Degree of biomass substrate solubilization

When evaluating the effect of pretreatment and especially enzymatic hydrolysis of biomass, we normally measure the monomeric sugar produced. At most time, quite a lot of soluble oligomers existing in the hydrolyzates that could not be measured by the standard HPLC analysis, which will lead to lower hydrolysis yield. This can be confirmed by post-dilute acid hydrolysis, showing several fold increase in  monomeric sugars (glucose and xylose) yield. The reason for this is that most of the commercial enzyme cocktails are not for global use and need customization for a specific biomass substrate. Some new technology ends up with strains than can work on low DP soluble sugars. Therefore, measurement of the degree of biomass solubilization after pretreatment or/and enzymatic hydrolysis will be useful information for the estimation of the treatments.  A weight loss is rough, but quick method.

Analytical Methods for Mass Balances

Biomass compositional data is essential to beginning any study, especially the carbohydrate contents (both soluble and  structural carbohydrates) that is the base to calculate theoretical ethanol and sugars yields, to close carbohydrate mass balances, to determine the enzyme loadings, and to calculate the process efficiencies.

1. Waxes and fats are extracted into either ether or hexane using sonication.
2. The soluble sugars are subsequently extracted in 80% (v/v) ethanol, a concentration which is selective for monosaccharides
3. Wateris then used to extract any water-soluble polysaccharides (e.g., fructans)
4. Starch is then removed by using amylase in acetate buffer

The final residual material is essentially pure plant cell walls, which is called "structural carbohydrates", including hemicellulose and cellulose. The structural carbohydrates composition will be analysized following two-step acid hydrolysis using H2SO4.

As a complete compositional analysis is labor-intensive, it is often more practical to measure selected components only. Soluble sugars can be extracted by sonication in water if the samples are not to be processed for determining starch contents. For total carbohydrate analysis (including soluble sugars), a two-stage acid hydrolysis procedure similar to NREL protocol can be directly applied to native samples

Non-structural carbohydrate in biomass

In extractives, a fraction of chemical components are those containing sugars, referred to as either water-soluble-carbohydrate, or non-structural carbohydrate, or free sugars, which is mostly composed of the monosaccharide of D-fructose, the disaccharide of sucrose, the trisaccharide of raffinose, and tetrasaccharide of stachyose as well as the group of polysaccharides known as fructans. In addition, arabinogalactan can be classified as among the extractives.

These non-structural sugars tend to be intermediates and primary products of photosynthesis and serve functions in storage, translocation, and metabolic utilization of carbon as well as protection against abiotic stresses. They also play an important role in the dynamics of piles of stored biomass.

The non-structural sugars tend to be present in much higher concentrations in grassy than woody feedstocks. As reported (Chen et at, 2007 and 2010),carbohydrates (primarily sucrose, glucose, and fructose) were found to be the predominant water-soluble components of corn stover and switchgrass, accounting for 18-27% of the dry weight of switchgrass extractives and 30−46% of the dry weight of corn stover extractives (4−12% of the dry weight of feedstocks). The actual level of free sugars in harvestable biomass depends on CO2 assimilation and demand for these assimilates, mainly for respiration and the production of protein and structural carbohydrates (White, 1973). Given that such needs vary greatly with growth cycle, nutritional state, tissue, season, and management regimes, the non-structural sugar concentrations tend to be much more dynamic than those of the structural polysaccharides (Wulfes et al., 1999).

Whether or not these sugars add to sugar yields in fractionation procedures will depend on whether or not they are retarded by process conditions.

Don't panic when your mass great than 100% for biomass composition analysis

When we do biomass chemical composition analysis, it often turns out the mass add-up of all the chemical components great than or less than 100%, which is very common.
1. Different chemical components are analyzed by different methods and instrument, which cause the errors
2. For sugar analysis, each sugar has different optimal post-hydrolysis conditions, which cause both under and over hydrolysis of the sugars
3. Klason lignin includes some ash and proteins, which are accounted for twice
4. Uronic acid and acid lignin should be included when doing mass balance.
5. The standard solution composition should be as close as possible the real samples