Calculation Results

Alright, so down to some number-crunching!

The magic formula I used for calculating percent starch was:

% Starch = [(∆Atest )(900) ]/  [(∆Astd)(sample weight in mg)]

(I won't bore you with details on where the 900 came from but it involves the multiplication of molecular weight of starch monomers, initial sample volumes from glucose sample volume assays, dilution factors, and whatnot.)

Okay. The values for the 1 mg sample, 5 mg sample, and 10 mg sample turned out to be 24.3%, 33.7%, and 56.7%.

Plotted on a graph, the relationship turns out to be somewhat linear, which is a good and expected:

Next week I will repeat the standardization experiment to make sure everything is correct (and to make sure it is repeatable), before moving on to the real samples.

Pink is My Favorite Color (Day 2 Standardization Continued)

Check out these beauts!

Fig. 1 (From left to right): Standard Blank, Standard, Reagent Blank, Cornstarch Samples "1", "5", "10"

The intensity of the pink color pictured above is proportional to the original glucose percentage. I got absorption readings of 0.02, 1.2, .185, .22, .41, and .95, respectively (at 540nm).

Also, this was my work area:

Fig. 2

...Cozy, no?

(That labeled bottle on the left is the sulfuric acid solution! And the paper on the right - that's my procedure bible.)

I'm going to take these measurements home and plug them into my calculations, and see what I'll get for the percent starch. 

This is so cool!!! :)

Day 2 of Starch Assay Standardization

So today I was back in 201P finishing up the remainder of last night's procedure. The first thing I did was turn on the Spectrophotometer (Spec 20) and let it warm up for 15 minutes. Then I retrieved my samples and pipetted the solutions - my Standard Blank (distilled water), Glucose Standard (water + the glucose that came in the kit), Reagent Blank (the blank from the Starch digestion - contains only the reagents), and the 3 cornstarch samples - into their respective cuvettes.

This step was all about timing. If we let the reaction for each sample go on for differing amounts of time, we aren't going to be evaluating them on the same standard. So upon adding the Glucose Assay Reagent to each sample, I had to incubate each tube for exactly 30 minutes in a 37˚C water bath; then, I had to stop each reaction by adding 2ml of the 12N Sulfuric Acid solution.

I figured the easiest, most efficient way I could do this was by adding the Glucose Assay Reagent to the first tube (standard blank) mix, and then put it in the incubator; wait 1 minute, before adding the reagent to each subsequent tube. Then, when time was up for the first tube, I would add the H2SO4, take that out, and then wait another minute; that's how I knew the 30 minutes was up for the second tube, and so on and so forth.

But I'm not going to lie - it was pretty nervewracking. I don't think my eyes ever left the screen of my i-phone timer.

An Elaboration

I thought I'd take a moment to clarify what this procedure is all about.

Essentially this starch assay is a reaction in 3 steps: 

I stopped right after the first reaction; after the fourth reaction, I will be left with samples that will be pink in color, in varying shades. I will be using a spectrophotometer to measure the absorbency of the samples, and this measurement will be proportional to how much starch is in each. 

IT'S GETTING REAL

My ordered starch assay kit came in the mail yesterday!
And so, today I stayed after school from 3 until 6pm to perform my first ever starch assay, but first on a control (cornstarch) to standardize my procedure. I used 1 mg, 5 mg, and 10 mg samples of cornstarch for this experiment.

Today basically involved a lot of precise massing, measuring, tube-cleaning, labeling, incubating, timing, diluting, pipetting, adding, mixing, waiting, and pipetting some more, transferring...and a lot of reconstituting materials beforehand - all the reagents, the 80% ethanol solution, and the 12N Sulfuric Acid solution... (<-- Yikes I know)

(Actually, after Dr. B finished making the 12 N H2SO4 solution, I felt the bottle, and it was hot. Like, hot hot. Hot like a stove. Or a steaming mug of hot chocolate. Or your favorite actor/actress! Or my school's theater when the heater goes on overdrive! ...You get the idea. )
(And that is why, folks, always remember to add acid to water, and not the other way around. #lessonsfromAPchem)

It was a 3 part procedure (one that I had typed up and tailored a month prior). The first part was the sample preparation; this time around it was extremely manageable as I was already working with finely ground cornstarch. The second part, was the starch digestion. Order matters here: I wound up adding first 80% Ethanol solution, then alpha-amylase (then heating the enzymatic reaction), then adding what was titled, "Starch Assay Reagent" (then incubating that), and finally diluting the samples and proceeding to the glucose determination, part 3.

ACTUALLY IT IS IMPORTANT TO NOTE that I stopped right after finishing Part 2 today, due to time constraints (Basically, it was 6pm and if I stayed any longer, the school would have probably kicked me out.)

So, after diluting each sample with distilled water, the last step of Part 2, I refrigerated the samples overnight at around 2-8˚C - not exactly the original plan, but hopefully it won't impact my results too much. Fingers crossed, and we'll see how tomorrow morning goes...

Good News and Bad News...

So after returning back from my wonderful, feast-filled, stomach-stuffing and much-needed Thanksgiving break, I came back to Prep Room 201P... and guess what  surprise was waiting for me:

Fig 1: 15s flask with unidentified cloudy substance

A STRANGE YELLOWISH-BROWNISH FOREIGN CONTAMINANT!!! Those pesky bacteria!

Or maybe it was a fungus... I'm not sure.

But, in any case, the good news was that the duckweed colony was proliferating!!! ;D

Originally only one frond was placed into the flask - now, there were 5. Go asexual reproduction, go!

The other samples also showed signs of growth:

Clockwise from upper left: 30s, 1m, 1m30s

For now, I am just going to let the samples grow and not worry about the mysterious intruder in yellow.

Observations for November 21-26

Actually all the flasks contained green fronds by now - including the 1m30s sample flask! All showed signs of budding, no contamination present whatsoever.

The Next Day...

The plants had no contaminant film surrounding them! 

*Cue celebratory music*

The 30s seemed to be the only one that was really healthy - the 1 min 30s seemed to be too long of a bleach time, as the frond had sunk to the bottom (although there was still a green bud zone.)

Sterilization Procedure

We were going to use 10% bleach solution to sterilize the duckweed fronds before placing them to grow in the sterile media. However, the problem with this method is that if you bleach the fronds for too long, the plants will die - if you bleach them for too short of a time, not all the bacteria will be killed. In order to combat this issue, we bleached each sample for a different amount of time: 15 seconds, 30 seconds, 1 minute, and 1 minute 30 seconds.

So the methods:
A few duckweed samples were transferred into a petri dish filled with tap water using an inoculating loop. An inoculating loop was then used to pick one clone and transfer it to a small watchglass filled with about 10 mL of 10%bleach solution; as soon as the plant was immersed in bleach, the timer was started. After 15/30/60/90 seconds, the plant was immediately removed with a new inoculating loop and placed into a new petri dish with sterile water for rinsing and observation (I was looking for a small green bud zone which would indicate that the sample was still alive) Then using a new sterile inoculating loop, the samples were transferred into their appropriate sterile flasks with minimal exposure time to air. A new sterile rinsing petri dish was used for each clone.

This Calls for a New Game Plan

To combat bacterial and fungal contamination, we were going to make the following changes:

1. Use giant cotton balls to stuff the flask openings instead of tinfoil (Dr. B said when she did research in her lab, this method worked quite well.)
2. Autoclave the media longer, with emphasis on listening for a very high whistle
3. Most important change: Sterilize the plants before transferring them to the media to grow

On November 18, I redid the autoclaving procedure:

8:18: started heating the pressure cooker
8:33: faint gas noise readily heard, first signs of bubbles foaming at the valve
8:37: started whistling
8:42: louder, but still no "chugging"
8:43: shut heat off
*Here I had to dash off to Multi class

10:01: turned heat on again
10:30-10:50: let it run

Ahhh, Contamination!!!

We let the duckweed sit for a few days. This was the 2 days after the media sterilization procedure; as you can see, signs of contamination are already apparent:

...This, is 9 days afterward:

Ick, I know!

... Clearly, sterilization is going to be a bigger challenge for us than I'd thought. 

(But hey! This is all part of the experience - might as well let it mold me! Ahahaha...)

"Autoclaves" and Trying Days

First thing to do when the SH media arrived was to reconstitute 1L of it, and get my samples growing. However, sterilization was going to be a big issue for us, as we had no autoclave. Fortunately, we were able to make do with a makeshift autoclave - Dr. B's pressure cooker.

Below is the methods write-up:

Wednesday, 10/30

Using a tin weigh boat and metal spatula, the entire contents of the SH Media bottle was massed on an electronic balance and determined to be 32.0 grams. If 32.0 g can be reconstituted to from 10L, then it follows that to make 1L would 3.2 g. Accordingly, all powdered media was placed back into the original container except for 3.2 g, and stored at 2-8˚C. To a 2 L Erlenmeyer flask was added 3.2 g of SH Media and 1 L of distilled water, and swirled until dissolved. 150 mL of the solution was apportioned into four 250 ml Erlenmeyer flasks each; the large Erlenmeyer flask containing the remaining solution was covered over the opening with tinfoil and left to stand at room temperature.

Then, the 250 ml flasks containing the media were each covered with tinfoil at their openings and placed into a small-sized safety pressure cooker. The pressure cooker was then sealed and placed onto a hot plate, with the heat setting turned on high. The pressure cooker was heated until whistling; the heat was then turned down to 8 and let sit for 20 minutes. After 20 minutes, the heat was turned off, and the flasks were left to cool.

Friday, 11/1

A jar of samples were taken out of the greenhouse. Forceps were used to pick up several colonies  by transferring them to a petri dish, and then isolating four separate fronds (each their own colony). One colony was transferred to their respective flask, and labeled. The flasks were then taken to the greenhouse, covered lightly with tinfoil, and exposed to normal intervals of sunlight and darkness.

Manual Labor Yay!! :D

It's late September; so it was imperative that I start gathering my duckweed before the weather becomes too cold and my precious would-be samples all form turions and sink to the bottom of the pond! So this weekend, I took a trip to my local canal, where duckweed blooms can be spotted annually, carrying in hand 3 jars, and one giant plastic Costco Fancy Almonds container (washed and rinsed thoroughly, of course.) I retrieved my samples from 3 different locations:

Fig. 1: A nice shaded spot; but good thing I wore boots here!

Fig. 2: An open but hard-to-reach spot; nearly dropped my container!

Fig. 3: Easiest place to collect duckweed

...Now I just have to figure out a way to transport my samples to school, and get them growing!

Why Duckweed is Awesome!

Here are 10 reasons why duckweed is AWESOME:

1. It is the world's smallest flowering plant.
2. It can accumulate biomass more rapidly than corn (that means more starch per acre!).
3. It is a bioremediator, meaning that it can remove pollutants from water like nitrogen, calcium, iron, and phosphorus.
4. It can be used for wastewater treatment.
5. It won't compete with land crops for space.
6. It is relatively easy to grow.
7. It is found all over the world.
8. It can reproduce sexually and asexually.
9. It has a growth doubling time of 2-7 days.
10. It can be used as low-cost feedstock for agricultural farming.

Sources: 

http://www.wired.com/wiredscience/2009/04/doubleduckweed/

http://www.mobot.org/jwcross/duckweed/duckweed.htm

http://www.isaaa.org/kc/cropbiotechupdate/article/default.asp?ID=4250

www.mdpi.com/journal/energies; article, Improving Production of Bioethanol from Duckweed (Landoltia punctata) by Pectinase Treatment

Twin Tasks to Tango With

After a couple of weeks of research, two objectives congealed right off the bat (well, three, but two immediate ones):

1. I would have to find a way to reliably grow the duckweed... to last throughout the winter.
2. I would have to assay the duckweed populations for d-glucose levels in order to determine which populations to cultivate.
3. Then there was physically inducing sexual reproduction in the plants.

Both John Cross's website, The Charms of Duckweed (http://www.mobot.org/jwcross/duckweed/growing-duckweed.htm) as well as Rutgers Duckweed Stock Cooperative's FAQ page have been incredibly helpful in addressing the first issue - I ended up ordering  Sigma Aldrich's SCHENK AND HILDEBRANDT BASAL SALT mixture (S6765) which could be reconstituted to make 10L of growth media. 

Number 2 posed a bit more of a challenge. I needed a legitimate starch assay kit - a simple Benedict's test would not be enough, because in order to determine percent starch in each sample, I would need something a bit more quantitative. The best deal I could find in the United States was Sigma Aldrich's Starch Assay Kit (STA20), which cost $129.50 per kit!

Luckily, my high school's Science Department Supervisor, Mrs. McLelland-Crawley, was there to the rescue!!! 

She helped me big time; she applied for a grant and I wound up receiving an amount that would cover the cost of two of these kits! It was amazing. Now, I can finally order the necessary materials, and get started! 

First Steps

When I was talking with my guidance counselor, Mrs. Fregosi (who is fabulous!), back in the Spring of 2013 to discuss my senior year schedule, I knew I wanted to spend my culminating year of high school pursuing an original inquiry; I was not new to scientific research. Since I was a freshman, I've participated in my school's chapter of Rutgers' University's Waksman Student Scholars Program, where, in analyzing the genome of duckweed, I first learned of its enticing potential; and in the summer of my junior year, I worked in a Team Project at New Jersey's Governor's School of the Sciences, using the facilities of Drew University to study the behavior of oculomotor behavior during reading. Both experiences have been thrilling and made me realize just how much I enjoy doing lab work. And, it is my firm, personal belief that doing hands-on research projects - going out there, in the undiluted spirit of scientific questioning - make for the best learning experiences; experiences, that tests or lectures wholly  cannot offer.

So, in order to do my much sought-after Independent Study course, I had to first meet with Dr. B to hash out the general backbone of the course, and then I submitted my proposal to my principal, Mr. Lepold. Needless to say, happily, it was approved; and then I got started on my preliminary research: browsing through scientific journals, looking at published academic papers on this subject, to see what other scientists were doing and in turn, get a better grasp of background concepts, what my project would entail, and what I could expect to encounter.

This meant lots of trips to the library - but I stumbled across cool research endeavors being carried out in the rest of the world. For example researchers at Arkansas State University (Ge, Zhang, et. al.) grew duckweed in agricultural wastewater and manipulated growth conditions through nutrient starvation and cultivation in the dark with the addition of glucose. And, halfway across the world, researchers Chen, Jin, et. al. increased glucose yield of their L. punctata samples through treatment with pectinase.

My approach is slightly different - I will be employing the concepts behind evolution I learned two years ago in AP Bio in my artificial selection of duckweed.

We'll see where that takes me this year! 

Hello! Welcome to my Research Project!

Hi there! My name is Celena Chen and I am a High School South senior who is currently doing a Research in the Sciences Independent Study this year on duckweed (aka Lemnaceae). 
Everyone knows that environmental sustainability and renewable sources of energy are major concerns that face our current and future generations. We need new, reliable sources of fuel, fast, to power our cities, populations, world; but at the same time, it is imperative that we look to minimize our ecological footprints by looking to environmentally-friendly sources of fuel.
This is where duckweed come in. Duckweed have gained recent headway for their potential as viable biofuel sources. They have two remarkable characteristics  that lend them well to being used as such: their rapid growth rate, and ability to accumulate high densities of starch (Xu, Zhao, Stomp, et. al., Biofuels Future Science Group Review, 2012) .What I am interested in is its latter quality. For my project, I am working with my mentor, AP Biology instructor Dr. Meenakshi Bhattacharya, who was also my Honors Bio teacher, in attempting to artificially select duckweed for higher starch content, as elevated D-glucose levels is a viable trait for large-scale production of ethanol. My hope is that the research I do this year at my high school will be relevant to the ongoing global hunt for better, smarter biofuels.