This protocol is a modified version of the DuBois assay (DuBois et al. 1956) for the quantification of total carbohydrate content of a sample.

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1 Carbohydrate Analysis 1. Purpose This protocol is a modified version of the DuBois assay (DuBois et al. 1956) for the quantification of total carbohydrate content of a sample. 2. Principle Sulphuric acid hydrolyses carbohydrates in the sample to their monomeric subunits (glucose/fructose/mannose etc.). The total carbohydrate monomer concentration is then measured by using the colorimetric reaction between phenol and sulphuric acid in the presence of carbohydrates. This is measured in a spectrophotometer at 485 nm. 3. Requirements 3.1. Equipment, materials and reagents Heater block or water bath Glass centrifuge vials (13 mm diameter) Glucose (laboratory grade) Phenol (laboratory grade) (Highly Toxic) Sulphuric acid, concentrated (laboratory grade) Sulphuric acid, 1M (can be made from concentrated) Spectrophotometer (capable of reading at 485 nm). Acid resistant cuvette or acid resistant 96 well plate depending on spectrophotometer type. Ultrapure water 4. Hazards and precautionary statements 1

2 Phenol is highly toxic and can be absorbed by the skin. Extra care must be taken to prevent any contact with it, especially in its concentrated form when weighed out and dissolving. It has an analgesic effect, so contact may not initially be felt. Wear a laboratory coat, eye protection and gloves (nitrile). When handling concentrated sulphuric acid or phenol double glove with nitrile gloves and use thick rubber gloves on top. All dispensing, pipetting, evaporation and disposal of solvents and acids should take place in a fume cupboard with the splash shield as low as possible. A fume hood should also be used for any work with phenol. Any waste solvents need to be disposed of in an appropriate container that clearly states what is contained. Further processing depends on local protocols. Care should be taken when disposing of phenol waste. For clean-up information or in cases of direct solvent contact please consult MS- DS information. Some information is shown below. Phenol Acutely toxic if swallowed or in contact with skin or if inhaled, causes severe skin burns and eye damage, suspected of causing genetic defects, causes damage to target organs, toxic to aquatic life with long lasting harmful effects. Has an analgesic effect as well as neurotoxic effect on central nervous system, so contact with it may not initially be felt. Sulphuric acid, concentrated Corrosive, causes severe skin and eye burns. 5. Procedure 5.1. Preparation of solutions and materials Phenol is dissolved in ultrapure water to make a 5% w/v solution. Take when weighing and dissolving as it is highly toxic. It is mixed thoroughly using a magnetic stirrer for at least 20 minutes. The stirrer remains in the phenol solution until it is depleted. A 0.01mg/ml solution of glucose is prepared from the glucose standard. A 1M sulphuric acid solution is created if required from concentrated sulphuric acid. Ensure the water is added to the glass vessel before the concentrated acid, not the reverse. 2

3 Glass vials and Teflon lined lids need to be soaked in Neutracon detergent (1%), then dried and the glass vials cleaned in a furnace at 500 C Preparation of standards and samples Before analysis samples of biomass are freeze dried and then ground to a powder using a pestle and mortar or similar equipment. This then kept in a -80 C freezer. Before use, samples are removed from the freezer, allowed to warm to room temperature then placed in a desiccator to remove any water and prevent samples hydrating (which would affect sample weight during weighing) Measurement of standards and samples Standard Curve Six boiling tubes were cleaned and dried. 1ml of glucose solution was pipetted into the first test tube. Into the subsequent test tubes 0.8ml, 0.6ml, 0.4ml, 0.2ml and 0 ml of the glucose solution was pippetted. This was then reversed and 0ml of de-ionised water into the first test tube, pipette 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1ml into the subsequent test tubes ensuring the total volume is 1ml. 0.5ml of 98% H2SO4 was added to the samples. 0.5ml of the 5% phenol solution was quickly added directly into the sample! (Not down the side of the glass tube). TAKE CARE- this reaction is exothermic and care must be taken to prevent the sampling bubbling over the top of the boiling tube. This was left for 30 minutes to cool. These samples were carefully poured into acid resistant cuvettes. This was read in the spectrophotometer using a UV/Vis at 485nm. These results were plotted using Microsoft Excel and the equation and r 2 value on the curve of the curve recorded. Sample Analysis 5mg (±10%) of sample is weighed into tubes suitable for containing 1M H 2SO 4 and that can be centrifuged, usually glass tubes with screw-closed lids. The sample weights were recorded in a notebook. 3

4 2mL of 1 molar H2SO4 is added to each tube containing sample. A blank can be added at this stage to ensure no extra carbohydrates or interferences are present in the analysis. This is then heated for 1 hour at 90 C in a water bath or heater block. The samples were then centrifuged for 10 minutes at 5500 rpm. 1ml of supernatant was pipetted into a boiling tube. 2.5ml of sulphuric acid was added to this boiling tube quickly followed by 0.5ml of phenol. This was then left for 30 minutes to cool and carefully poured into acid resistant cuvettes or into an acid resistant 96 well plate. This was then read again using the UV/Vis at 485nm, with each sample being read three times to check there is low variability between readings. The carbohydrate content was calculated using the equation from the standard curve and the average of the three absorbance values for each sample. If a sample gives an absorbance value above 1 then the analysis needs to be repeated from pipetting the supernatant, using a lower volume of the supernatant as appropriate. A colour in the boiling tubes while cooling that is more intense than the standards is likely to produce an absorbance value above Calculation of results The standard curve is generated by plotting absorbance on the x-axis, and glucose concentration on the y-axis. This gives an equation of the form y = Ax + B. The R 2 value should be more than 0.95, though above 0.99 is better. Inputting the absorbance as x gives the carbohydrates as compared to glucose. 7. Quality Control Standards should produce a linear graph when plotted, with an R 2 ideally above Blanks included with the samples should give absorbance values at the background level of the machine (i.e. the reading given when no sample or DI water is present in the spectrophotometer). 4

5 Each sample is read in the spectrophotometer three times to identify any variance of readings, and the average absorbance value used where no great variance is found. 8. Errors and interferences Every sample should be duplicated, or triplicated where sample volume permits, to counter variation in readings. 9. Waste stream and proper disposal All chemicals need to be disposed of according to local regulations and protocols. Placing waste chemicals into a waste chemical bottle for collection and proper disposal. Care should be taken with any waste containing phenol. 10. References DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anlytical Chemistry, 28: Other points If samples are stored in a plastic container then they will become statically charged. This may cause difficulties when biomass is removed from the tubes for weighing. 5