Identification and Quantification of Sugars and Oils in Algae

Blake Johnson, Ching-Shuan Lau and Dr. Danielle Julie CarrierDepartment of Chemical Engineering, Department of Biological Engineering

April 26, 2010

Background:• Plant material wall is composed of cellulose and hemicellulose

(Figure 1).• Cellulose and hemicellulose have to be broken down into their basic

monomers (glucose and xylose) before being fermented into any biofuel or biobased product.

Figure 1: Molecular Structure of cellulose and hemicellulose• Currently, ethanol production through lignocellulose is not cost

effective. The process is very delicate and more expensive than producing ethanol through corn or producing gasoline. The process needs to be reduced in cost before lignocellulosic ethanol production can be considered.

• Green algae (Figure 2) could be a possible source of carbohydrates that can be transformed into various biobased products.

Figure 2: Dried green algae harvested from Noland Water Treatment Facility (Fayetteville, AR)

Project Objectives:• Release the cell wall material contained in algae through dilute acid

pretreatment and enzymatic hydrolysis. • Quantify the carbohydrates, the by-products (like formic acid,

hydroxymethylfurfural (HMF) and furfural) and the crude oil present in the Noland Water Treatment Plant algae.

• Determine if the carbohydrate profile is dependent on seasonal growth (May – September 2010)

Research Impact:• Increase ethanol and biodiesel production from algae by refining the

process of extracting sugars and oil.

Materials:

Procedure:Sugar extraction

1. Measure 2 gram of dried algae and mix in reactor tube with 20 mL of 1% sulfuric acid solvent.

2. Reactor tubes was placed in sand bath at 160 oC for 1 h.3. After the reaction, allowed the sample to cool, neutralized to pH 7 and filtered.4. Analyze the samples by HPLC to identify and quantify the sugars as well as the by-

products (formic acid, HMF and furfural) in pretreated algae samples.

Oil Extraction5. Place 3 g of dried algae in cellulose Thimble. 6. Extract with hexane in Soxhlet for 2 h to extract oil from algae.

*Keep cooling water running to condense hexane vapor and prevent Soxhlet from running dry.

7. Allow hexane to evaporate, leaving only extracted oil in flask.*Let flask and algae sit for 2 days to vaporize the remaining hexane.

8. Measure the gain in mass of the flask after the extraction to determine the amount of oil collected on the flask after extraction.

Figure 3: Reactor tubes used to hydrolyze algae in dilute acid hydrolysis by targeting the break down of hemicellulose into xylose monomer.

Figure 4: Sand bath used for the dilute acid hydrolysis pretreatment at 160o C

Figure 5: HPLC (High Performance Liquid Chromatography) used to identify and quantify the sugars and by-products in algae.

Figure 7: Soxhlet used to extract oil from algae. The sample shown on the right turned yellowish indicating the presence of oil, as compared to the sample on the left that remained clear.

Results:

Discussion:• Algae samples contained xylose and glucose in very small concentration (<10% of

the overall mass). • Algae samples harvested in June and July contained oil, as shown by the yellowish

color of hexane solvent (Figure 7). However, the mass gained from the oil is not significantly differently from algae of other months that had no visible oil present (Table).

• The oil content in algae is less than 3% mass of the overall algae mass, which is lower than algae used for biofuel that typically contains 30% oil.

• Based on the amount of oil and sugar extracted, this particular type of algae is not recommended for use in biofuel production.

Conclusion:• Algae sample used in this study contains very small quantity of oil and sugars in

the form of xylose and glucose.

Mg present in 2000 mg of Algae May June July August SeptemberLiquid Fraction Glucose 69.69 24.63 24.47 - 24.21After dilute acid Xylose 33.63 43.96 40.83 - 40.15pretreatment Formic Acid 120.45 75.07 82.00 42.67 169.40

Acetic Acid 55.03 64.34 35.94 15.37 88.13Furfural 1.83 1.42 0.89 0.20 1.50

Hydroxyl methyl furan (HMF) 1.98 2.25 1.52 0.70 2.97

Solid Fraction Glucose 37.79 47.75 36.57 - 45.74After enzymatic Xylose 2.33 35.30 27.42 - 32.12hydrolysisTotal Glucose 107.48 72.38 61.04 - 69.96

Xylose 35.96 79.25 68.25 - 72.27Formic Acid 120.45 75.07 82.00 42.67 169.40Acetic Acid 55.03 64.34 35.94 15.37 88.13

Furfural 1.83 1.42 0.89 0.20 1.50

Hydroxyl methyl furan (HMF) 1.98 2.25 1.52 0.70 2.97

Yield (g sugar / g algae) Glucose 5.4% 3.6% 3.1% - 3.5%Xylose 1.8% 4.0% 3.4% - 3.6%

May June July August

SeptemberMass of oil (mg) in recovered in 3 gram of algae 30.00 62.60 ± 10.75 21.00 51.30 ± 0.42 40.50 ± 7.78

% Oil Recovered / gram algae 1.0% 2.1% 0.7% 1.7% 1.3%

Figure 6: Water bath used for the enzymatic hydrolysis of pretreated algae pellet ( 55 oC for 24 hrs).

* Only has only valid set of results. The second set of results has an inaccurate flask reading.

3rd Annual FEP Honors Research Symposium