Biomass Flocculation Study_IP-Free

Technical Report
All proprietary information has been removed from this document
All proprietary information has been removed from this document.
Title Biomass Flocculation Study
Author Kirk Oler
Organization S2G Biochem
Date November 28, 2014
Revision 2
Project Client X
Acknowledgements Bryan Gene for writing the Biomass Characterization Memo.
Mark Lewis for assistance selecting flocculants.
Bill McKean for technical consultation about flocculation.
Abstract
Flocculation was investigated as a potential unit operation to aid the conditioning of Biomass
prior to catalytic hydrotreating. Three different flocculating agents were investigated along with
operating parameters such as time, flocculating agent concentration, and pH. Efficacy of
flocculation was measured qualitatively by UV-Vis spectroscopy. Flocculation was at least half
as effective as granular activated carbon in removing color from solution.
Contents
Abstract........................................................................................................................................... 1
Contents .......................................................................................................................................... 1
Introduction..................................................................................................................................... 2
Statement of Problem...................................................................................................................... 3
Approach......................................................................................................................................... 3
Operating Conditions: Flocculant............................................................................................... 5
Various Concentrations of Flocculant and Coagulant ................................................................ 5
Comparison of Flocculant and GAC........................................................................................... 6
Operating Conditions: Coagulant ............................................................................................... 6
Results............................................................................................................................................. 7
Operating Conditions: Flocculant............................................................................................... 7
Various Concentrations of Flocculant and Coagulant ................................................................ 9
Comparison of Flocculant and GAC........................................................................................... 9
Operating Conditions: Coagulant ............................................................................................. 11
Summary and Conclusions ........................................................................................................... 11
References..................................................................................................................................... 12
Appendix....................................................................................................................................... 12
Appendix A. SOP for Analysis of Lignocellulosic Biomass ............................. 12

Technical Report
All proprietary information has been removed from this document Page 2 of 17
Appendix B. Photographs of Cationic Starch Coagulant Trials 1-8 .................. 16
Appendix C. Photographs of Comparison of Flocculant and GAC................... 16
Appendix D. Photographs of Experimental Controls......................................... 17
Introduction
Pretreatment is an important step in preparing biomass feed streams for catalytic hydrotreating.
It is important to remove substances which can precipitate on process equipment (e.g. lignin) as
well as those which can poison the catalyst (e.g. chloride). Granular activated carbon (GAC) has
been used by S2G to remove high molecular weight organic compounds such as lignin, fatty
acids and proteins which can contribute to solution color. However, very dirty feed streams
require large amounts of GAC to obtain sufficient purification. Flocculating agents are
commonly used in a variety of industries to remove contaminants from solution. Not only are
they readily available but they are effective at low concentration. For example a typical amount
in the pulp and paper industry is 0.5 pounds flocculant per ton of dry pulp (250 ppm, dry basis)
(McKean, 2014). Normal flocculant concentrations in settling basins is 0.1 - 0.5 g per cubic
meter of suspension (0.1 - 0.5 ppm, wet basis). For dewatering sludge with centrifuges and
filters, 2 – 8 kg flocculant per ton of dry sludge (2200 – 8800 ppm, dry basis) is used (Burkert &
Hartmann, 2012). This study found that with Biomass, flocculation removes solution color with
at least half, if not much more than, the efficacy of GAC. This could reduce the amount of GAC
needed to condition feed streams at the Pilot Plant meaning substantial cost savings in materials
and labor.
The objective of this study was to screen a few flocculating agents from the hundreds which are
commercially available. Due to the time constraints of this study, minimal experimental
replication was performed; consequently statistical analysis was not performed.
In this report the term “flocculating agent” refers generally to any material used to make a floc
within a suspension, such as a polyelectrolyte, flocculant, or coagulant. The term “flocculant”
refers to the specific cationic polyacrylamide flocculating agent tested in this study.
Streaming potential can theoretically be used to measure the effectiveness of flocculation
((AWWA), 2012). However, the instrument used to measure streaming potential for this study
was only precise enough to give qualitative not quantitative information about the net solution
charge. So streaming potential measurements were useful in determining that Biomass had a net
negative (anionic) charge which indicated the need for a cationic flocculating agent. However,
streaming potential measurements were not precise enough to determine how much of that
flocculating agent should be used (McKean, 2014).
This study determined flocculating agent efficacy via their ability to reduce supernatant
absorbance at 420 nm. UV-Vis spectroscopy was chosen because it was quick, available, and
affordable; however other methods may be more illuminating. For example, several documented
methods have measured the rate at which the floc settles or the rate at which it can be separated
from supernatant via various types of filtration (Burkert & Hartmann, 2012).

Technical Report
To account for sample dilutions, Beer’s Law was assumed to be applicable. That is:
𝐴 = 𝑎𝑏𝑐
Where 𝐴 is absorbance, 𝑎 is absorptivity (extinction coefficient), 𝑏 is path length through
solution, and 𝑐 is concentration of absorbing species. This is usually true for analyte
concentrations up to about 10 mM (Penner, 2010). Because the composition of Biomass is
somewhat uncertain and all the readings taken for this study were qualitative, minimal effort was
made to validate this assumption (see section “Approach: Operating Conditions: Coagulant”).
This study began with determining the streaming potential (i.e. net charge) of the Biomass. The
solution was found to be net anionic so cationic flocculating agents were investigated. First, a
cationic polyelectrolyte was used to see if flocculation was even possible. This was a highly
characterized, expensive, flocculating agent typically used to characterize the net charge of a
solution. This was used while the next flocculating agent was being selected. Second, a
commercial cationic polyacrylamide flocculant was used. The material had a relatively high
molecular weight and low degree of substitution (McKean, 2014). This flocculant was
investigated in three phases: (1) Investigating the operating conditions of time, presence or
absence of flocculant, and pH; (2) Comparing various concentrations; and (3) Comparing
flocculant and GAC. Third, a commercial cationic starch coagulant was used. This had a
relatively low molecular weight and intermediate (0.38) degree of substitution (McKean, 2014).
It was investigated relative to the operating conditions of initial sample dilution, concentration,
and pH.
Statement of Problem
Feed stocks have a substantial amount of color in solution which must be removed prior to
catalytic hydrotreating. Flocculation may be an additional unit operation to aid in such color
removal.
Approach
Biomass was prepared for testing by diluting with deionized water, centrifuging at 1700 g’s, and
decanting. Samples treated with cationic polyelectrolyte were diluted by a factor of six and
centrifuged for 20 minutes. Samples treated with cationic polyacrylamide flocculant were
diluted by a factor of 10 and centrifuged for 20 minutes. Samples treated with cationic starch
coagulant were diluted by factors of five and 10 and centrifuged for 40 minutes. Ten mL
supernatant samples were used for all tests.
These dilution factors were chosen based on previous experiments and analytical equipment
needs. Dilution factors of five and six were used because such were previously used for
preliminary Biomass testing at the Pilot Plant in Vancouver, B.C. Such dilutions were also
thought to be what might be used if flocculation were eventually performed at a pilot plant scale.
A dilution factor of 10 was used because it provided a suitable percent total solids for measuring
streaming potential with the Mütek PCD-04.

Technical Report
These centrifuge durations were chosen to minimize suspended solids in the Biomass
supernatant. Slight variation in the suspended solids content was visually observed between
batches centrifuged for 20 minutes. This variation was reduced when processing time was
increased to 40 minutes.
Total solids content of Biomass was determined by evaporation in a convection oven at 115° C
for at least nine hours. The average of duplicate samples of undiluted Biomass was 41% total
solids. The average of triplicate samples of 5x dilute Biomass was 6.3% total solids. The
average of nine replicate samples of 10x dilute Biomass was 3.2% total solids. After accounting
for dilution, the average total solids of all these 5x and 10x dilute Biomass samples was 32%.
This is nine percent less than the total solids in the undiluted Biomass samples. This difference
accounts for the solids removed during centrifugation and subsequent decanting.
Streaming potential (i.e. net charge) of Biomass was measured with a BTG Mütek PCD-04.
The cationic polyelectrolyte was poly-DADMAC (polydimethyl diallyl ammonium chloride),
sample #808, manufactured by BTG. A 10 mL sample of Biomass was adjusted to 0.667 mN
polyelectrolyte in a 20 mL glass vial as an initial test of flocc-ability. An experimental control
was made by adding deionized water to 10 mL Biomass 6x dilute supernatant until it was the
same volume as the test sample.
The cationic polyacrylamide flocculant used was BUFLOC 5031, date 02-11-08, lot: GMO,
manufactured by Buckman Laboratories, Memphis, Tennessee. This sample was used because it
was available and obtaining a fresh sample would have been time prohibitive. This was diluted
with deionized water to 0.1% v/v and used to make experimental samples with 16.7 – 34.9 µL
flocculant per g Biomass total solids (16,700 – 34,900 ppm, dry basis).
The cationic starch coagulant used was WESCAT C, manufactured by Western Polymer
Corporation, Moses Lake, Washington. This had a 0.38 degree of substitution. A 2% w/v
solution was prepared in 22° C deionized water which was then heated and stirred until the
solution viscosity visually increased and then decreased. It was used to make experimental
samples with 1.53 – 30.4 mg coagulant per g Biomass total solids (1,530 – 30,400 ppm, dry
basis).
Prepared flocculating agents and Biomass supernatant were stored covered and refrigerated.
They were brought to 22° C prior to use.
The pH meter was calibrated prior to use each day.
Because of the small sample volumes used, floc volumes were too small to quantify or
characterize directly.

Technical Report
Operating Conditions: Flocculant
A full-factorial design was used to investigate conditions which might impact the effectiveness
of color removal by cationic polyacrylamide flocculant. These conditions were time,
concentration, and pH. Low and high levels for these factors were selected as follows: 1 hr. vs.
24 hrs., no flocculant vs. 28.8 µL flocculant per g Biomass solids, and pH 5 vs. pH 11. See
Table 1 for complete experimental design. Trial 8 was used as the experimental control. All
trials were conducted at 22° C and performed in a random order.
Table 1: Full-factorial experimental design for cationic polyacrylamide flocculant
Trial
Time
(hr.)
Flocculant
Concentration
(µL flocc. / g
Biomass 4
solids)
pH
1 24 28.8 11
2 1 28.8 11
3 1 0.0 11
4 24 0.0 5
5 1 28.8 5
6 24 0.0 11
7 24 28.8 5
8 1 0.0 5
Eight 10 mL samples of 10x dilute Biomass were added to 50 mL glass beakers. Sample pH was
measured and adjusted using 0.275 N sodium hydroxide; flocculant addition followed
immediately thereafter. After the specified duration, pH was re-measured and samples were
passed through 0.45 µm filters into 20 mL glass vials in preparation for UV-Vis spectroscopy.
Effectiveness of each trial was measured by absorbance at 420 nm with a Shimadzu UV-1700
PharmaSpec spectrophotometer. This wavelength was useful for a qualitative comparison of
sample color as it appeared to the naked eye. Taking measurements at 240 nm for acid soluble
lignin was considered but abandoned because it would have been time prohibitive, especially
given that the readings would be qualitative anyway. Prior to use, the instrument was zeroed
with deionized water in an acrylic cuvette. Samples were measured at the pH specified in the
full-factorial experimental design. Samples were diluted such that absorbance readings were in
the range of 0.1 - 1.0.
Various Concentrations of Flocculant and Coagulant
Eight 10 mL samples of 10x dilute Biomass were added to 50 mL glass breakers. To four
samples was added 16.7 – 34.9 µL flocculant per g Biomass total solids. To the other four
samples, deionized water was added in volumes corresponding to the flocculant volumes added
to the other samples. These served as experimental controls. Samples were passed through 0.45

Technical Report
µm filters (those with deionized water immediately, those with flocculant after one hour) into 20
mL glass vials. Absorbance was measured with the method previously described.
See “Approach” section “Operating Conditions: Coagulant” for details about Biomass samples
treated with various concentrations of coagulant.
Comparison of Flocculant and GAC
Four 10 mL samples of 10x dilute Biomass were added to 20 mL glass vials.
The first sample was treated with just flocculant. To it was added 22.8 µL flocculant per g
Biomass total solids. After standing for one hour, the sample was passed through a 0.45 µm filter
into a clean 20 mL glass vial.
The second sample was treated with just GAC (DARCO 12x40, manufactured by Norit). The
sample was treated with 2 g. GAC, shaken for 30 seconds, let stand one hour, and decanted
through a 0.45 µm filter into a clean 20 mL glass vial.
The third sample was treated with flocculant followed by GAC. To it was added 22.8 µL
flocculant per g Biomass total solids. After standing for one hour, the sample was decanted
through a 0.45 µm filter into a 20 mL glass vial. Ten mL of this filtrate was treated with 2 g.
GAC, shaken for 30 seconds, let stand one hour, and decanted through a 0.45 µm filter into a
clean 20 mL glass vial.
The fourth sample was treated with GAC followed by GAC. The sample was treated with 2 g.
GAC, shaken for 30 seconds, let stand one hour, and decanted through a 0.45 µm filter into a
clean 20 mL glass vial. Ten mL of this filtrate was treated with a fresh 2 g. GAC, shaken for 30
seconds, let stand one hour, and decanted through a 0.45 µm filter into a clean 20 mL glass vial.
Each of the four samples were replicated three times.
An experimental control was made by passing 10 mL 10x dilute Biomass through a 0.45 µm
filter into a 20 mL glass vial.
Amounts, treatment durations, and procedures concerning GAC were taken from the SOP used
to prepare dirty samples for HPLC analysis, “Analysis of Carbohydrates in Lignocellulosic
Biomass.” A copy of this SOP is located in Appendix A.
Operating Conditions: Coagulant
A full-factorial design was used to investigate conditions which impact the effectiveness of color
removal by cationic starch coagulant. These conditions were Initial Dilution Factor, Coagulant
Concentration, and pH. Low and high levels of these factors were selected. Levels for Initial
Dilution Factor were 5x dilute Biomass and 10x dilute Biomass. Levels for Coagulant
Concentration were 1.53 – 30.4 mg coagulant per g Biomass total solids. Levels for pH were
two and five. See Table 2 for complete experimental design. Two experimental controls were

Technical Report
used; one 5x dilute Biomass and one 10x dilute Biomass. Temperature and coagulant reaction
duration were held constant for all samples at 22° C and one hour, respectively. All trials were
performed in a random order.
Table 2: Full-factorial experimental design for cationic starch coagulant
Trial
Initial
Dilution
Factor
Coagulant
Concentration
(mg coag. / g
Biomass
solids)
pH
1 10 30.4 5
2 5 15.3 5
3 5 1.53 5
4 10 3.04 2
5 5 15.3 2
6 10 3.04 5
7 10 30.4 2
8 5 1.53 2
Dilute Biomass samples were added to 50 mL glass beakers. Sample pH was measured and
adjusted using 0.4 N sulfuric acid; coagulant addition followed immediately thereafter. After
one hour, samples and controls were passed through 0.45 µm filters into 20 mL glass vials in
preparation for UV-Vis spectroscopy. Absorbance was measured with the method previously
described. See Appendix D for photographs of experimental controls.
To make sure the light color observed for Trials 4, 5, 7, 8 was not due to low pH alone, these
samples were adjusted with 0.275 N sodium hydroxide to pH 5. During this adjustment, sample
color remained constant as determined by visual inspection. This indicated that their color
change was indeed the result of removing colored compounds from solution. This was the only
validation of Beer’s Law performed in this study.
Results
The sample of dilute Biomass adjusted to 0.677 mN cationic polyelectrolyte became cloudy upon
adjustment and formed visible floc within 20 minutes. After settling for 12 hrs. at 22° C, the
supernatant was lighter colored than that of the control sample. This indicated that flocculation
was in fact possible with 6x dilute Biomass.
Operating Conditions: Flocculant
For the full-factorial design with cationic polyacrylamide flocculant, absorbance readings were
adjusted for dilution during treatment (e.g. flocculant addition and/or pH adjustment) and
dilution during measurement. Absorbance reduction values were calculated by comparing these

Technical Report
adjusted absorbance values to the absorbance of Trial 8, the experimental control. See Figure 1.
The more positive the absorbance reduction value, the lighter the sample color.
Pairs of trials gave similar results (e.g. Trials 1, 2; Trials 3, 6; Trials 4, 8; and Trials 5, 7). The
trials in each pair only differed by whether they were tested for 1 hr. or for 24 hrs. This pairing
indicated that time did not impact the color removal of cationic polyacrylamide flocculation. See
Figure 1.
The cationic polyacrylamide flocculant removed a similar amount of color from solution at pH 5
as at pH 11. In Trials 5 and 7, flocculant in pH 5 samples produced absorbance reductions
around 45% (see Figure 1). Elevated pH added substantially to the color of Trials 3, 6. In spite
of this, flocculant addition in Trials 1 and 2 showed absorbance reductions of 55 to 75% relative
to Trials 6, 3, respectively.
All trials with native pH 5 (Trials 4, 5, 7, 8) did not change pH when re-measured after filtration.
However, all trials adjusted to pH 11 (Trials 1, 2, 3, 6) showed decreased pH after filtration.
After one hour, Trials 2, 3 decreased to pH 10.43 and 10.39, respectively. After 24 hours, Trials
1, 6 decreased to pH 9.06 and 8.99, respectively.
Only Trials 5, 8 had sample colors lighter than the control. Because time did not impact color
reduction, Trial 5 represents the best conditions of those studied for cationic polyacrylamide
flocculant in 10x dilute Biomass. Those conditions were 28.8 µL flocculant per g Biomass
solids at pH 5 for one hour.
Figure 1: Absorbance reduction for full-factorial design with cationic polyacrylamide flocculant
-140
-120
-100
-80
-60
-40
-20
0
20
40
60
1 2 3 4 5 6 7 8
AbsorbanceReduction(%)
Trial

Technical Report
Various Concentrations of Flocculant and Coagulant
For varying flocculant and coagulant concentrations, absorbance readings were adjusted for
dilution during treatment (e.g. flocculant addition and/or pH adjustment) and dilution during
measurement. Absorbance reduction values were calculated by comparing the absorbance of
each sample with that of its control. Each data set was linearly regressed. Data and regressions
are plotted in Figure 2. The higher the absorbance reduction, the lighter the sample color. The
correlation between flocculant concentration and absorbance reduction was perfect (R2
= 1.00)
over the tested range of 16.7 – 34.9 µL flocculant per g Biomass total solids. Flocculant addition
did not change the pH of these samples. In Figure 2, the coagulant curve for pH 2 is very similar
in shape to that of pH 5, only shifted upward. This indicates that low pH promoted color
removal with all coagulant concentrations tested (1.53 – 30.4 mg coagulant per g Biomass
solids). Based on this trend, it seems likely that testing 16.7 – 34.9 µL flocculant per g Biomass
total solids at pH 2 would yield color removal higher than any observed so far.
Figure 2: Absorbance reduction due to cationic polyacrylamide flocculant and cationic starch coagulant
See Appendix B for photographs of coagulant Trials 1 – 8.
Comparison of Flocculant and GAC
For comparing flocculant with GAC, absorbance readings were adjusted for dilution during
treatment (e.g. flocculant addition) and dilution during measurement. Absorbance reduction
values were calculated by comparing the absorbance measurements of the four sample treatments
y = 0.62x + 25.23
R² = 1.00
y = -0.05x2 + 2.14x + 20.07
R² = 0.92
y = -0.05x2 + 1.87x - 3.14
R² = 0.91
-5
0
5
10
15
20
25
30
35
40
45
50
0 5 10 15 20 25 30 35 40
Flocculating Agent Concentration (mg or µL flocculating agent per g Biomass solids)
Absorbance Reduction as a Function of
Flocculating Agent Concentration
Cationic Polyacrylamide Flocculant, pH 5 (µL/g) Cationic Starch Coagulant, pH 2 (mg/g)
Cationic Starch Coagulant, pH 5 (mg/g)

Technical Report
with that of the experimental control. In Figure 3, absorbance reduction is plotted as a function
of the total volume of GAC and flocculant used to treat each sample. Average absorbance
reduction values were 41% for samples treated with just flocculant (22.8 µL flocculant per g
Biomass solids), 74% for samples treated with just 2 g GAC, 89% for samples treated with
flocculant (22.8 µL flocculant per g Biomass solids) followed by 2 g GAC, and 97% for samples
treated with 2 g GAC followed by another 2 g GAC.
See Appendix C for photographs of representative samples from each of the four treatments.
In these trials, GAC was clearly more effective than flocculant at removing color. The samples
treated just once with GAC removed almost twice as much color as those treated with just
flocculant. By treating with flocculant then GAC, color removal was halfway between single
GAC treatment samples and double GAC treatment samples. However, the GAC concentrations
used were three orders of magnitude greater than the flocculant concentrations (compare x-axes
of Figure 3 with that of Figure 2) These GAC amounts used were taken from “Analysis of
Carbohydrates in Lignocellulosic Biomass,” reprinted in Appendix A. This is the SOP used to
prepare dirty sugar streams for HPLC analysis and as such is the SOP used for laboratory GAC
treatment. In light of this, these tests should be redone using GAC concentrations that represent
those used in a production environment such as the pilot plant. In spite of this, flocculation
seems promising because it is moderately effective at concentrations much lower than those used
here for GAC.
Figure 3: Absorbance reduction due to cationic polyacrylamide flocculant and granular activated carbon
0
10
20
30
40
50
60
70
80
90
100
0 5000 10000 15000 20000 25000 30000 35000
Total Treatment Concentration (µL GAC + flocc. per g Biomass solids)
Flocculant, 22.8 µL per g Product 4 solids GAC, 2 g Flocculant + GAC GAC + GAC

Technical Report
Operating Conditions: Coagulant
For the full-factorial design with cationic starch coagulant, absorbance readings were adjusted
for dilution during treatment (e.g. Initial Sample Dilution, Coagulant Concentration, and pH) and
dilution during measurement. Absorbance reduction values were calculated by comparing these
adjusted absorbance values to those of the experimental controls. These are plotted in Figure 2.
The more positive the absorbance reduction value, the lighter the sample color.
Second order polynomials were fit to the data (see Figure 2). The curve for pH 2 is very similar
in shape to that of pH 5, but shifted upward. This indicates that low pH promoted color removal
at all concentrations tested (1.53 – 30.4 mg coagulant per g Biomass solids). Based on these few
data, the curves for pH 5 and pH 2 have maximums at 17 and 21 mg coagulant per g Biomass
solids corresponding to 13% and 44% absorbance reduction, respectively.
In Figure 2, the curves for pH 2 coagulant and pH 5 flocculant intersect at 28.5 mg coagulant per
g Biomass solids. Given that the flocculant curve is a line with a shallow positive slope which
intersects on the right side of the broad coagulant curve, slightly increased color removal in
flocculant samples requires a considerable concentration increase. For example in Figure 2, for
the flocculant samples to surpass the coagulant performance by 4% absorbance reduction
requires an increased flocculant concentration of 67%.
See Appendix B for photographs of coagulant Trials 1 -8.
Summary and Conclusions
This flocculation screening study yielded helpful results and further research opportunities.
Biomass was characterized as having a net anionic charge. Consequently, three cationic rather
flocculating agents were tested: (1) cationic polyelectrolyte, (2) cationic polyacrylamide
flocculant, and (3) cationic starch coagulant. The polyelectrolyte tests indicated that flocculation
of Biomass was possible. The best color removal overall was 47% of the control using pH 5,
34.9 µL polyacrylamide flocculant per g Biomass solids, 22° C, and one hour reaction time. The
best color removal by starch coagulant was 42% of the control color using pH 2, 15.3 mg
coagulant per g Biomass solids, 22° C, and one hour reaction time. Although the best coagulant
trial removed 5% less color than the best flocculant trial, it did so using less than half as much
flocculating agent.
Comparison between GAC and flocculant showed flocculant to be at least half as effective as
GAC. However, these results were actually inconclusive because of a concentration mismatch
due to using an SOP designed for laboratory analysis rather than production manufacturing.
Next steps for investigating flocculating agents includes process economics with ASPEN,
addition experimentation, and researching legal concentration limits for food applications.
Assuming favorable process economics, further research could proceed in a number of ways.
Probably the most beneficial would be to obtain a sample of a cationic starch flocculant with a
degree of substitution similar to the cationic polyacrylamide flocculant already tested. This
would be tested over a wide range of concentrations (e.g. 0 – 35 mg flocculant per g Biomass

Technical Report
solids) at pH 2. Such a starch flocculant would be less expensive than its polyacrylamide
counterpart and at pH 2 it could provide better color removal than any flocculating agent
described in this report.
Further research could include replicating this study and performing statistical analysis on the
data. Due to the time constraints of this study, minimal experimental replication was performed;
consequently statistical analysis was not performed.
Characterizing the floc itself is another potential avenue for further research. Because of the
small sample volumes used in this study, floc volumes were too small to directly measure
amount or composition. Larger sample could provide enough material for these analyses.
Further research could also investigate alternate methods of quantifying the effectiveness of
flocculating agents. This study measured efficacy via absorbance of the supernatant at 420 nm
because UV-Vis spectroscopy was simple and readily available; however other methods may be
more illuminating. For example, several documented methods measure the rate at which the floc
settles or the rate at which it can be separated from supernatant via various types of filtration
(Burkert & Hartmann, 2012).
Further research is needed to understand the legally permissible concentrations of flocculating
agents for food applications.
References
(AWWA), T. A. (2012). MIXING, COAGULATION, AND FLOCCULATION. The American
Society of Civil Engineers (ASCE): Water Treatment Plant Design, Fifth Edition.
McGraw-Hill Professional, AccessEngineering.
Burkert, H., & Hartmann, J. (2012). Flocculants. In Ullmann's Encyclopedia of Industrial
Chemistry (pp. 199-206). John Wiley and Sons, Inc.
McKean, W. (2014, November 5). Personal Communication. Seattle, Washington.
Penner, M. H. (2010). Chapter 22: Ultraviolet, Visible, and Fluorescence Spectroscopy. In S.
Nielsen, Food Analysis (pp. 389-391). New York: Springer Science+Business Media.
Appendix
Appendix A. SOP for Analysis of Lignocellulosic Biomass
Title Analysis of Carbohydrates in Lignocellulosic
Biomass
Author Joshua Davies, W.T. McKean, and Tye Dunham
Organization S2G and IPCI
Date 2014/03/25

Technical Report
Revision Draft 2.0
Preparation
A. Collect minimum 10mL, maximum 20mL sample of Lignocellulosic Biomass
a. Ensure that liquid level is about the same as the heater block level
B. Prepare 72% H2SO4
C. Locate CaO (should be in HPLC room)
Procedure
A. Take initial pH reading of sample
B. Use Table 1 (Appendix) to bring sample acid concentration to 4% (round to
nearest 5mL)
a. Take pH reading after 10 minutes
C. Heat sample to temperature specified by supervisor for hydrolysis
a. Typical hydrolysis is conducted at 90ºC for 24 hours or 120ºC for 1 hour
b. If Hydrolysis is done at 120ºC for 1 hour, heating setting should be set at
between 3 and 4 on High Setting
B. After Hydrolysis, let sample cool to room temperature or to touch
C. Add CaO until the sample pH is between 6 and 9
D. Run sample in centrifuge for 10 min
E. Using syringe and filter (small pink filter) remove all supernate from vial and store
in clean vial
F. Add 2 grams of GAC per 10mL of supernate sample, shake it up
G. Allow GAC and supernate to sit for 30 minutes minimum
H. Remove supernate from GAC using syringe and filter (small pink filter) remove
supernate from vial and store in clean vial
I. Add 2 grams of anion resin per 10mL of supernate sample and 1 gram of cation
resin per 10mL of supernate sample

Technical Report
J. Allow resins and supernate to sit for 30 minutes minimum
K. Remove supernate from GAC using syringe and filter (small pink filter) remove
supernate from vial and store in clean vial
L. Take sample of pretreated Lignocellulosic Biomass for HPLC, see HPLC SOP
APPENDIX

Technical Report

Technical Report
Appendix B. Photographs of Cationic Starch Coagulant Trials 1-8
Appendix C. Photographs of Comparison of Flocculant and GAC
A representative replicate from each of the four trials comparing flocculant and GAC is shown here. “F1” was treated with only
flocculant. “G3” was treated with only GAC. “FG1” was treated with flocculant followed by GAC. “GG1” was treated with GAC
followed by GAC.

Technical Report
Appendix D. Photographs of Experimental Controls
These control samples are simply Biomass 10x dilute and 5x dilute, respectively which were filtered through 0.45 µm filters.

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