdf

(142.41 KiB) Downloaded 444 times

[irregular]

Hello Donna,

When I blend the newspaper with water, what ratio should I have?

I willl have to use HCI and the sodium hydroxide for my base experiment, we couldn't obtain sulfuric acid.

I tried using the glucose strips last night. We made a solution of sugar and water and figured out the mmoles/g, but then when we went to test them we got abnormal results, we kept getting a "LO" sign. We used up 4. Today I realized that I've been using the wrong glucose test strips - the ones for blood, not urine. However, I found out that I can find the sugar level in a solution using the hydrometer instead of using glucose strips for urine. Will the sugar level (sucrose) be the same as the glucose level?

http://mtngrv.missouristate.edu/MWFHU/Brix.htm

Thanks!

[donnahardy2]

Hi Irregular,

Let’s see. If you want to make a 1: 8.5 dilution of your acid, you could try using 100 grams of newspaper blended with about 300 ml of water. You may have to experiment to get mixable slurry of newspaper and water, so if this is too thick, then add more water. Then pour about 265 ml of the slurry into the flask and add 35 ml of the 31% HCl. Measure everything carefully so you will be able to reproduce your experiment. Use safety glasses and gloves when handling the acid and wear old clothes. A drop of acid will eat a hole in fabric. How will you be measuring your materials?

HCl and NaOH are perfect.

Glucose strips are usually based an enzyme reaction that converts glucose to another compound that can be measured with a color change reaction. I will have to look up the chemistry to explain to you if you use these strips in your project. Please tell me what brand of strips you are using because there are different chemistries used.

Glucose is a 6-carbon sugar and sucrose is a disaccharide (2 sugars), composed of glucose and galactose. The chemistry of glucose and sucrose are different, so sucrose cannot be detected by the glucose strips.
However, if you want to use the glucose strips, you should be able to buy glucose to use as a control, either in a pharmacy as glucose tablets or in the baking section of a grocery store (or maybe a specialized baking supply store). The glucose strips are nice because you can get a result with just a small volume of your sample.

http://en.wikipedia.org/wiki/Glucose
http://en.wikipedia.org/wiki/Sucrose

However, both glucose and sucrose can be measured by a hydrometer, which is based on specific gravity, so this should work for you. However, there are different hydrometers used for sugar solutions and ethanol. What type of hydrometer will you be using?

http://en.wikipedia.org/wiki/Hydrometer

Donna Hardy

[irregular]

Hi Donna,

Thank you very much for the proportions. When I add the NaOH to neutralize, will I have to add little by little and test pH until it's neutral, or can I add a specific quantity? I will be adding 20g of yeast after that; does that sound fine?

If I can detect the sucrose and glucose with a hydrometer, then I'll use it. My hydrometer indicates the sugar in grams per liter, so I'm assuming it's a saccharometer too (it doesn't indicate if it is in writing).

Thanks.

EDIT: I obtained a weighing scale from my school which measures starting 0.1g. I also have small, old measuring instruments used to give medicine to children.

[donnahardy2]

Hi Irregular,

Your measuring devices are excellent for this project.

If you have 300 ml of a 1:8.5 dilution (1 part HCl plus 7.5 parts water) of 31% HCl, this is equivalent to a 1 M solution of HCl. To neutralize this, you will need to add 12 grams of the Roebic drain cleaner (NaOH). You need to measure everything very precisely, I would recommend adding 11.5 grams of the drain cleaner in case there are a few extra hydrogen or hydroxide ions in any of your other materials that we haven't accounted for, and then add the rest if needed. If the pH is between 4 and 7, the yeast will grow well.

You can test your hydrometer with a sucrose solution.

I haven't mentioned the water. If you have very acid or very hard tap water, you should use distilled water for your experiment. If you don't have it, then use tap water. But contact your local water company tomorrow and find out the composition of your water so you will know what else you are adding to your sample.

Donna Hardy

[irregular]

Hi Donna,

Thank you very, very much, you cleared up my questions about the proportions.

My dad brought distilled water from work, so I have some I can use.

I am currently testing my hydrometer and working out the sugar measurement and all the conversions.

Do let me know if you find any useful information about alkaline hydrolysis, organosolv or oxidative delignification. I will continue to look on my own, tonight I will reread all my papers relating to those three processes, including the paper you sent.

Thanks!

EDIT: For the past few days, when looking at the temperature of my water bath, my mercury thermometer indicates a different temperature than my LCD aquarium thermometer. Right now, it is constant at 30 C for the LCD and 25 C for the mercury. Which should I follow?

[donnahardy2]

Hi Irregular,

You are welcome. I will explain the exact chemistry behind the proportions when I get a chance and then you will be able to do the calculations next time yourself.

I have printed out two articles on the organosolv process and will read these tonight and let you know if I can find the information you need.

When you are at school tomorrow, ask if there is any deionization, also called mixed bed resin that you can have. This would be useful for removing the NaCl from your sample if you find your yeast is inhibited.

Donna Hardy

[irregular]

Hi Donna,

Unfortunately my school didn't have the deionization resin. Today, I started my experiment by creating three samples - of newspaper, orange peels and sawdust. I thought that I might as well run three at the same time, instead of just one. Now these three aren't my variables, they will just indicate which is the best source of lignocellulosic biomass. I will proceed to do my experiments with the independant variable with the best source.

I added 100g of orange peel with 400 ml water, 100g newspaper with 800 ml water and 50g sawdust with 800 ml water. Is it bad if I have different concentrations?

I couldn't proceed to the addition of HCI because:
1) I cannot use beakers to put in my water bath since they are too short for the hydrometer to fit into. The cylinders I have are only 250ml, and I only have 2 of them, not 3. A total of 300ml plus bubbles won't fit in the cylinders.
2) My water bath quite tall, and bottles/cylinders/beakers won't stay down there by themselves, they float.
3) The solutions are very very thick, especially newspaper and orange peels. The hydrometer won't be able to take a reading.

Possible solutions?
1) Reduce sample size
2) Reduce water amount in bath, from 3/4 - 4/5 full to 1/2 full
3) Put large bricks underwater to hold bottles/cylinders/beakers down.

What suggestions and advice do you have as to my problems? It's late now, so I'd like to figure out a solution by experimenting different ideas tomorrow after school and start the hydrolysis that evening.

I haven't been able to spend as much time as I would've liked trying to decide on my variables, and I know I must reach a decision very soon. I read the paper you sent me, but I haven't completely understood it yet. What exactly do they mean by "alkaline conditions"? Is that simply when they add the alkali to the sample? I will review it again. How did you find those two papers on the organosolv process? I found a paper on the organosolv process. Please let me know if you found anything interesting/useful/a potential variable.

Thanks!

[donnahardy2]

Hi Irregular,

You have a good selection of cellulose sources, and this is such a complicated experiment that it’s really good that you are doing a trial run. Ir’s perfectly OK to use different quantities of sample and water as long as you write down what you did so you will remember what happened when the experiment is complete. When you set up your final experiment, you will want to make sure that the ratio of cellulose: water is the same, as this should be a controlled parameter. I think the purpose of this initial experiment is to optimize the sample set-up and get some experience in working with the chemicals.

You can transfer part of your sample to the hydrometer to measure the sugars; you don’t have to test the whole volume. However, I would not try to measure sugars until after the hydrolysis and neutralization steps to minimize handling of the acidic samples. The hydrometer readings probably won’t work with the freshly prepared samples anyway. After the samples are hydrolyzed and neutralized, hopefully the cellulose will be broken down to the glucose and won’t be so viscous. If the viscosity does not decrease, then you definitely need to add more water. You can use the hydrometer to monitor the fermentation of the glucose to ethanol.

All of your ideas to secure the containers so they won’t tip over in the water bath are good. I think you should not use containers that are more than two-thirds full and I really like your idea of using the bricks to support the containers.

I will think about the remaining questions and post again later today.

Donna Hardy

[donnahardy2]

Hi Irregular,

I am attaching an article that describes the organosolv method. The procedure involves a series of filtration steps, which you cannot include, so you would just do the part that involves heating the sample at a high temperature under pressure in an ethanol: water mixture to break down the cellulose. For your project, you could select one of your samples, perhaps the newspaper, and compare the standard acid hydrolysis method and the organosolv method for cellulose hydrolysis and determine which one is better for ethanol production. I’ll check and see if anyone has published using this method for newspapers, so maybe you could be the first one to report these results. How does that sound to you? Were you thinking about anything else?

The paper is the first one where I’ve noticed that a ratio of sample to liquid is mentioned. In this case they used 1 part of cellulose material to 7 parts of liquid. So the samples you are using may be a little too concentrated. You will need to experiment with this parameter.

Here is a news article that I found today that includes a very good explanation on the background information for you project, why the hydrolysis step is so critical. You should save this for inspiration for your background information section of your project.

http://www.eurekalert.org/pub_releases/ ... 020311.php

Alkaline hydrolysis uses high pH conditions and heat to break down the bonds between the glucose molecules in cellulose. You haven’t had chemistry yet, but you do need to understand about pH for this project. The Wikipedia description of pH is pretty good. The pH is the inverse log of the hydrogen ion concentration; acidic or low pH samples contain a higher concentration of hydrogen ions and high pH, or basic sample have a lower concentration of hydrogen ions.

http://en.wikipedia.org/wiki/PH

The HCl you have has a very low pH. The NaOH you have has a very high pH, and would be suitable for an alkaline hydrolysis experiment, or for neutralizing the HCl in your acid hydrolyzed samples. The yeast cells you will be using grow best between pH 4 and 6 (slightly acidic).

Please read the Wikipedia article and let me know if there’s anything that does not make sense, or if you need another explanation on this topic.

Donna Hardy

Organosolv pt I.pdf

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[donnahardy2]

organosolv pt II.pdf

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[irregular]

Hello,

I started the hydrolysis today. I reduced the amount of water in the water bath, just a little higher above the pilot light. My dad obtained a conductivity meter from work which measures conductivity, salinity and TDS. I used 3 jars, which held about 500ml. Then I tested the pH, conductivity, salinity and TDS for all three biomass samples. Next, I took out 265 of each sample and added 35ml of HCI with each sample. Again, I tested the conductivity, pH, salinity and TDS. THen, I put the closed jars in a plastic bag, and then in the water bath. I will leave them for 24 hours. Over the 24 hours, other than visual change do you think I should me measuring something at a certain interval?

Thank you for the paper and the links.

For your project, you could select one of your samples, perhaps the newspaper, and compare the standard acid hydrolysis method and the organosolv method for cellulose hydrolysis and determine which one is better for ethanol production. I’ll check and see if anyone has published using this method for newspapers, so maybe you could be the first one to report these results. How does that sound to you? Were you thinking about anything else?

Yes, this would be a good idea, thanks. Since my experiment has started, I have some extra time. I will focus more on my variables now.

Thanks!

EDIT: Last night I added the NaOH for neutralization, and then the yeast as well. Before neutralizing I had to add more water to the orange peel and newspaper samples. I will start using the hydrometer to test for fermentation today.

[donnahardy2]

Hi Irregular,

How is your experiment going? Did you notice a visual change in the samples after the hydrolysis step? I'm sorry I didn't answer your last question on a timely basis, but no, I don't think you need to measure anything during the hydrolysis step.

I thought of one more control you could try, in this experiment, or the next one, if you have time. After the hydrolysis step, you could measure the concentration of sugar and then make up a sample that contains the same amount of table sugar for your yeast to grow in. The difference in results between the sugar sample and your hydrolyzed samples would be due to the inhibitors due to the sample itself or to the hydrolysis method.

What is your deadline for this project?

Donna Hardy

[irregular]

Hi Donna,

The deadline for my project is April 1st, but I was hoping that I can finish my experiments towards the start of March. I didn't really see a visual change in the hydrolysis, only that the colours darkened and deepened.

Thank you for the suggestion, that's a great idea! I will discuss the idea with my dad.

After neutralizing and testing for the first time this morning, I saw that the pH hasn't changed, and it's still around 1-3. We even added 4g more of NaOH, but still nothing changed.
Also, I've added a total of 1 270ml to the newspaper solution, and it's still too viscous for the hydrometer to read. Do you think that I have to still add more water, how much?

Thanks!

[donnahardy2]

Hi Irregular,

Oh good, you have a month to do experiments, and hopefully get some really good results, with plenty of time to write up the results.

I do not have any experience in hydrolyzing newspaper, so I’m not sure how thick it should be. The dissolved sugars would not make it too think for a hydrometer. You’ve added plenty of NaOH, but maybe you should add 1-2 grams more. If the sample is really pH 1, then it’s too acidic to work with. Newspaper has lots of lignin in it, so maybe you need to filter all of the solids out of the sample and test the filtrate. If the newspaper was in the acid overnight, I’m sure the cellulose is hydrolyzed so the sample should contain glucose. Do you have something to filter the sample through? A colander lined with coffee filter, cheesecloth or something similar?

Please note that handling the sample with non-sterile utensils will contaminate it, and something will start growing in it right away if you leave it at ambient temperature. After you measure the filtrate with the hydrometer, boil the sample for 10 minutes and store it in the refrigerator until you are ready to add the yeast.

I wonder if your pH paper is giving the right reading. Do you have a pH meter at school or does your Dad have access to one at work so you could verify the pH of the sample before you inoculate the yeast?

Donna Hardy

[irregular]

Hello,

I used an old filter at home (we won't be using it again so I don't have to worry about the contamination), and I was able to take samples using the liquid that came out.

I have been using pH strips from my dad's work, so they should be correct. I took another pH sample an hour or two back and I noted - the pH of the sawdust is 10-12, the pH of the newspaper is 1-2, the pH of the orange peels is 6. I did not add another 1-2 grams of NaOH for this reason. It seems as if the newspaper isn't being affected, and then obviously the sawdust is too basic. What do you think I should do? Do you think it would be appropriate to add some HCI to the sawdust, and if yes how much? What about the newspaper?

Thanks!