4. Set all water baths or incubators to the correct temperature prior
to commencing the assay as attemperation for the 100
C water
bath may take >2 h.
5. Example dilution of acid from 12 to 1 M would involve careful
addition of 11 mL RO water.
6. If using the 1000Â dilution factor for samples (for HPAE-PADbased total sugar analysis) and the subsequent detection response
is poor (poor signal-to-noise ratio through inadequately small
peak sizes), then a more concentrated sample dilution can be
evaluated such as 100Â.
7. The reaction of H 2 SO 4 with phenol is highly exothermic; therefore,
care should be taken. We advocate that all additions of reagents
(reactions) are conducted in a fume hood.
8. A large stock of HPLC and HPAEC-PAD standards may be produced, filtered through <0.45 μm filters, labelled, and stored at
À20
C for up to 3 months if frequent analysis is required. The
stock can be removed from the freezer, thawed, and vortexed prior
to use.
References
1. Sluiter A, Hames B, Ruiz R, Scarlata C,
Sluiter J, Templeton D, Crocker D (2008)
Determination of structural carbohydrates
and lignin in biomass. NREL Laboratory Analytical Procedure 1617
2. van der Wal H, Sperber BL, Houweling-Tan B
et al (2013) Production of acetone, butanol,
and ethanol from biomass of the green seaweed
Ulva lactuca. Bioresour Technol 128:431–437
3. Ge L, Wang P, Mou H (2011) Study on saccharification techniques of seaweed wastes for
the transformation of ethanol. Renew Energy
36(1):84–89
4. Trivedi N, Gupta V, Reddy C, Jha B (2013)
Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva
fasciata Delile. Bioresour Technol 150:106–112
5. Dubois M, Gilles KA, Hamilton JK et al (1956)
Colorimetric method for determination of sugars
and related substances. Anal Chem 28(3):350–356.
https://doi.org/10.1021/Ac60111a017
6. Kostas ET, Wilkinson SJ, White DA, Cook DJ
(2016) Optimization of a total acid hydrolysis
based protocol for the quantification of carbohydrate in macroalgae. J Algal Biomass Utln 7
(1):21–36
7. Wilkinson S, Smart K, Cook D (2014) A comparison of dilute acid and alkali catalysed
hydrothermal pre-treatments for bioethanol
production from brewers spent grains. J Am
Soc Brew Chem 72(2):143–153
8. Wilkinson S, Smart K, Cook D (2014) Optimisation of alkaline reagent based chemical
pre-treatment of brewers spent grains for
bioethanol production. Ind Crop Prod
62:219–227
9. Kostas ET, White DA, Du C, Cook DJ (2016)
Selection of yeast strains for bioethanol production from UK seaweeds. J Appl Phycol 28
(2):1427–1441
10. Wilkinson S, Smart KA, Cook DJ (2015) Optimising the (microwave) hydrothermal pretreatment of brewers spent grains for bioethanol
production. J Fuels. Doi: https://doi.org/10.
1155/2015/369283
190
Emily T. Kostas et al.
to commencing the assay as attemperation for the 100
C water
bath may take >2 h.
5. Example dilution of acid from 12 to 1 M would involve careful
addition of 11 mL RO water.
6. If using the 1000Â dilution factor for samples (for HPAE-PADbased total sugar analysis) and the subsequent detection response
is poor (poor signal-to-noise ratio through inadequately small
peak sizes), then a more concentrated sample dilution can be
evaluated such as 100Â.
7. The reaction of H 2 SO 4 with phenol is highly exothermic; therefore,
care should be taken. We advocate that all additions of reagents
(reactions) are conducted in a fume hood.
8. A large stock of HPLC and HPAEC-PAD standards may be produced, filtered through <0.45 μm filters, labelled, and stored at
À20
C for up to 3 months if frequent analysis is required. The
stock can be removed from the freezer, thawed, and vortexed prior
to use.
References
1. Sluiter A, Hames B, Ruiz R, Scarlata C,
Sluiter J, Templeton D, Crocker D (2008)
Determination of structural carbohydrates
and lignin in biomass. NREL Laboratory Analytical Procedure 1617
2. van der Wal H, Sperber BL, Houweling-Tan B
et al (2013) Production of acetone, butanol,
and ethanol from biomass of the green seaweed
Ulva lactuca. Bioresour Technol 128:431–437
3. Ge L, Wang P, Mou H (2011) Study on saccharification techniques of seaweed wastes for
the transformation of ethanol. Renew Energy
36(1):84–89
4. Trivedi N, Gupta V, Reddy C, Jha B (2013)
Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva
fasciata Delile. Bioresour Technol 150:106–112
5. Dubois M, Gilles KA, Hamilton JK et al (1956)
Colorimetric method for determination of sugars
and related substances. Anal Chem 28(3):350–356.
https://doi.org/10.1021/Ac60111a017
6. Kostas ET, Wilkinson SJ, White DA, Cook DJ
(2016) Optimization of a total acid hydrolysis
based protocol for the quantification of carbohydrate in macroalgae. J Algal Biomass Utln 7
(1):21–36
7. Wilkinson S, Smart K, Cook D (2014) A comparison of dilute acid and alkali catalysed
hydrothermal pre-treatments for bioethanol
production from brewers spent grains. J Am
Soc Brew Chem 72(2):143–153
8. Wilkinson S, Smart K, Cook D (2014) Optimisation of alkaline reagent based chemical
pre-treatment of brewers spent grains for
bioethanol production. Ind Crop Prod
62:219–227
9. Kostas ET, White DA, Du C, Cook DJ (2016)
Selection of yeast strains for bioethanol production from UK seaweeds. J Appl Phycol 28
(2):1427–1441
10. Wilkinson S, Smart KA, Cook DJ (2015) Optimising the (microwave) hydrothermal pretreatment of brewers spent grains for bioethanol
production. J Fuels. Doi: https://doi.org/10.
1155/2015/369283
190
Emily T. Kostas et al.