3 Methods
3.1 Sample
Preparation for
Hydrolysis
1. Label 10 mL heavy-wall glass tubes for each of the samples and
quality control samples (see Note 4).
2. Record the weight of all labeled tubes to the nearest 0.1 mg.
Written labels should be dry before the weight is recorded.
3. Weigh 25 Æ 2.5 mg of freeze-dried algal biomass into a labeled
and tared 10 mL glass tube (see Note 5). Record the weight of
the sample to the nearest 0.1 mg.
4. Dry samples in vials overnight to obtain an oven-dry weight
before proceeding with the hydrolysis (see Note 6).
3.2 Sample
Hydrolysis
1. Add 250 μL of 72% (w/w) sulfuric acid to each tube (see Note
7).
2. Place tubes in a 30 Æ 3
C water bath with a water level set to
just above the sample/acid level in the tubes. Incubate samples
for 1 h (see Note 8).
3. After 1 h, remove tubes from the water bath, and add 7 mL of
18.2 MΩ water to each tube. This will bring the sulfuric acid
concentration to 4% (w/w) (see Note 9).
4. Autoclave the sealed samples for 1 h at 121
C using the liquids
(slow) setting (see Note 10).
5. After completion of autoclave cycle, allow samples to cool (see
Note 11).
3.3 Spectrophotometric
Determination of
Monomeric Sugars
1. Hydrolyzed samples for spectrophotometric analysis should be
prepared by separating the solids from the hydrolysate (see
Note 12). See Fig. 1b for a typical hydrolysate.
2. Preheat a digital dry block that fits 13 Â 100 mm glass test
tubes (or equivalent reaction vials) to 80 Æ 3
C (see Note 13).
3. Prepare standard and sample dilutions directly in the 13 Â 100 mm
glass reaction vials. Keep batches to no more than 40 reaction vials
(see Note 14).
4. Prepare the MBTH working solution in a beaker or plastic tube
(see Note 15).
5. Add 500 μL 0.5 M NaOH to each glass tube and vortex.
6. Add 500 μL MBTH working solution (from Step 4 above) to
each glass tube, vortex carefully to mix, and cover the tubes
with a glass marble or cap. Immediately place the tubes with
solutions in the preheated dry block at 80
C for 15 Æ 1 min
(see Note 16).
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
195
3.1 Sample
Preparation for
Hydrolysis
1. Label 10 mL heavy-wall glass tubes for each of the samples and
quality control samples (see Note 4).
2. Record the weight of all labeled tubes to the nearest 0.1 mg.
Written labels should be dry before the weight is recorded.
3. Weigh 25 Æ 2.5 mg of freeze-dried algal biomass into a labeled
and tared 10 mL glass tube (see Note 5). Record the weight of
the sample to the nearest 0.1 mg.
4. Dry samples in vials overnight to obtain an oven-dry weight
before proceeding with the hydrolysis (see Note 6).
3.2 Sample
Hydrolysis
1. Add 250 μL of 72% (w/w) sulfuric acid to each tube (see Note
7).
2. Place tubes in a 30 Æ 3
C water bath with a water level set to
just above the sample/acid level in the tubes. Incubate samples
for 1 h (see Note 8).
3. After 1 h, remove tubes from the water bath, and add 7 mL of
18.2 MΩ water to each tube. This will bring the sulfuric acid
concentration to 4% (w/w) (see Note 9).
4. Autoclave the sealed samples for 1 h at 121
C using the liquids
(slow) setting (see Note 10).
5. After completion of autoclave cycle, allow samples to cool (see
Note 11).
3.3 Spectrophotometric
Determination of
Monomeric Sugars
1. Hydrolyzed samples for spectrophotometric analysis should be
prepared by separating the solids from the hydrolysate (see
Note 12). See Fig. 1b for a typical hydrolysate.
2. Preheat a digital dry block that fits 13 Â 100 mm glass test
tubes (or equivalent reaction vials) to 80 Æ 3
C (see Note 13).
3. Prepare standard and sample dilutions directly in the 13 Â 100 mm
glass reaction vials. Keep batches to no more than 40 reaction vials
(see Note 14).
4. Prepare the MBTH working solution in a beaker or plastic tube
(see Note 15).
5. Add 500 μL 0.5 M NaOH to each glass tube and vortex.
6. Add 500 μL MBTH working solution (from Step 4 above) to
each glass tube, vortex carefully to mix, and cover the tubes
with a glass marble or cap. Immediately place the tubes with
solutions in the preheated dry block at 80
C for 15 Æ 1 min
(see Note 16).
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
195