Extracting cellulose from pencil shavings?

[scientist_on_the_run]

Hello,
I am in the research stage of my science project on extracting cellulose from pencil shavings (yes, bet you've never heard that before). I am in the eighth grade, but this is my first experience with a science fair project. I have a few questions I would really like answers to.
The main problem I've been running into is that I don't have the materials; I'm encountering long names of chemicals that I have no idea how to obtain. Also, all of the cellulose extraction processes I've read about require pressure heaters and other factory-like equipment. How should or could I go about conducting my experiment? Is there any way to do it in my own kitchen, or will I have to find a willing lab or university?
Second, I have been trying to find a way to degrade/otherwise break down the lignin in the wood of the shavings so I can get to the cellulose. In addition to the complex chemicals, I've read about white rot fungi that degrades lignin (specifically in a thread on this site from user Irregular). I know this is a developing field, but what materials would be best to use? Should I modify or expand my research topic at all to compensate for anything?
I really appreciate anything you can tell me. Thank you!
-scientist_on_the_run

[SciB]

Hi and welcome to Scibuddies!

Could you please explain the purpose of your project? For us to answer your questions accurately, we need to know what hypothesis you are proposing and what experiments you plan to do to test that hypothesis. It is a good idea to let Scibuddies help you at the very beginning of a project because we can sometimes suggest better experiments, proper controls and statistical tests. This is especially important when this is your first science project.

If your purpose is to test different ways to use biomass such as pencil shavings to make some useful product like methane or biodiesel, then I think you are on the right track thinking about fungi to digest the shavings. There are certain fungi that break down cellulose and lignin. It isn't necessary to extract the cellulose first. Just moisten the pencil shavings and add a piece of wood that has some of the white rot fungal threads in it and I think it will digest them.

Once the wood is broken down by fungi it can be used by certain types of algae or bacteria to produce biofuel. Here's an article about this from Scientific American and I can send you more information if you are interested: http://www.scientificamerican.com/artic ... ream-team/

Let us hear back from you and we can help you create a winning project.

Sybee

[scientist_on_the_run]

Thank you for your prompt response. I realized I didn't really know what direction I wanted to take my project; I guess I'd never gotten past the "if I can do this at all" stage.

I thought it over, though, and I think I would like to go the troute of biofuels. The process discussed in the Scientific American article about the bacteria actually sucking up carbon dioxide fascinated me. I will definitely do more research in this area so I know what I'll be dealing with and can make a better hypothesis. I do know, however, that I would like to do several iterations with, if possible, different types of fungi or degrading materials and then analyze which ones worked best and why. It also occurred to me to use regular wood pulp or shavings (not from pencils) as a sort of control. That way I could get a feel for the fungi's baseline properties. Would that benefit my experiment? Or could it just make the idea of using pencil shavings seem ridiculous?

Again, I am wondering how I would obtain these fungi. Especially since it is February right now and we currently have three feet of snow on the ground where I live! Also, would it be necessary to separate the graphite and wood coating from the shavings? Thanks again!

[SciB]

Hi,

Biofuel from biomass is a really hot area of research now and would make a great science project—if we can design something doable. It is easy to imagine all kinds of experiments that you would like to do but unfortunately you have to stay within the resources you have or can get easily and inexpensively.

As you read in the SciAm article, the idea now is to use a fungus to break down the wood shavings or switch grass or corn husks—whatever biomass is available—into compounds that are digestible by bacteria or algae. So, you really are looking at two different things here and I think trying to do both would be a bit much for your first attempt at a science project.

The other constraint on your project is time. Fungi are very good decomposers of organic matter such as wood but the process takes months in nature. Scientists at the Department of Energy are trying to speed things up by testing a variety of different species of fungi but this is still in the early stage (http://jgi.doe.gov/treading-gray-area-a ... cay-fungi/).

On the bacterial side of the process there are several species that are being tested as producers of biofuel. In the SciAm story they describe a specially engineered human gut bacterium Escherichia ( ‘E’) coli that has been genetically modified to produce the fuel isobutanol. It would be great if you could get this bug but they probably are not giving it away to anyone who wants it.

Bacteria are not the only microbes able to make fuel. Ethanol can easily be produced by yeast, although that is not considered a ‘drop-in’ gasoline replacement like isobutanol. The other organism I like as a primary fuel producer is algae. They grow quickly and only require a few nutrients, carbon dioxide and sunlight or artificial light. They produce oil that can be used as biodiesel. Here’s a long list of some of the algae species that can be used, but a common one like Chlorella would be easier to get (http://www.oilgae.com/algae/oil/yield/yield.html).

So, you have a lot to think about and choose from! Let us know what you want to try and we’ll see if we can help you achieve it. I understand getting active fungi from under 3 feet of snow might be difficult, but setting up bacterial or algae cultures indoors is doable.

Good luck!

Sybee

[scientist_on_the_run]

Thanks for getting my wheels turning. If fungi is not an option, are you saying that algae or bacteria could work to both break the shavings into compounds and then digest them to produce some type of biofuel? I like the idea of growing algae to use. Doing so would produce oil, correct? That seems much more doable than the other options I had been considering.

I'm trying and failing to identify any variables for my experiment. Am I measuring...what, exactly? Would my independent be the type of algae/bacteria/fungi and the dependent be the amount or quality of biofuel created? As much as possible, I'd like to keep the pencil shavings in the picture and not just wind up making oil or other energy from wood. What appealed to me about biofuel was the idea of a "closed loop"; you grow the trees, the trees suck up carbon dioxide, you cut down the trees and turn them into fuel, the fuel puts carbon dioxide back into the atmosphere. Unlike gasoline, which just pollutes. I want to be able to emphasize in my conclusion/analysis at the end that my source of fuel was formerly occupying space in landfills (I'm actually in the process of calculating the volume of pencil shavings thrown away per kid, classroom, school, etc each year).

But back to the actual experiment. If I'm not getting ahead of myself, I think I need to nail down the materials I will be needing. My science teacher wants a detailed material list, if not the procedure itself, soon. Thank you for your guidance!

[SciB]

You are welcome! Sorry if I misled you. Bacteria and algae would be the SECOND stage of the process since they aren’t able to use cellulose or lignin. I wasn’t sure what you were interested in so I didn’t want to overload you with a lot of experimental details. Let’s see if we can make a testable hypothesis out of all this!

1. Fungi + wood shavings = carbohydrates from cellulose + algae = biodiesel
2. Can’t use fungi, so how to make carbs from wood?
3. Can wood cellulose be broken down chemically into sugars? Yes, by digesting with 5% hydrochloric acid. See the following reference:

Chemical and enzymatic degradation of cellulose
http://www.eng.umd.edu/~nsw/ench485/lab4.htm

4. Can bacteria break down cellulose into sugars? Yes. Termites can’t digest wood. They have a species of bacteria in their gut that does the digesting and they use the resulting carbohydrates as food.

Bacteria that degrade cellulose
http://www.hindawi.com/journals/ijmicro/2012/578925/

OK. Now where can you go with all this? Bacterial digestion would be the closest to fungal, and it is a natural, renewable, ecofriendly process—but I don’t know where you could get cellulose-digesting bacteria that wouldn’t cost several hundred dollars. Like fungi, these bacteria are present in rotting wood and vegetable matter, but that doesn’t help you since you can’t get to them in the winter time.

I’m thinking there are two possibilities—one rational and the other crazy. The reasonable way to digest your pencil shavings would be to heat them in 5% hydrochloric acid for several hours and then neutralize the acid with sodium hydroxide. Have you had basic chemistry lab yet? Strong acids and strong bases are corrosive to skin and eyes [ALWAYS wear safety glasses in the lab!!] and you would have to work with them in your school chemistry lab but it could be done.

Ok, now for the crazy idea. Go to your garden supply store and ask someone if they have Compost Starter http://www.homedepot.com/p/Jobe-s-4-lb- ... /203992573

If not you can order it online. Compost starter is a blend of bacteria and fungi and nutrients that are the same kind of decomposers found in soil but in a concentrated form. If you mix the pencil shavings with some compost starter in a clay flower pot and keep it moist and dark and warm for a couple weeks, I think you can digest at least some of the shavings into carbs and sugars. Afterwards, you can add some distilled water to the mixture and filter the whole thing through a coffee filter. The liquid you collect can then be added to an algae culture to feed it.

Phew! That’s a lot of writing. I’ll quit now and let you ‘digest’ all this information. You can let us know what you want to try and we will help you plan it out.

Good luck!

Sybee

[scientist_on_the_run]

Haha! Love your ideas, crazy or not. I think I understand the process now. I had no idea you experts would be so thorough and so attentive! Usually with these types of things you post your question and the due date passes without ever hearing a response. Anyway.

Not that I'm not adventurous, but for the sake of time I think I might have to kindly decline the Compost Starter idea. Would it really take a couple of weeks to digest the shavings into carbs? We have to have at least some of our data before then. It sounds really cool though...

I haven't had chemistry yet (haven't gotten past rolling stuff down ramps to learn about force and motion...aye yai yai), but my science teacher said she could get in touch with the high school if any of us needed. If they don't have the 5% hydrochloric acid and sodium hydroxide, there's a great technology facility nearby that offers a lot of physics and chemistry classes that would probably let me work there.

About those acids, actually: I clicked on the link about the degradation of cellulose but wasn't sure where (if at all) you got that procedure. If you could direct me there or provide a few more details ("several hours"...hmmmm) that would be greatly appreciated.

[SciB]

Oh, sorry! Forgot to copy the url from the acid page into the post. Here it is--http://www.eng.umd.edu/~nsw/ench485/lab4.htm

I've never done this kind of a digestion so i can't help you much with the details. Hopefully you can do it in a lab with a fume hood because HCl vapors are very irritating to the nose and eyes. I think heating the 5% HCl with the shavings would make the reaction go faster but I wouldn't go as high as 90C. Try 60C.

I don't have a clue how you would tell when the shavings are all digested. I guess if they turn into mush.

You had asked once about the graphite. Don't worry about that. You will be filtering the glop before you use it anyway. Oh, one more thing I just thought of you could try. Do you have or can borrow any glucose test strips like Diastix? You can get them at any pharmacy. After you do the acid digestion and neutralization you could test the solution to see if you produced any glucose. I don't know if this digestion would take cellulose all the way to glucose but it would be worth checking it.

And one more thing...
After you do the acid digestion you need to neutralize the acid but you don't want to overshoot and make the liquid too alkaline. You can check the pH with pH paper, but the best way to do it since [hopefully!] you will be working in a lab, is to use a pH meter that continuously registers the pH. Using the meter with the digest on a magnetic stirrer you can add just enough sodium or potassium hydroxide to make the pH about 8.

Hope this answers all your questions for now.

Good luck!

Sybee

[scientist_on_the_run]

Would you mind taking a look at a drawing/flow chart I made just to check that I have the process correct? I did it on Google Docs and you should find it here: https://docs.google.com/a/fivetowns.net ... sp=sharing I'm about to put together my procedure and material list. Thanks again!

[scientist_on_the_run]

Oops this is the correct link: https://docs.google.com/document/d/15dS ... sp=sharing

[SciB]

I'm not sure if i am reading your diagram correctly but the way you have it drawn it looks like you are saying you will neutralize the acid BEFORE you add the shavings. You want to cook the wood with acid alone first and then after some number of hours, you cool the liquid to room temp, put it on a magnetic stirrer, put in the pH meter probe, turn the stirrer on and slowly add the alkaline solution until you bring the pH up to about 8. Then test it with the Diastix to see if you have glucose.

Watch this video for an example of how to use a pH meter: https://www.youtube.com/watch?v=vwY-xWMam7o

Post your materials list and I'll go over it to make sure you haven't omitted something. Don't forget the Chlorella algae culture. This is available from Carolina Biologicals: http://www.carolina.com/catalog/search- ... SearchForm

Sybee

[scientist_on_the_run]

Ok, sorry for the delay. Below I have composed what is hopefully a mostly-complete material list and procedure draft. Then I made a list of some of the things I'm a little confused on. Thanks for all your input!


Materials:

Equipment
- 1ml and 10ml pipettes
- scale/balance
- thermometer
- fume hood
- test tube with cap, large enough to hold 10ml solution and 0.2g shavings
- magnetic stirrer
- pH paper or pH meter
- glucose test strips (ie Diatrix)
- safety equipment (goggles, gloves, etc)

Reagents
- 0.2g pencil shavings
- 10ml 5% hydrochloric acid
- sodium or potassium hydroxide
- Chlorella algae culture


Procedure:

1) Add 0.2g pencil shavings to 10ml of 5% hydrochloric acid in a lightly capped test tube
2) Bring solution to 60°C (140°F)
3) Allow reaction to last for 2 hours, taking 1ml samples at _____ intervals
4) Stop hydrolysis reaction in sample by neutralizing the acid. Add a small volume of a sodium or potassium hydroxide solution to make pH about 8, checking with pH paper or meter.
5) Measure the glucose concentration of the alkaline sample with Diatrix strips.


Unsure about...
- amount of sodium/potassium hydroxide
- what to use to heat the 5% hydrochloric acid to 60C
- if should heat that solution before or after adding in pencil shavings
- at what time intervals to take samples
- how to filter the "glop"
- which algae culture to use (is it the first one on that link from carolina.com?)

[SciB]

Hi,
Good job getting all this stuff organized from my notes! Now to make it work…

The one big question that jumped out at me is HOW MUCH pencil shavings to use. My first impression is that you are way too low with only 0.2 g. That’s probably like a teaspoon—not enough, imho. I have no idea how much usable ‘food’ [carbs, glucose, disaccharides, etc.] this acid digestion is going to produce, so if anything you need to err on the side of MORE not fewer shavings. I understand that you were following the cellulose degradation procedure that I sent you, but for your project you need to scale this up.

You said you’d been collecting shavings so you must have lots by now. I would use ½ cup of shavings, weigh them of course, and take pix of them and all your apparatus for the record. If you happen to have an old coffee grinder you could shred up the shavings finer so that they would be digested faster but if you can’t do this, don’t worry about it.

Now, as to the ratio of acid to shavings, you want to have enough acid to do the job but not so much that you end up with liters of liquid that may be too dilute. The ratio in the method you read was 10 ml of acid to 0.2 g of shavings so weigh ½ c of shavings and let me know the weight. Then I can help you better to decide how much acid to use.

Let me say right here that you will HAVE to have a person in the lab to help you and supervise what you are doing. Working with chemicals requires knowledge and care and protective equipment like goggles, gloves and a lab coat.

For the sake of example, let’s say that the volume of acid plus shavings is about 500 mL. You would put this into a 1.5 or 2 L flask, add a magnetic stir bar and put the flask onto a stirring hot-plate, which hopefully your lab has. Put a thermometer in the flask, turn on the stirrer on low and make sure the magnet isn’t knocking the thermometer around. If the lab has Parafilm or plastic wrap, use it to seal the top of the flask. Bring the temperature up to around 60C and check it several times to make sure the thermostat is keeping it around that temperature.

Now, I don’t know how long it will take for the acid to break down the wood fibers and the cellulose, but I would guess several hours. I don’t know what the wood will look like after it is digested but the chips and bits should have broken down and turned to something like jelly I would think. You could remove a 1 mL sample after one hour and test it with a glucose strip, but remember that you have to neutralize the acid first. Ask your lab supervisor how to do this. The lab probably has 5M sodium hydroxide that could be used, but you need to have someone show you how to neutralize the sample and check the pH.

When you have decided that the wood is digested enough, add sodium hydroxide solution to bring the pH to about 8 then filter it with Whatman #1 filter paper. Ask your lab supervisor how to do this.

The rest of the project has to do with algae growing and I’m sure you will have more questions about that! The algae that you want to use is Chlorella vulgaris and you can order a live liquid culture from Carolina Bio (#152069). They sell a culture medium for this but don’t buy it because you have to make your own using the pencil shaving extract that you prepared in the first part of your project.

Items to add to your Materials list:
1. Six 12 oz water bottles for growing algae in
2. Fluorescent light [natural daylight or GroLite] large enough to light the algae cultures
3. Large funnel and coffee filters for filtering the algae

I think that’s all for now. You see now how a fairly simple-sounding project can have a ton of details to work out! Planning everything ahead of time is the key to good research outcomes.

Good luck!

Sybee

[scientist_on_the_run]

Thank you, that helped a lot. I did as you said and measured out a half cup of shavings. I got the weight to be 40g. I crunched the numbers and came up with 2L of acid. What size flask should I use for this?

Is Whatman #1 filter paper a likely thing to have in high school a lab? I looked online and it seemed quite pricey. If my lab does not have it, do you think anyone would give me a discount for education purposes?

I have now divided my experiment into two parts to make it more manageable: Part I, Acid Digestion; and Part II, Algae. Below is my revised material list for both parts and procedure for Part I...I want to be sure I have been as thorough as possible up to this point before diving into the procedure for Part II.

Materials

Equipment
safety equipment* (goggles, gloves, lab coat, etc)
scale/balance
2 L flask
magnetic stir bar
magnetic stirring hot plate
fume hood* (preferable safety measure, not entirely necessary)
thermometer, if one is not included on hot plate
plastic wrap
timer
pH paper or pH meter
glucose test strips (ie Diatrix)
Whatman #1 filter paper
6 12-oz water bottles (for growing algae)
Fluorescent light source
Large funnel
Coffee filters

Reagents
40g (approximately 1/2 cup) pencil shavings
2 L 5% hydrochloric acid
sodium or potassium hydroxide
Chlorella vulgaris algae culture


Part I Procedure

1) In the flask, put 40g pencil shavings and 2 L 5% hydrochloric acid. Let this be Solution A.
2) Place magnetic stir bar and thermometer in flask, then cover with plastic wrap.
3) Position the magnetic stirring hot plate in the fume hood, if using one, and place the flask on top of the hot plate.
2) Bring Solution A to 60°C (140°F), making sure the magnet is not interfering with the thermometer, and start timer.
3) Allow reaction to take place. After one hour, take a 1ml sample. Let this be Sample B. Leave the rest of Solution A as is, permitting the reaction to continue.
4) Stop hydrolysis reaction in Sample B by neutralizing the acid: add a small volume of a sodium or potassium hydroxide to make the pH about 8. Check this with pH paper or meter.
5) Measure the glucose concentration of Sample B with glucose test/Diatrix strips.
6) After 2 hrs or when digestion appears complete (shavings broken down, should resemble something like jelly), remove flask from hot plate and allow to cool to room temperature.
7) Repeat steps 4 and 5 for Solution A.

Thoughts? If my lists are all set, I would appreciate some guidance with Part II. I noticed the Chlorella culture will come with instructions for growing the algae, but I know what I'm doing is not exactly typical...

[SciB]

Hi,

Looks good! But since I can’t write down every detail of the procedure and I can’t be there to supervise, I want to be sure you will have an experienced lab partner with you to make sure everything is done correctly and to answer any questions you have, ok?

You don’t have to use Whatman filter paper. A coffee filter will probably work just fine. Whatman filter papers are the lab standard for quality and accuracy but you just need to separate the undigested bits from the liquid part. I am hoping that your lab will have a vacuum line so you can do vacuum filtration of the digest. If not, it may take a while for all the liquid to drip through. Ask your lab partner if they have a filtering funnel and side-arm flask with vacuum hose. If so, you can add these to your materials list.

You calculated the right volume of acid according to the paper on cellulose digestion, but I’m concerned that 2 L might be too much. I’d like to make the solution a little more concentrated so I am going to suggest that you use 1 L of 10% HCl instead of 2 L of 5%. If you have a 1.5 L flask use that, otherwise use a 2 L flask.

When you take the 1 mL samples for glucose testing be sure to turn off the stir bar for a minute or so to let the debris settle and then pipet your sample from the clear liquid. Also, you should take a zero-time sample right after you mix the acid with the shavings [let them settle before taking the 1 mL sample] to establish a baseline.

I don’t know how long this digestion is going to take and you may have to leave it overnight to get a complete breakdown of the shavings. When you are doing an experiment that you haven’t done before you have to be prepared to make mid-course changes in the procedure. Just remember to write down everything in your lab book with the date and time. Take lots of photos and maybe make a video of some parts of the process.

When you neutralize the 1 mL sample, keep track of the volume of the NaOH solution that you added. Then you will know how much to add to the next sample, and also it will allow you to calculate how much to add at the end to the whole solution to bring the pH to 8.

Ok, now as to growing the Chlorella, here are the instructions: http://www.google.com/url?sa=t&rct=j&q= ... XY&cad=rja

I would order the culture right away so you can get it growing in order to have enough to do your experiments with at least 3 bottles of algae for the control and 3 for the experimental. You will also want to have a culture in reserve as a back-up in case you need to repeat the experiment. The culture of Chlorella that you receive from Carolina Bio will be in 10 mL of STERILE medium and they emphasize the need to use sterile medium and culture vessels for growing the algae. I am going to assume that the bottles used for water that you buy in the supermarket are nearly if not absolutely sterile so they can be used as is as long as you don’t contaminate the mouth of the bottle by touching it with something non-sterile like your fingers.

You can use the water from the water bottles to make the algae culture medium. You will have two types of medium—one without wood digest [‘WD’—you don’t have to use my term. You can call it whatever you want!) and one with WD. Chlorella can make their own carbohydrates from sunlight and CO2 but they do need some other compounds—nitrate, phosphate and some micronutrients. These can be supplied by adding a synthetic plant food such as Miracle-Gro (http://www.miraclegro.com/smg/goprod/mi ... /prod70342) used at 1/3 the recommended amount.

You will want to grow 8 oz of algae in each bottle so you need to have 24 oz of each growth medium for 6 bottles [3 controls and 3 experimentals]. At this point I do not know how much WD you will be adding. That will depend on what you see from the Diastix readings. To start the culture you will need to add 9 ml of culture to 6 oz of growth medium without WD so you will need to make 30 oz of this medium.

To sterilize the growth medium get a 4 cup [32 oz] pyrex glass measuring cup and fill it with the 30 oz of medium without WD. Cover the top with plastic wrap and poke a small hole in it with a thin knife to let the steam out. Put the measuring cup in a microwave and set it for 6 minutes on High. This will kill most of the bacteria and yeasts and give you a growth medium that is sterile enough for the purposes of this project. Using a couple of oven mitts carefully take the measuring cup out of the microwave and let it cool until you can pour it into the bottles without melting them. LEAVE THE PLASTIC WRAP ON! This maintains the sterility. When you are ready to pour pull back enough of the plastic to expose the pour spout and put approximately 8 oz of medium into each bottle, replacing the cap immediately after you finish filling the bottle. Put the remaining 6 oz into another bottle and when it has cooled to room temperature [20-24C] add 9 mL of the algae culture [shake the tube gently to resuspend the algae before you pipet it] to this bottle as a starter culture. Take 3 mL out of each of the other cooled bottles and put it into the algae culture tube to replace the liquid you removed. This will guarantee you have an uncontaminated reserve culture in case anything goes wrong.

Put the 6 oz starter culture and the culture tube under the lights with the caps on loosely and leave the lights on for about 18 hours, then put them in the dark for 6 hours. Shake the bottle and the culture tube gently several times a day to move the algae around so they all get light. I don’t know how long it will to grow up to a suitable density. Take a photo of it every day for comparison. Once this culture becomes nice and green you will be able to use it to start your actual experiment by adding exactly 1 oz of culture to each of the 6 bottles.

Phew! That was a lot of details! I hope you can follow all that. I probably forgot something so you can post again with more questions.

Good luck!

Sybee

[scientist_on_the_run]

Clarification(s)...
1) I am digesting one round of 40g shavings. Or should I plan on needing twice or triple those materials?
2) I am ordering one Chlorella culture (one $7.50 unit).
3) Would it contaminate the water bottles or other materials if I handle them with latex or rubber gloves on?
4) For my growth medium, I will combine 30 oz of water from the bottles and 1/3 the recommended amount of Miracle Gro.
5) How do I pour 8 oz of the sterilized medium into each bottle? Isn't there only 30 oz?
6) Do I add the WD to the three experimental bottles when I am ready to put the 1 oz algae culture in?

Also, I am still unsure what my variable situation is. This experiment isn't testing what kind of acid or algae works best or what the effect of x is on y; it's just kind of being done. How do I formulate a hypothesis and identify independent and dependents?

[SciB]

Hi,

Sorry for the confusion. Let me try to clarify what I meant to say. I’ll take your questions one by one:

1. 40 g of shavings in 1 L of 10% HCl will provide more than enough WD for the 3 experimental bottles

2. Yep, that’s good. They don’t tell you how much algae is in the culture so I can’t say how long it is going to take for your starter culture to grow up. If you give it light for 18 hr, keep the bottle at a temp of 20-24C and swirl it a few times a day, the algae should grow pretty fast.

3. Touching the outside of the bottles is not a problem. You just don’t want to touch the top or inside the cap.

4-6. I was afraid this might be confusing. You need to make TWO growth mediums—one for the control without WD and one for the experimental with WD. The reason I am having you make 30 oz of medium without WD is so you can have 6 oz to use for the starter culture and 24 oz for each of the three 8 oz control cultures. After you make the control medium [water plus plant food] you will make 24 oz of the WD medium—water + plant food + WD. You will microwave this medium for 6 minutes the same as you did for the control medium.

There is one other thing I just thought that you should test and that is the pH of the growth media BEFORE you sterilize it. You can use pH paper or a pH meter for this. Just put a drop of each medium on pH paper and if it is not 8.0 then you will need to add some sodium hydroxide solution. If the lab has a pH meter, use it, because it makes adjusting pH MUCH easier.

I would say that your hypothesis is: A digest of wood shavings can be used to increase Chlorella biomass. Your independent variables would be the type of algae and the growth medium, the hours of light, temperature, etc. The dependent variable is what you will measure and that will be the mass of dried algae in each culture at the end of the growth period.

You might also say that you have another hypothesis--that acid digestion of wood shavings produces sugars that can be used for growth by Chlorella. There your dependent variable would be the production of sugar in the form of glucose that you measure using the Diastix. The problem with this second hypothesis is that I don’t know that acid digestion will produce glucose. I have read a little about it and I know that acid will cleave cellulose, which is a polymer of glucose units, into smaller pieces, but I don’t know if it will digest it all the way to individual glucose molecules. Also, I don’t know if the Diastix will react with sugar units that contain 2 or 3 or more glucose molecules in a linear array.

Science is all about curiosity and wanting to know. If you knew all the answers there would be no need to do experiments! You want to know if you can make food for algae out of wood and I am betting that you can. Whether you can do it by using acid digestion rather than a fungus as you first thought of I don’t know—but that is what you are going to find out!

I hope this makes more sense now, but if you have any more questions, don’t hesitate to ask. Just because I’m an ‘expert’ doesn’t mean I know everything! I’m always learning and struggling to make sure that what I think is true really is. Science never stands still. That’s one of the coolest things about it. New data comes out and knocks the old theory on its head—and that’s great! I want to know what is TRUE not what supports my pet theories.

Good luck!

Sybee

[scientist_on_the_run]

Thank you so much, Sybee. I hope you know what a help you've been. ONE more question though..."the mass of dried algae in each culture at the end of the growth" does somehow relate to biofuel, right? I went back and read through our loooong conversation and was reassured to see that you'd said it would produce oil. Is this still true? Or did that kinda get lost in the process?

[SciB]

No, we haven't lost that thread! The problem is, as I said, extracting the oil from the dried algae This is mechanically difficult as you need a high pressure extruder to squeeze the oil out. The amount of oil in Chlorella is around 30% of the dry weight so I am suggesting that you use that as your estimate of how much oil that you could get if you pressed the algae. You could print a picture of a press to display on your board and explain why you had to omit this from your experiment. It would be great if you could get a sample of algae oil from somebody but I have no idea who you could ask.

Good luck getting everything to work out and keep us posted on your progress!

Sybee

[scientist_on_the_run]

Hey Sybee. Quick question regarding Miracle Gro...should I go by the recommended amount for outdoor plants (1 tablespoon per 1 gallon>>0.23 tbs per 30 oz>>0.08 tbs which is 1/4 tsp) or indoor plants (1/2 tsp per 1 gallon>>0.12 tsp per 30 oz>>0.04 tsp)? Hopefully those calculations are correct. And if it is indoor, how would I measure or even approximate 0.04 of a teaspoon?! Thank you!

[scientist_on_the_run]

Sorry for multiple posts in one day, but I have more clarifications...
1) Once the algae in the starter culture bottle is ready and I have added 1 oz to each of the other 6 bottles, do I repeat the 18 hrs light/6 hrs dark cycle for their growth?
2) Is it okay to just use sunlight and then a cardboard box for the light cycle, or should I find a way to hook up some fluorescent bulbs to the inside of the box? Or is this totally wrong?
2) When do I know my experiment is "done" and I can analyze the results- will the algae be dried up?

[SciB]

Hi! I wondered how it was going and I see you are getting ready to grow the algae. What happened with the wood shavings? Did they appear to digest with the acid? How long did you let the digestion go? Did you detect any glucose with the Diastix?

To answer your questions:

1. I think 1 tsp Miracle-Gro per gallon will do it. I know, this works out to 1/3 rather than ¼ strength, but I want the algae to grow fast and fully. It will be easiest to measure 1 tsp, so make a gallon of the fertilizer then just measure out the volume you need for the experiment. This way you’ll have some extra in case you need it.

2. Yes, keep the same light/dark cycle for all cultures.

3. I would use a daylight fluorescent lamp indoors because you can control the light better and also the temperature. The cultures do need to be fully dark for 6 hours to grow properly. Sunlight works fine, of course, but you need to be careful that the sun does not heat the water too much because the growth of Chlorella will slow down at temperatures above 40C (104F).

4. When to stop the experiment depends on how fast the algae in the fastest-growing culture grows and I have no idea how many days that it will take to reach maximum growth. Why I said to watch the fastest-growing culture is because the growth rate slows down as the nutrients get used up and you don’t want the slower-growing cultures to catch up. Take a picture of all the cultures each day at the same time of day and try to estimate the density of the color. There should be noticeable increases in color as the algae grow and divide every day. The best way to measure the density is by building a colorimeter like this one: https://www.sciencebuddies.org/science- ... p075.shtml but you probably don’t have time to get the parts, put it together and test it before you need it so it would be easiest to just estimate the culture density by eye and to make good photos every day at the same time.

5. No--the algae better not be dried up!! I think you are confusing what I said about collecting the algae at the end of the experiment and measuring the weight of each culture after it has been dried. When you decide that it is time to stop the experiment, filter each culture thru a coffee filter to collect all the algae then let it dry completely before you record the weight. Be sure to write a code number on each filter so you know which is which!

Let me know what happened with the wood shavings. Did you see any glucose? Keep posting about the algae growth. Remember to keep a reserve algae culture going (NO wood extract!) to use as a backup if you have to do an experiment over again.

Good luck!

Sybee

[scientist_on_the_run]

Sorry for such a delay! At the time of your post, I had not done the digestion portion of the experiment, so didn't have anything to report. My teacher and I went to the lab yesterday and performed it. The results have been a little inconclusive so far. Each of our samples got ruined because, no matter how few drops of NaOH we used as increments, the pH would jump from 1 (super acidic) to 12 (super basic). We thought it might have been a problem with the pH paper we were using, but we tested water and vinegar and got accurate, mid-scale readings for those. At that point we knew we couldn't get correct sample data, so we played around with the HCL/NaOH ratio trying to reach a pH of 8 and still couldn't get it. So I was unable to get readings on the Diastix.

As for the main solution, it is still digesting 25+ hours after we put it on the magnetic stir. Part of this may be due to the fact that I had to remove it from the equipment to take it home to let it continue (now it is not being stirred constantly nor at 60°C temp). Oddly enough, it has turned a vibrant shade of green (?!). By now most of the shavings have sunk to the bottom, though there are still some floating around on the top of the acid, and as far as I can tell they have only wilted a little and taken on a green hue. I've taken glucose readings several times, both at the lab and since taking it home, and they have always shown a shade between negative and the lowest amount (100 mg/dL). This is not such a big deal, but what I am worried about is adjusting the pH once (if!) the shavings are further digested. Is this step necessary for not killing the algae?

[SciB]

Hi,

Please try to communicate more frequently. If you have a problem with something like adjusting the pH I may be able to help you—so ask!

If you are overshooting the pH it means that you are using too strong an NaOH solution. What was the molarity or percentage of the solution you used? Try diluting it with distilled water until you can neutralize the acid and get the pH to about 8. Try to find someone who has a pH meter you can use—it is SO much easier to do pH changes using a meter. Even an inexpensive handheld meter is better than pH paper.

You are using 10% HCl to digest the wood, correct? I don’t know why the solution is green but I would suspect maybe there was paint on the pencil and that may be where the pigment is coming from. Is the solution green or just the wood?

You will have to adjust the pH to about 8 before you add the digest to the algae or you will definitely kill them. Try neutralizing some 10% HCl with diluted NaOH as I suggested and let me know if that helps.

How are the algae growing? Are you able to maintain a temperature of around 24C [75F]? What about light? Are you growing the algae inside under fluorescent lights? The algae need to be growing and dividing well or the experiment won’t work.

Keep us posted on your progress.

Sybee

[scientist_on_the_run]

My teacher is checking on the molarity/percentage of the NaOH. I remember it was pretty strong, though, so diluting it will probably help a lot. If I can tell you its concentration, do you know roughly how much distilled water to add to it?

The solution itself is green! It sure looks nice, but it does make it kind of difficult to observe the shavings. From what I can tell, at nearly 48 hrs after starting the reaction, the shavings have all sunk to the bottom and most of them have broken down into small dust-like pieces but some are still in shaving form. They seem rather transparent.

There was a little mix-up with the algae and for some reason my order didn't go through the first time, but we worked it out and it should be arriving any day now. I have my set-up ready with water bottles marked and a fluorescent lamp. Hopefully I won't be too crunched for time.

I'll try to post more frequently now that the experiment's really underway. Thanks again for all your help.

[SciB]

Hi,

In order to neutralize a strong acid like HCl you need to add an equal volume of the same molarity of a strong base such as NaOH. For example, if you had 1 mL of 3 M HCl, you would neutralize it by adding 1 mL of 3 M NaOH.
Adding an equal volume of base to the acid will dilute the wood extract by half, however, and you don’t want to do that. If my calculations are correct, 10% HCl [1 part concentrated HCl + 9 parts water] is 3.5 M.

Question 1: Are you absolutely sure that the 10% HCl was made correctly?

A 50% solution of NaOH is 19.4 M. If that is the solution that you have, I would dilute it to 10 M and try neutralizing 10% HCl with that. It should take about one-third volume of 10 M NaOH to dilute 3.5 M HCl.

Question 2: Do you have a pH meter that you can use? Carolina Bio sells a decent handheld meter for less than $50 (http://www.carolina.com/environmental-s ... &question=)

I’m glad to hear that at least some of the shavings seem to be breaking down. When they become transparent that means the cellulose polymer is separating from the fibrous lignin and that is what you want to happen.

Question 3: Have you been taking photos of the pencil shaving digest at various times, say every 4 hours?

When you get the right concentration of NaOH and succeed in neutralizing a sample of the wood digest, let me know if the green color changes. The color could be coming from a reaction between the wood fibers and the acid or possibly from paint on the pencil shavings. Was there paint on the shavings?

Question 4: Is the area where you are going to grow the algae at least 24C (75F)? The warmer it is the faster they will grow. Also, give them light for 14-16 hours a day.

Good luck!

Sybee

[scientist_on_the_run]

Q1: Yes, it is definitely a 1 liter solution of 10% HCL.
My teacher says that the NaOH we were using was a 2.0 molarity solution, and that for the second sample we actually diluted it with water to a 1 molarity solution. That evidently still didn't work, and she thinks that diluting it further to a 10% molarity solution so it has the same strength as the HCL would be best. I seem to be getting mixed messages here...would that be a good next step or should I go more your route and not dilute it so far? I was surprised how low of a molarity it had to begin with (at least compared to 19.4 or 10) and that it still overshot the pH.

Q2: I do not have access to a pH meter. My folks don't feel like it's worth it, even with Carolina's great price. There is no shortage of pH paper, though, so it will just have to take a little longer to calculate.

Q3: Yes, I have been taking lots of photos and jotting down observations. The only regret I have is not getting a good picture of the shavings in the flask before the digestion started. I don't think they had any paint on them, but it is definitely possible that some shavings from a colored pencil got in there. I'll be sure to make note of any changes in color after getting 8 pH in a sample.

Q4: This was a bit of dilemma as it is quite cold where I live. With the help of a portable heater with a thermostat and a small room, it should be possible to keep the temperature at least 24C. I also have a box I will put over the bottles for the given time each day. I actually have a question about this...what is the ideal light/dark cycle? I have heard everything from 18 to 14 hours of light.

[SciB]

Hi,

Q1: I think there might be some confusion as to what is meant by ‘10%’ here. Concentrated HCl is 10-12M and contains 32-36% HCl by weight. If you made a 10% solution by weight it would be about 3.2M; but if you made a 10% HCl solution by volume it will be about 1.1M.

Q: How was your 10% HCl made?
If you take equal volumes of HCl and NaOH solution of the same molarity and mix them the resulting pH should be around 7.0. You want a pH of 8.0-8.5 so you do need to overshoot a little. I don’t know why your previous experiment resulted in a very alkaline solution.

Q: Did you mix the solutions really well before you tested them with the pH paper?
Q: What volumes of solution did you mix? How are you measuring the volume?
Q: Are you putting the pH paper in the solution or transferring a drop to the paper?

You must get the acidic extract to pH 8.0-8.5 before you can add it to the algae, so if your NaOH solution is too strong then keep diluting it and trying it with the HCl until you get the correct pH reading.

Q2: I did not mean for you to buy a meter. This is something that every school science lab should have. I know high schools have tight budgets but this is one instrument that is extremely useful and important and not that expensive.

Q3: Do you have any shavings left? If not, get a pencil sharpener and make some more. Then you can make another mixture of the same amount of shavings in the same volume of acid. It won’t be the identical solution but it should look the same and you can photograph it so you will have a picture of the starting mixture.

Q4: I had a feeling temperature might be a problem but if you can keep it reasonably close to 24C the algae should grow fine. I think 18 hours of light is the maximum you could use. I suggested 14-16 hours as being perhaps more convenient for your schedule. You do want the algae to use the nutrients from the wood digest so I would suggest at least an 8 hour dark period during which they will be using the digest as a carbon source rather than CO2 from the air. The algae that don’t have the extra nutrients from the digest should grow slower because they have to use some of the food they made during the day from photosynthesis. Here’s a paper that compares the growth of Chlorella with or without supplementary glucose: https://www.google.com/url?sa=t&rct=j&q ... XY&cad=rja

The paper is rather technical so just look for the main points and conclusions and if you have questions, make a list and post it. The growth rate of Chlorella with added glucose was substantially greater than the control cultures. This is what the biofuel researchers are trying to do—increase the mass of algae produced while spending the least amount to grow the algae. If they can grow algae in the dark then they will save the cost of artificial lighting.

I hope all this is helpful and not too confusing. Make a list of the answers to my questions so I have the information I need to help you, and if you think of more questions then post them too.

Good luck!

Sybee

[SciB]

Hi,

I happened to see this article on the current great interest in Chlorella as an ingredient in human food and thought you might be interested: http://www.nutraingredients.com/Manufac ... garis-line

Algae culture seems to be poised to be one of the next big industries in this century. You picked a good subject for a science project!

Sybee

[scientist_on_the_run]

Thanks for the article. I had no idea what a market there was for chlorella and other algae, especially as a health ingredient!

I did a test today with the diluted NaOH (1 ml to 20 ml of water), and even after 91 drops into HCL (21 at first and then 14 increments of five drops) when class was over the pH was still 1. To answer your questions, I did mix the solution thoroughly after every five drops and I dipped the paper into the solution. My teacher suggested I try just soaking the shavings in water until I'm fairly certain it's not too acidic--maybe I could mush one of the pH strips against a shaving?--instead of spending more time and more supplies on the acids. This seems...I don't know. Clarification question though: it is the physical shavings/wood digest I'm putting in the experimental algae bottles, not the liquid, right?

My algae has arrived and I am going to get the starter culture growing. May it grow quickly!!! The fair is approaching fast.