the biggest challenge to quantification of protein in whole biomass
with a colorimetric method is the observation that the Folin
reagent will react with other reducing substances in solution
(including reducing sugars in solution), making this assay susceptible to algae species- and growth condition-specific interferences.
These interferences also cause an incorrect high bias (up to twofold) to the calculated protein concentration data [9, 10].
Perhaps the most direct method for protein determination is to
measure the constituent amino acids (AA) released after a harsh acid
hydrolysis (often a 6 M HCl digestion for 24 h) followed by HPLC
amino acid analysis [11]. This method has the advantage of breaking down the biological matrix and does not depend on selective
removal of protein from the biomass. Free amino acids can also be
detected in the hydrolysate along with AAs hydrolyzed from proteins. However, for complete AA analysis multiple (up to six)
hydrolyses for each sample are needed to completely quantify the
chemically diverse amino acids found in proteins [12]. Direct
amino acid quantification after several hydrolyses per sample is
useful for comprehensive total protein content and compositional
analysis; it is also expensive and time consuming and thus less
applicable for screening or processing a large number of samples.
There are literature reports that contain the amino acid profiles of a
large number of species of algae and represent different growth
conditions [4, 13–15]. In the absence of a dedicated custom
nitrogen-to-protein conversion factor, an average factor based on
literature data can be used instead.
Nitrogen analysis methods (%N) have been used to quickly
determine crude protein values, though nonprotein nitrogen can
interfere with this calculation (see Note 8). Reference methods are
available to determine nitrogen in feedstocks such as animal feed,
cellulosic biomass, and grains though the nitrogen-to-protein value
used for this calculation tends to overestimate algal protein content
[16]. Both a combustion (Dumas [17]) or a Kjeldahl method is
acceptable [18], with the combustion method typically being preferred because of simplicity. In brief for a combustion-based analysis of elemental nitrogen weighed samples are combusted in oxygen
at temperatures between 950 and 1100
C. The combustion products (including N and NO x ) were swept by a helium carrier gas
through combustion catalysts, scrubbers, and a tube filled with
reduced copper. The copper removes excess oxygen and reduces
the NO x to N 2 . The N 2 was then separated from other gases on a
chromatography column and measured with a thermal conductivity
detector (TCD). By utilizing the completeness and specificity of
direct AA analysis to determine algae-specific nitrogen-to-protein
conversion factors and combining with a simpler %N method a
useful high throughput algae protein method emerges. Once a
234
L. M. L. Laurens et al.
with a colorimetric method is the observation that the Folin
reagent will react with other reducing substances in solution
(including reducing sugars in solution), making this assay susceptible to algae species- and growth condition-specific interferences.
These interferences also cause an incorrect high bias (up to twofold) to the calculated protein concentration data [9, 10].
Perhaps the most direct method for protein determination is to
measure the constituent amino acids (AA) released after a harsh acid
hydrolysis (often a 6 M HCl digestion for 24 h) followed by HPLC
amino acid analysis [11]. This method has the advantage of breaking down the biological matrix and does not depend on selective
removal of protein from the biomass. Free amino acids can also be
detected in the hydrolysate along with AAs hydrolyzed from proteins. However, for complete AA analysis multiple (up to six)
hydrolyses for each sample are needed to completely quantify the
chemically diverse amino acids found in proteins [12]. Direct
amino acid quantification after several hydrolyses per sample is
useful for comprehensive total protein content and compositional
analysis; it is also expensive and time consuming and thus less
applicable for screening or processing a large number of samples.
There are literature reports that contain the amino acid profiles of a
large number of species of algae and represent different growth
conditions [4, 13–15]. In the absence of a dedicated custom
nitrogen-to-protein conversion factor, an average factor based on
literature data can be used instead.
Nitrogen analysis methods (%N) have been used to quickly
determine crude protein values, though nonprotein nitrogen can
interfere with this calculation (see Note 8). Reference methods are
available to determine nitrogen in feedstocks such as animal feed,
cellulosic biomass, and grains though the nitrogen-to-protein value
used for this calculation tends to overestimate algal protein content
[16]. Both a combustion (Dumas [17]) or a Kjeldahl method is
acceptable [18], with the combustion method typically being preferred because of simplicity. In brief for a combustion-based analysis of elemental nitrogen weighed samples are combusted in oxygen
at temperatures between 950 and 1100
C. The combustion products (including N and NO x ) were swept by a helium carrier gas
through combustion catalysts, scrubbers, and a tube filled with
reduced copper. The copper removes excess oxygen and reduces
the NO x to N 2 . The N 2 was then separated from other gases on a
chromatography column and measured with a thermal conductivity
detector (TCD). By utilizing the completeness and specificity of
direct AA analysis to determine algae-specific nitrogen-to-protein
conversion factors and combining with a simpler %N method a
useful high throughput algae protein method emerges. Once a
234
L. M. L. Laurens et al.