Fermentation of Brown Sargassum Seaweeds using Pichia

angophorae: Effect of pH Reduction to Bioethanol Yield

Experimental Plan

Balagtas, Christylene
Bañares, Maria Niña

This Experimental Plan for the study entitled “Fermentation of Brown Sargassum Seaweeds
using Pichia angophorae: Effect of pH Reduction to Bioethanol Yield” prepared and submitted by
Christylene S. Balagtas and Maria Niña C. Bañares, in partial fulfillment of the requirements for the
course in Plant Design and Project Study has been examined, and endorsed for submission.

_________________________
Engr. Melba T. Mendoza
Mentor

Noted by:

Engr. Richel de Villa_______________________________

College of Engineering Administrative Assistant

_________________________________________________ _June 25, 2010_

Signature Date
 Fermentation of Brown Sargassum Seaweeds using Pichia angophorae: Effect of pH Reduction to Bioethanol Yield
General Statement of the Problem: If brown Sargassum seaweed is a good marine biomass for bioethanol production through
fermentation which is affected by operating conditions like pH, a study is necessary to identify what condition(s) is essential to have
high yields.
Plant Design Objective(s)
1. To determine the effect of pH at
fermentation to ethanol
yield
Experimental Parameters to be
Facilities Requirements Schedule
Objective(s) Measured
I. To obtain the Equipment or apparatus:
July 2 – August 27,
seaweeds needed Mass (kg) Balance, refrigerator (for
2010
for fermentation storage purposes)
Equipment or apparatus: August 27-
pH meter, fermentor, September 26, 2010
centrifuge, blender, glass
vials, flasks, beakers,
II. To determine the graduated cylinder, and
pH of the liquid in volumetric pipettes
pH,
the fermentor at
volume of slurry (L)
sampling Materials/chemicals/enzymes:
High-temperature (HT) a-
amylase, glucoamylase
(Allcoholase II), 1 M HCl, 5
mM H2SO4, urea, water bath,
and active dry yeast
III. To determine final ethanol Equipment or apparatus
amount of ethanol concentration (g/mL) HPLC, glass vials, flasks,
produced beakers, graduated cylinder,
and volumetric pipettes
Materials/chemicals/enzymes:
 Heptane, Cyclohexane, 1-
Methylnaphthalene, o-Xylene
(1,2-Dimethylbenzene), and
deionized water
III. To monitor the Equipment to be used:
composition of the Spectrophotometer or
sample (remaining Concenration (g/mL) refractometer, glass vials,
sugars, laminarin, flasks, pipettes and beakers
ethanol, methanol,
etc.) Materials/chemicals/enzymes:
n-Pentane or Toluene, 1-
Bromonaphthalene, distilled
or deionized water, 2,2,4-
trimethylpentane, and
Methylcyclohexane

V. To monitor the Temperature (⁰C) Equipment or apparatus:

temperature every Thermometer
sampling
VI. Submit samples Total yeast cell
to Biology counts and viable
Department for counts
analysis

IV. To monitor Apparatus:

dissolved oxygen Dissolved oxygen DO meter, beakers, and
(DO) every (ppm) flasks
sampling
Project Gantt chart:

WEEK NUMBER
JUNE JULY AUGUST SEPTEMBER OCTOBER NOVEMBER DECEMBER JANUARY FEB
TASKS 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2
Preliminaries (seek approval for proposal)
Prepare Experimental plan
Secure equipments to be used
Seek permission from Chem and Bio Department
Allocate budget
Secure seaweeds source
Purchase chemicals
Harvest and storage of seaweeds
Start experimental procedure
Acquire samples for tests
Subject samples for testing
Gather data
Evaluate data and results
Continue paper preparation
Seek advice and approval from mentor
Revise final paper
Prepare presentation for defense
MILESTONES
Preparations cleared for experiment x
Data analyzed x x x
Conclusions derived x x x
Final paper revised x x
Presentations organized x x x
Research paper defended x x
Final paper revised and approved x x x x
 APPENDIX G

STANDARD METHOF FOR DETERMINATION OF GLUCOSE CONCENTRATION USING THE NELSON-SOMOGYI

METHOD

The glucose concentration of any substrate will be measured using a spectrophotometric technique. This test determines all sugars
present in the sample as glucose, which is expressed in g/L of solution by taking absorbance readings of the actual sample and
standards at 660 nm using a spectrophotometer.

GENERAL PROCEDURE:

1. A set of standard glucose samples with the following concentrations (g/L): 0.05, 010, 0.15, 0.20, 0.25 is usually prepared.
Analyze directly the samples using the standard calibration curve.
2. Collect the samples from the fermentation effluents.
3. Take an aliquot of the samples and dilute it to the desired concentration. (Note: One may only need to perform several
trials in order to obtain the correct dilution.)
4. Take 2 mL of the sample and mix it with 2 mL of the Copper-Carbamate-Tartrate Reagent (CCTR) in a test tube.
5. Place the mixture in a water bath for a period of 15 minutes and allow it to cool.
6. Add 2 mL of the prepared indicator (BPR).
7. Mix thoroughly using a vortex mixer.
8. Read absorbance at 660 nm.
9. Establish the standard calibration cirve by plotting the absorbance readings against standard glucose concentration.
10. From the plot, obtain a linear relation between the absorbance and the glucose concentration.
11. Determine the glucose concentration of the sample determined from the linear relation of the standard curve. (Note: The
actual glucose concentration of the samples should be corrected for dilution.)

PREPARATION OF THE Copper-Carbamate-Tartrate Reagent (CCTR):

1. Mix approximately 10 g of sodium bicarbonate, 15 g of sodium carbonate, 90 g of sodium sulfate and 7.5 g Rochelle salt
and dilute to the mark in a 500 mL volumetric flask.
2. Mix about 45 g of sodium sulfate and 5 g of copper sulfate monohydrate and dilute to the mark in a separate 250 mL
volumetric flask.
3. Mix the two solutions only upon analysis with a volume ration 4:1.