Observations Nov/2/2009

2-Nov-2009

1. In half of the beakers the algae had sediment during the weekend.

2. In a few they had rose up from the beakers and felt into the tank, minimizing its volume.

3. After shaking them manually they all look bright green and healthy.

4. The pH had risen in all, but the one with 1.8g/L urea had rise even more going from ~6.15 to ~7.10.

5. The algae in all 3 concentrations are growing nicely.

Biodiesel

Estas son algunas de las imagenes del material despues de ser floculado y presto para ser procesado.

First lipid test

Ya hicimos nuestra primera corrida para la extracción de lípidos a nuestras algas. Esta fue todo un exito. Con esto logramos varias cosas. Primero, validamos el procedimiento a seguir para este análisis. Segundo superamos todos los problemas relacionados al mismo variando muchos de los pasos a seguir. Y tercero pero no menos importante, cuantificamos la cantidad de lípidos dentro de esta muestra de cultivo.

New Flocculation test

As part of the validation of our procedure, we prepared a test run in which we became familiar directly with all the procedure. Although already we know this in a theoretical form, is for us important also to know it form practical and to identify the areas of greater difficulty, thus to take precautions. And we already found the first one.

The first step after collecting the sample of the culture was to flocculate it. This, as we mentioned in the flocculation test, would help us to conglomerate the algae and to discard the present water for several things, first to work with a smaller volume, second to be able to calculate the amount of hexane and isopropyl that we will use to break the cells and finally not  have interferences with substances that are present, like surpluses of the nutrients.

But this was not so easy. The flocculation process is highly affected by the acidity of the substance to be flocculate and that was the factor that we had to vary. At the time of testing firstly our cultures were in acid means, pH 5.3. At the time of making this test  the pH was on 8 affecting directly the behavior of the substance to be flocculate.

According to the Handbook book of Microalgal culture, Biotechnology and applied phycology, A. Richmong 2007; pp 217, they mention that in algae cultures with a pH from 11.8 to 12 a flocculation of  a 95% of efficiency can be given. According to this information we decided to test several times to observe the behavior of our algae on different degrees from acidity. For this we tested to pH of 12 - 8 and 5.4. This, to try to see the specific behavior of our algae at the time of being floculate. The pH of 8 is the original one from the culture without being manipulated, 12 are an adjustment with NaOH and 5,4 were fit with acetic acid. For our surprise the  culture with more acidity was the best one to be flocculate, this as much by the amount of cells suspended like by the rapidity, followed by which it was basic means. It is possible to indicate, they can appreciate and it in the image next, the cultures in the middle basic slightly changed of color becoming a little yellow, although this color is not function of amount of cells given to that the majorities of these were in the part inferior of the test tube agglomerate.

Inicialmente                                                                      Finalmente

Como parte de la validación de nuestro procedimiento, nos dimos a la tarea de llevar a cabo una corrida en la cual nos familiarizáramos directamente con el mismo. Aunque ya conocíamos el este de forma teórica, es para nosotros importante también conocerlo de forma práctica y identificar las áreas de mayor dificultad, para así tomar precauciones. Y ya encontramos la primera.

El primer paso después de colectar nuestra muestra proveniente del cultivo era flocular la misma. Esto, como mencionamos en el escrito prueba de floculación, nos ayudara a conglomerar las algas y descartar el agua presente para varias cosas, primero trabajar con un volumen menor, segundo para poder calcular la cantidad de hexano e isopropil que usaremos para romper las celular y por ultimo para no tener interferencias con sustancias que estén presentes, como remanentes de los nutrientes.

Pero esto no fue tan fácil. El proceso de floculación es altamente dependiente de la acides de la sustancia a flocular y ese fue el factor que tuvimos que variar. Al momento de hacer la primera prueba nuestros cultivos se encontraban en un medio acido, pH 5.3. Al momento de hacer esta corrida de corroboración de procedimiento el pH se encontraba sobre los 8 trastocando directamente el comportamiento de la sustancia a flocular.
Según el libro Handbook of Microalgal culture, Biotechnology and applied phycology, A. Richmong 2007 ;pp 217, ellos mencionan que en cultivos de algas con un pH de 11.8 a 12 se puede dar una floculación de hasta un 95% de eficiencia. Dado a esta información decidimos hacer varias pruebas para observar el comportamiento de nuestras algas a diferentes grados de acides. Para esto hicimos pruebas a pH de 12 - 8 y 5.4. Esto para tratar de ver el comportamiento especifico de nuestra alga al momento de ser floculada. El pH de 8 es el proveniente del cultivo sin ser manipulado, 12 es un ajuste con NaOH y 5.4 fue ajustado con acido acético. Para nuestra sorpresa el cultivo con una acides mayor fue el mejor en flocular, esto tanto por la cantidad de células suspendidas como por la rapidez, seguido por el que estaba un medio básico. Cabe señalar, y lo pueden apreciar en la imagen a continuación, los cultivos en medio básico cambiaron ligeramente de color tornándose un poco amarillos, aunque este color no es función de cantidad de células dado a que la mayorías de estas se encontraban aglomeradas en la parte inferior de la probeta.

Biodiesel culture

Nuestro cultivo sigue dando frutos. Este cultivo crece vertiginosamente. Hemos decido que este sera utilizado para nuestra primera tanda de biodiesel.

Antes

Solving problems with the research

Doing our research we have encountered several problems. The CO2 tank was used up completely by the first 3 days, so we needed to come up with another type of agitation since buying CO2 tanks are expensive.
We have purchased one is coming soon, but it will only be use for 2 cultures for now on. As our project we need to find out if CO2 is beneficial to algae growth so we have 2 cultures with CO2 and 2 without, we are measuring the cell growth and after 17 days determine which is better. Since the algae were left without agitation for some time, we plan to start over again with the 2 cultures that need CO2 while the other 2 (Non CO2) that are in the Shaker we will continue to monitor them and post there progress.

We also want to determine the best Nitrogen Concentration and Nitrogen deprivation period we use Urea as our Nitrogen source, for this experiments we have 15 cultures divided into 3 groups of 5 cultures:

1. Group 1 (green) has an Urea concentration of 1.2g/L
2. Group 2(red) has an Urea concentration of 1.8g/L
3. Group 3(blue) has and Urea concentration of 2.4g/L

For them we have been measuring there pH and cell growth daily and we will also measure there Urea consumption using an Spectrophotometer.

The problem that arise is that we were using CO2 as a source of agitation so when the tank ended they concentrated at the bottom and have not grown as we expected. We are going to start over with them too. We will use Air for a source of agitation. Another problem we identified is that the Nutrients we feed had a very acidic pH(~3). To regulate a pH at ~6.8 we have a new culture that we are investigating now for this one we pour in the nutrients of a 3 pH and use NaOH 0.1M and added it until the pH was ~6.8 then we poured in the algae its source of agitation is Air and we are measuring its pH and cell count each day.

If they grow successfully, we want to regulate the pH of all the 15 cultures once we start up again with them.

Stock Culture Non CO2 With CO2

Cell disruption

After floculate ours first sample of algae we get ready to break the cells in them. This, to obtain lipids contained for its measurement. For this by each gram of biomass we respectively used a mixture of hexane and isoporopyl 3/2. The hexane roll is to break the cell whereas the one of the isopropyl is to catch greasy compounds, lipids.

In the left image our mixture at the beginning of the process can be appreciated. In the part top it is possible to observed the lacks of color. This is the organic phase of the mixture. It is where is the mixture of hexane with isopropyl. Whereas in the part inferior and of a green color the watery phase is observed where are the algae and the water.

After a vigorous agitation of several minutes and letting rest the mixture, its being to be appraised as they changed the phases. We see how the part inferior lose its greenish color and the superior reddened. This, due to the lost of the chlorophyll of the algaes. These were catched by the present organic agents in the mixture. In the second image, the right, can be appreciated that difference in colors bought with the left image. That change in color in both phases is appraised. Demonstrating this that the disription of the cells was carried out.


Luego de flocular nuestra primera muestra de algas nos dispusimos a romper las células en ellas. Esto para obtener los lípidos contenidos para su medición . Para esto por cada gramo de biomasa utilizamos una mezcla de hexano e isoporopil 3/2 respectivamente. El papel del hexano es romper la celula mientras que el del isopropil es atrapar los compuestos grasos, lípidos.

En la imagen izquierda se puede apreciar como lucia nuestra mezcla al inicio del proceso. En la parte superior de la misma se puede observar que carece de color. Esta es la fase orgánica de la mezcla.Es aqui donde se encuentra la mezcla de Hexano con isopropil. Mientras que en la parte inferior y de un color verde se observa la fase acuosa donde se encuentran las algas y el agua.

Luego de una agitación vigorosa de varios minutos y dejando reposar la mezcla, se comenzo a apreciar como cambiaban las fases. Se podia ver como la parte inferior del envase perdia su color verdoso y el superior se coloreaba. Esto, debido a la perdida de la clorofila de las algas. Estas eran atrapadas por los agentes orgánicos presentes en la mezcla. En la segunda imagen, la derecha, se puede apreciar esa diferencia en colores comprada con la imagen izquierda. Se aprecia ese cambio en color en ambas fases. Demostrando esto que el ropimiento de las células se llevo a cabo.