Industrial Crops and Products 49 (2013) 648–658

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Industrial Crops and Products

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Pilot plant clarification of sweet sorghum juice and evaporation of

raw and clarified juices
Brett Andrzejewski a , Gillian Eggleston a,∗ , Randall Powell b
a
USDA-ARS Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, USA
b
BioDimensions Delta Renewables LLC, Memphis, TN, USA

a r t i c l e i n f o a b s t r a c t

Article history: One of the fundamental processing areas identified by industry for the commercial, large-scale manufac-
Received 22 March 2013 ture of liquid biofuels and bioproducts from sweet sorghum (Sorghum bicolor L. Moench) is the clarification
Received in revised form 28 May 2013 of juice to make it suitable for concentration into syrup for long-term storage, year-round supply, effi-
Accepted 19 June 2013
cient transport, and acceptable fermentation yields. Pilot plant studies were conducted to evaluate the
clarification of juices (80 ◦ C; target limed pH of 6.3; 5 ppm polyanionic flocculant) from a sweet sorghum
Keywords:
hybrid and cultivar M81E on three sample dates across a 3-month (September–November) processing
Sweet sorghum
season in 2011. Turbidity removal across pilot plant clarification was 95–98% after only 30–50 min reten-
Sugar feedstocks
Pilot plant
tion time (Rt ). The higher Rt at the pilot than laboratory scale caused a slight loss of total fermentable
Clarification sugars (sucrose + glucose + fructose) to acid degradation, thus a slightly higher target limed pH of ∼6.5
Vacuum evaporation is recommended to preserve sugars during clarification and downstream thermal evaporation. Under
Syrup non-optimized fermentation conditions (Saccharomyces cerevisiae yeast 10% (w/w); 35 ◦ C; 14 h; 18 Brix),
Ethanol yields higher and less variable bioethanol yields with less foam formation occurred under sterile than non-
sterile conditions for both raw and clarified syrups. Ethanol yields ranged from 7.1 to 8.2% (56.0–64.7 g/L)
and 5.8 to 8.4% (45.8–66.3 g/L) and sterile and non-sterile conditions, respectively. Moreover, under ster-
ile conditions, there were no significant differences at the 5% probability level for ethanol yields between
the raw and clarified syrups, indicating clarification did not impede fermentation. Overall, clarification
of the juices reduced the loss of fermentable sugars during the evaporation stage, and allowed for better
syrup storage.
Published by Elsevier B.V.

1. Introduction
While leading projects have been announced in Brazil and China,
several private-sector groups in the USA, including BioDimensions
Delta Renewables LLC, Memphis, TN, EnviroFuels, Riverview, FL,
and Myriant Technologies LLC, Lake Providence, LA, are pursu-
ing the development of new domestic industrial sugar feedstocks
to supply the anticipated bioprocessing demand (Andrzejewski
et al., 2013). Sweet sorghum has been widely recognized as a
promising sugar feedstock crop due to its efficient C-4 photosyn-
thetic pathway, easy cultivation from seed, low fertilizer and water
requirements (Teetor et al., 2011), possibility of multiple crops per
Abbreviations: Rt , retention time; MOL, milk of lime; MW, molecular
weight; NTU, nephalometer turbidity units; CJ, clarified juice; EDTA, disodium
ethylenediaminetetraacetate dehydrate; HPAEC, High Performance Anion Exchange
Chromatography; ODR, Oscillatory Deformation Rheology; PSA, particle size analy-
sis; ICP, inductively coupled plasma spectroscopy; HTC, heat transfer coefficient.
∗ Corresponding author. Tel.: +1 504 286 4446; fax: +1 504 286 4367.
E-mail address: gillian.eggleston@ars.usda.gov (G. Eggleston).
season, wide geographic suitability across the USA, directly fer-
mentable sugars, and huge breeding potential.
For the large-scale, commercial manufacture of biobased fuels
and chemicals from sweet sorghum, fundamental processing ques-
tions urgently need to be addressed, especially those associated
with seasonal production. Fig. 1 illustrates the different processing
pathways for conversion of sweet sorghum sugars to fermentation
products. Each processing pathway has an optimal storage time
to prevent loss of fermentable sugars ranging from hours for raw
juice to months for syrup. Identified processing areas that need
to be addressed include, but are not limited to: (1) stabilization
of the raw juice; (2) clarification of the raw juice to make it suit-
able for concentration; and (3) concentration of the juice into stable
syrup for efficient transport, storage, and year-round supply. Often,
fermentation is the rate limiting step in conversion of the sweet
sorghum sugars to value added products. Manufacture of storable
sweet sorghum syrup would also help offset the problem that can
arise when the crushing capacity is greater than the distillery capac-
ity and would also allow for timely harvest of the field crop (Fig. 2).
The goal of clarification is to stabilize juice with respect
to microbial deterioration and remove suspended and turbid

0926-6690/$ – see front matter. Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.indcrop.2013.06.027
 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658
Fig. 1. Process flow diagram showing the multiple pathways for fermentation of
sweet sorghum sugars. Each pathway has an optimal storage time (hours to months)
to minimize consumption of sugars. Pathway 1 represents direct fermentation of the
juice at the process plant. Pathway 2 represents transportation and short-term (a
few days) storage of clarified juice. Pathway 3 represents long-term storage, efficient
transportation, and year-round use of syrups at a processing facility.
particles through precipitation, and to allow subsequent concen-
tration in a viable liquid sugar product using standard commercial
evaporation technologies (Fig. 1). Our research team recently devel-
oped a clarification method in the laboratory for sweet sorghum
juices (Andrzejewski et al., 2013), that was based on the hot lime
clarification of sugarcane juices (Eggleston et al., 2003) but with
significant variations to take into account the differences in sweet
sorghum and sugarcane juices, i.e., sweet sorghum juice contains
much higher amounts of glucose and fructose that are susceptible
to alkaline degradation. Sweet sorghum juice is first heated to allow
colloidal particles, particularly proteins, to coagulate and form nat-
ural flocs. Hydrated lime (calcium hydroxide) is then added in the
form of milk of lime (MOL), to the heated sweet sorghum juice
to increase the pH to 6.3 for (i) neutralization of acids, (ii) reduc-
tion of unwanted acid degradation of invert sugars in downstream
thermal evaporation, (iii) formation of calcium phosphate flocs, and
(iv) introduction of positively charged particles in the juice solution
(Andrzejewski et al., 2013). Calcium, from the juice and MOL, and
phosphorous that occurs naturally in the sweet sorghum juice are
necessary in the formation of calcium–phosphate bridges that aid in
the flocculation process (Eggleston et al., 2012b). Floc precipitation
is further aided by HMW polyanionic acrylamide flocculants which
agglomerate particles and add weight increasing settling rates.
The overall objective of this study was to evaluate the viability
of the laboratory clarification method (Andrzejewski et al., 2013)
at the pilot plant scale which is more representative of the com-
mercial scale. An optimal commercial clarification method will use
a minimum of energy and costly clarification aids while allowing
the preservation of fermentable sugars and other important macro-
and micronutrients in the liquid sugar syrup on storage, and pro-
ducing the maximum yields of downstream fermentable sugars.
2. Materials and methods
2.1. Chemicals
Powdered, hydrated lime (Carmeuse, Pittsburgh, PA, USA)
and flocculant (Praestol 2640 z, ∼2,700,000 MW copolymer of
acrylamide and sodium acrylate with a medium charge density
[Stockhausen, Krefeld, Germany]) were kindly provided by staff of
Lafourche Sugars, LLC (Thibodaux, LA, USA). A solution of milk of
649
lime (MOL) was created by thoroughly mixing 120 g of hydrated
lime with 1 L of distilled water. The solution was verified to be
10 Baumé with a hydrometer. Flocculant (1 g) was added to 1 L
of distilled water (0.1% solution) and allowed to thorough mix for
1 h and sit for 24 h before use. Analytical grade CeliteTM filter aid,
cupric sulfate pentahydrate, hydrochloric acid, potassium sodium
tartrate, sodium hydroxide, triethanolamine, ammonium molydate
tetrahydrate, 1-amino-2-napthol-4-sulfonic acid were acquired
from Sigma–Aldrich (St. Louis, MO, USA). Disodium ethylenedi-
aminetetraacetate dehydrate (EDTA) was acquired from Bio-Rad
(Hercules, CA, USA). Sodium acetate trihydrate came from Fisher
Scientific (Fair Lawn, NJ, USA). Nitric acid and hydrogen peroxide
(trace metal grade) were acquired from J.T. Baker (Phillipsburg,
NJ, USA). Distillers yeast in dried form (Saccharomyces cerevisiae)
(Crosby & Baker Ltd., Westport, MA, USA) was donated by Delta
BioRenewables LLC.
2.2. Sweet sorghum
Sweet sorghum juice was kindly provided by BioDimensions
Delta Renewables, Memphis TN. Sweet sorghum hybrid-A was
planted on June 1, 2011 and harvested on September 14, 2011.
Commercial sweet sorghum cultivar M81E was planted on July 8,
2011 and June 15, 2011 and harvested on October 20, 2011 and
November 10, 2011, respectively. Sweet sorghum billets (∼25 cm)
were harvested with a Case New Holland (Burr Ridge, IL) sugarcane
combine harvester. Seed heads were removed by the sugarcane
harvester topper. Sweet sorghum billets were crushed by passing
through custom built 4-roll mill (Jeffersonville, KY, USA) followed
by a Laurel Machine and Foundry Co. (Laurel, MS, USA). The col-
lected first and second expressed juice was mixed and filtered
through a 0.6 mm pore size screen. Biocide (∼150 mL; Busan 881TM ,
Buckman Labs., USA) was added to each of the three 114 L drums
containing juice on each sampling date. The juice was transported
in a cargo tote (1.2 m × 1.0 m × 1.2 m) containing a salt-water ice
bath, with ice and salt added as necessary to maintain a temper-
ature below 6 ◦ C during transport (∼6 h) to the USDA-ARS-SRRC
sugar processing pilot plant in New Orleans. Preliminary experi-
ments were first conducted to ensure the storage conditions did
not cause deterioration of the juice on transit. Aliquots of juices
were collected in Memphis and New Orleans, just before and after
transport, respectively. As the juice Brix and pH did not change
dramatically on transport, and only trace amounts of mannitol
formed (an indicator of sugarcane and sweet sorghum deteri-
oration; Eggleston, 2002; Lingle et al., 2013), the conditions of
transportation did not cause juice deterioration during transport
prior to processing.
2.3. Pilot plant clarification and syrup production
2.3.1. Raw syrup production
On the same day of harvest and transport, the sweet sorghum
raw juice was processed directly into raw syrup in the USDA-
ARS-SRRC pilot plant “mini” evaporator unit (210 L full capacity)
operated under vacuum, which is based on a Robert’s type calandria
(rising film) evaporator frequently operated in sugarcane factories
(Eggleston et al., 2011). The mini-evaporator was heated by in-
house steam regulated to 69 kPa, and vacuum was produced from a
rotary type Elmo Reitschle (Schopfheim, Germany) vacuum pump
capable of 95 kPa and a 34 m3 h−1 volumetric flow rate (for full
details on design and operation see Eggleston et al., 2011). Raw juice
was fed into the evaporator at the bottom by suction until it cov-
ered just above the calandria tubes (30.3 L), and then boiled under
∼51 kPa vacuum at ∼82.2 ◦ C to ensure no or minimal entrainment.
The Brix was allowed to steadily increase and boiling continued
under these conditions until the syrup was ∼45 Brix. The vacuum
650 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658

Fig. 2. Particle size analysis for (A) September 14 (hybrid-A) raw (orange) and clarified (black) diluted syrups, (B) October 20 (M81E) raw (orange) and clarified (black) diluted
syrups, and (C) November 8 (M81E) raw (orange) and clarified (black) diluted syrups. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of the article.)

was then increased to 67.7 kPa and the temperature lowered to
∼74 ◦ C to simulate multiple-effect evaporators. Eventually the vac-
uum pressure was increased to 85 kPa at 60–66 ◦ C with the raw
juice continuously fed into the evaporator until the Brix was 70,
which was then dropped out of the evaporator at the bottom.
2.3.2. Clarified syrup production
Clarification of sweet sorghum raw juice into clarified juice (CJ)
was conducted in the USDA-ARS-SRRC pilot plant, and the CJ was
then evaporated into clarified syrup the same as for raw syrup
production described above. For full details on design and oper-
ation of the pilot plant juice clarification equipment see Eggleston
et al. (2011). Raw juice (∼151 L) was mechanically pumped into a
265 L juice tank where the juice was heated (∼1.3 ◦ C/min) to 80 ◦ C
with 69 kPa steam and constant stirring. Temperature and pH of
juice inside the tank were monitored using sensors. When the juice
reached 80 ◦ C it was immediately limed to a target pH of 6.3 with
MOL and constantly mixing. Once the target pH was obtained the
mixer was turned off, and flocculant (StockhausenTM polyanionic
solution 0.1%; 5 ppm) added with stirring by hand using a paddle for
1 min. The flocculated, heated limed juice (FHLJ) was then imme-
diately gravity fed into a settling or clarification tank below, and
allowed to settle. The rate of settling was monitored via test port
valves on the side of the clarification tank (Eggleston et al., 2011). A
sample was collected in a test-tube at specified time intervals and
settling visually observed in front of a bright lamp. Mud was gravity
drained from the bottom valve and stored in a −40 ◦ C freezer until
analyzed. The CJ formed (∼114 L) was fed into stainless steel hold-
ing tanks (19 L) and stored overnight in a walk-in cooler at 4 ◦ C. The
next morning the CJ was evaporated under the same conditions as
for raw juice (see Section 2.3.1). Syrups once produced were stored
in a −40 ◦ C freezer until analyzed for fermentation performance.
 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658
2.4. Laboratory fermentations
Frozen syrups were thawed, and simple fermentations of the
sweet sorghum diluted syrups were undertaken under both ster-
ile and non-sterile conditions. For the non-sterile conditions, the
syrup, water, and equipment used were not sterilized, whereas
under sterile conditions they were autoclaved (121 ◦ C; 103 kPa;
20 min). Distillers yeast (S. cerevisiae) was first hydrated with
deionized water (1:10, w/w) at 35 ◦ C for 30 min. The yeast cream
formed was then centrifuged for 10 min at 1000 rpm (170 × g)
and the supernatant decanted and discarded. The centrifuged
yeast was re-suspended (1:10, w/w) in either non-sterile or pre-
autoclaved diluted sweet sorghum syrup (35 ◦ C; 18.0 Brix) in an
Erlenmeyer flask (125 mL) with a foam stopper, and placed into
a New Brunswick Scientific G24 Environmental Incubator Shaker
(Edison, NJ, USA) at 35 ◦ C, with no shaking for 14 h. Fermentations
were run in triplicate. After fermentation, the fermented samples
were stored in a −40 ◦ C freezer until analyzed.
2.5. Brix (percent dissolved solids), pH, color, and turbidity
Sample Brix was measured using an Index Instruments (Kissim-
mee, FL, USA) TCR 15–30 temperature controlled refractometer
accurate to ±0.01 Brix, and results expressed as an average of
three measurements. Juice pH was measured on a Metrohm
Brinkman 716 DMS Titrino (Riverview, FL, USA) with a Met-
tler Toledo (Columbus, OH, USA) xerolyte electrode. Color of the
raw and clarified juices and syrups were measured following
ICUMSA method GS2/3-9. Samples (25 mL) were diluted in tri-
ethanolamine/HCl buffer at pH 7.0 (1:1, v/v) and filtered through a
0.45 ␮m syringe filter. Absorbance was then measured at 420 nm on
a Shimadzu 1240 UV–Vis spectrophotometer (Nakagyo-ku, Kyoto,
Japan). Nephalometer turbidity (NTU) measurements were mea-
sured on a Hach 2100N turbidimeter (Loveland, CO, USA); results
are an average of five measurements.
2.6. Reducing sugar titration
Total reducing sugars were measured with a TechnalTM Reduc-
ing Sugar Titrator (Piracicaba, Sao Paulo, Brazil) following the
method reported in Andrzejewski et al. (2013).
2.7. Sugars and sugar alcohols measured using High Performance
Anion Exchange Chromatography (HPAEC)
Glucose, fructose, sucrose, and mannitol in juice and syrup sam-
ples were determined by HPAEC using CarboPac PA1 analytical and
guard columns (Dionex Corp., Sunnyvale, CA, USA) following the
method reported in Andrzejewski et al. (2013).
2.8. Ethanol and ethanol yields
Ethanol in the fermented sample was determined using a Dionex
ES50 HPLC system (Sunnyvale, CA, USA) with refractive index (RI)
detection. Fermented juice was first filtered through a 0.45 PVDF
filter and separated on Phenomenex Rezex ROA analytical acid and
guard columns (Torrance, CA, USA) over a 30 min isocratic method
with 0.005 N sulfuric acid eluent at 1 mL/min, and a column tem-
perature of 30 ◦ C. Separated fermented juice components were
detected using a Shodex refractive index detector RI-101 (Yoko-
hama, Japan). Samples were maintained at 4 ◦ C using a Dionex AS-1
refrigerated autosampler (Sunnyvale, CA, USA).
The ethanol yield coefficient (%) was calculated as the con-
centration of ethanol produced (g/L)/initial concentration of sugar
651
×1/0.51 × 100, where 0.51 indicates the theoretical ethanol yield
(0.51 g ethanol/g hexose; Nofemele et al., 2012).
2.9. Total protein and phosphate determinations
Total protein was determined using the Bradford dye-binding
assay from Bio-Rad (Hercules, CA, USA), with bovine serum albu-
min as the standard. Free phosphate was determined following the
SASTA Method 3.8 – Phosphates (P2 O5 ) (2005).
2.10. Viscosity using Oscillatory Deformation Rheology (ODR)
Mechanical spectra were recorded on an AR-1000 Advanced
[Oscillatory deformation] rheometer (TA Instruments, Houston, TX,
USA), using cone and plate geometry of angle 2◦ and diameter
4.0 cm. Sample temperature was 25.0 ◦ C controlled within ±0.01 ◦ C
by a peltier cell. Readings were taken 1 min after the sample had
attained thermal equilibrium. A frequency sweep of 0.1–1000 rad/s
was applied to each sample.
2.11. Particle size analysis (PSA)
Particle Laser Scattering Analysis (PSA) was conducted on a Par-
tica Laser Scattering Particle Size Distributor Analyzer LA-950V2
by HoribaTM (Kyoto, Japan). Diluted syrups (18 Brix; 1–5 mL) were
added in the liquid mode of the instrument. Using Horiba LA-950
software (ver. 5), it was ensured that the % transmittance was in
the valid range. After final use, the instrument was washed with
de-ionized water.
2.12. Metal ion determinations
Metal ions were analyzed by inductively coupled plasma (ICP)
spectroscopy using a Leeman Labs Profile sequential ICP (Hud-
son, NH, USA). Juice samples (1 mL) were digested with nitric acid
(4 mL) and hydrogen peroxide (1 mL) using a Milestone Ethos EZ
microwave digestion system (Shelton, CT, USA). The digested sam-
ples were diluted to 25 mL with deionized water. The method
incorporated a 15 min ramp to 200 ◦ C and then a 10 min hold at
200 ◦ C.
2.13. Digital microscopy
Digital microscopy images were taken with an Olympus MIC-
DTM digital microscope (Center Valley, U.S.). One drop of diluted
syrup (18 Brix) or mud was placed on the microscope slide and at
least five random images were taken for each sample. A medical
iodine tincture solution (2%) from Walgreen Co. (Deerfield, IL) was
filtered twice through a 0.22 ␮m syringe filter then used to stain for
starch granules. Micrographs of the starch were examined before
and after staining with 1 drop of 2% tincture of iodine.
2.14. Statistics
Student’s t-test, independent, two-tail, was performed to com-
pare the difference of the raw and clarified juices or syrups using
Microsoft Excel 2007 (Redmond, WA, USA).
3. Results and discussion
3.1. Pilot plant clarification of sweet sorghum juices
Pilot plant clarification of sweet sorghum juices was performed
at 80 ◦ C with a target limed juice pH of 6.3 and the addition of 5 ppm
polyanionic flocculant, because these were the optimum conditions
observed in our previous laboratory studies (Andrzejewski et al.,
652 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658

Table 1
Pilot plant clarification of hybrid-A sweet sorghum juice on September 14, 2011. The error is the standard deviation of sample measurements.*

Parameter Sweet sorghum hybrid-A raw juice Sweet sorghum hybrid-A clarified juice % change
‡
pH 5.08 ± 0.07b 6.84 ± 0.10a 34
Brix 13.97 ± 0.15a 13.10 ± 0.12b −6
Turbidity (NTU) 6554 ± 20a 131 ± 2b −98
Color (ICU) 7089 ± 665a 3858 ± 122b −46
Phosphate (mg/L) 158.5 ± 1.2a 6.9 ± 0.1b −96
Total protein (mg/L) 116 ± 12a <20b† >−82
Calcium (mg/L) 8.15 ± 0.02b 8.62 ± 0.04a 6
Starch§ (mg/L) 1120 ± 55a 913 ± 41a −18
Sucrose (g/L) 56.2 ± 4.8a 57.5 ± 0.3a 2
Glucose (g/L) 35.5 ± 3.0a 37.7 ± 0.2a 5
Fructose (g/L) 14.9 ± 1.8a 16.7 ± 0.6a 13
Total sugars (S + G + F) (g/L) 106.7 ± 9.6a 111.9 ± 1.2a 5
Titratable reducing sugars (g/L) 45.9 ± 0.6b 49.4 ± 0.4a 7.6
*
Different lowercase letters represent significant differences between the raw and clarified juices at the 5% probability level. Those parameters that were significant
different are shaded.
‡
The target pH was inadvertently over limed at the 80 ◦ C processing temperature.
†
Below detectable limits.
§
Starch concentrations are a function of the starch method used. Recent results in our laboratory indicate that the method used only measures a portion of the soluble
starch and is an underestimation.

2013). Clarification results for sweet sorghum hybrid A juice (Sept
14) are listed in Table 1. The CJ pH (6.84 measured at room tem-
perature) was higher than the limed pH of 6.3 mostly because this
target pH was inadvertently overshot at the processing tempera-
ture of 80 ◦ C. It must also be noted, that the pH of the CJ is always
higher at room temperature (∼25 ◦ C) than 80 ◦ C because there is
less dissociation of sugars and water at lower temperatures (Clarke
et al., 1997). The Brix value decreased (P < 0.05) slightly from the
raw to the CJ. Excellent turbidity removal (P < 0.05) of 98% occurred
across pilot plant clarification of the hybrid-A raw juice yielding a
transparent, yellowish-green clarified juice (131 NTU). This turbid-
ity removal was slightly better than the approximate 95% turbidity
removal for laboratory clarification performance of sweet sorghum
juice (Andrzejewski et al., 2013). Furthermore, the team noted that
the time for clarification was ∼30 min which is considerably faster
than the typical 1.5 h for sugarcane juice in the same pilot plant and
in sugarcane factories (Eggleston et al., 2012a). This may be due
to the higher protein and turbidity values in sweet sorghum than
sugarcane juices (Andrzejewski et al., 2013). Approximately half
of the juice color was removed across clarification (Table 1), most
likely because of precipitation with other impurities. Phosphate
decreased by 96% after the raw juice was clarified. Some phosphate
removal is expected due to the formation of calcium–phosphate
bridges that aid in flocculation and precipitate during clarification
(Eggleston et al., 2012b).
Total protein in the hybrid-A sweet sorghum juice decreased to
below detectable limits after clarification. This should not adversely
affect downstream fermentation as yeast do not typically utilize the
nitrogen in protein, but require assimilable nitrogen and nitrogen
from amino acids. Calcium levels increased (P < 0.05) after clarifica-
tion of the hybrid-A juice (Table 1) because of the addition of MOL.
Starch decreased after clarification (not significant) in the hybrid-A
juice. Importantly, within experimental error, there were no signif-
icant changes in total sugars (sucrose, glucose, and fructose) after
the raw hybrid-A juice was clarified when measured by HPAEC and
even when the sugar concentrations were calculated on a Brix basis.
This was further evidenced by reducing sugar measurements with a
reducing sugar titrator (Table 1). The team previously observed that
HPAEC and reducing sugar titrator results were similar and within
experimental error (Andrzejewski et al., 2013), although in Table 1
there was a significant difference in titratable reducing sugars in
raw and clarified juices (this was not observed in Tables 2 and 3).
Clarification results for sweet sorghum cultivar M81E in Oct are
listed in Table 2. CJ pH measured at room temperature was 6.42 and
only slightly higher than the target pH of 6.3 at 80 ◦ C (Table 2). Brix
increased (P < 0.05) in the CJ after clarification, which is most likely
attributable to the addition of processing aids. Turbidity removal
across clarification was 95%, which was slightly lower than for
hybrid A (Table 1) but still very acceptable. Laboratory clarifica-
tion of M81E juice was typically more difficult than hybrid juices
because initially the M81E raw juice was more turbid and less pro-
tein was available for floc formation (Andrzejewski et al., 2013). In
this study, however, the M81E raw juice value for protein in October
was higher than for hybrid A raw juice protein values in September.
In comparison, M81E raw juice protein was very low in Nov indi-
cating that protein is affected by planting date and/or maturity
factors. The team previously observed a sweet sorghum cultivar
effect for protein in raw juices (Andrzejewski et al., 2013). Unlike
hybrid-A juice (Table 1), the color did not significantly decrease
after clarification. Interestingly, phosphate which is required for
effective clarification did not show a dramatic change even with
high turbidity removal. It may be that the protein, which showed
a decrease across clarification to below detectable levels and coag-
ulated to form visibly large flocs, aided the clarification process
leaving considerable phosphate. Calcium did increase significantly
(P < 0.05) after clarification. Starch was removed during the clari-
fication process and to a greater degree than the hybrid-A sweet
sorghum clarification (Table 1).
There was a slight decrease in total sugars (especially sucrose
and fructose that are more labile than glucose in thermal, acidic
conditions) across clarification (Table 2). Furthermore, when the
soluble solids concentration effect was removed, i.e., the sug-
ars were calculated on a % Brix bases, the total sugars decrease
across clarification was worse from 1428 to 1350 g/L/Brix, although
this was not significantly different statistically. The pH of the CJ
was 6.42 at room temperature which is evidence that the target
limed pH of 6.3 at 80 ◦ C was not exceeded in October (Table 2)
whereas it was in September (Table 1). As the goal is to preserve
as much fermentable sugar as possible, the target pH of 6.3 may
have been slightly low for pilot plant clarification across ∼40 min
Rt .
Clarification results for sweet sorghum cultivar M81E on
November 8 are listed in Table 3. The M81E sweet sorghum in
November was more mature than in October (Table 2) as indi-
cated by the higher raw juice Brix, total fermentable sugar, and
starch values, and the lower pH, calcium, and total protein values.
It took slightly longer for clarification of the M81E mature juice
in November than October (∼50 min) most likely because of the
lower protein content, but as slightly higher mud was observed
as well this could also have slowed down clarification (Eggleston
 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658 653

Table 2
Pilot plant clarification of M81-E sweet sorghum juice on October 20, 2011. The error is the standard deviation of sample measurements.*

Parameter Sweet sorghum M-81E raw juice Sweet sorghum M-81E clarified juice % change across clarification

pH 5.93 ± 0.02b 6.42 ± 0.04a 8

Brix 13.18 ± 0.07b 13.51 ± 0.01a 3
Turbidity (NTU) 6891 ± 85a 362 ± 2b −95
Color (ICU) 6055 ± 273a 5956 ± 41a −2
Phosphate (mg/L) 106.7 ± 12.4a 97.6 ± 4.3a −8
Total protein (mg/L) 149 ± 6a <20b† >−87
Calcium (mg/L) 6.11 ± 0.01b 8.14 ± 0.04a 33
Starch§ (mg/L) 914 ± 51a 575 ± 19b −37
Sucrose (g/L) 127.5 ± 2.6a 122.4 ± 1.1a −3
Glucose (g/L) 41.5 ± 1.6a 41.8 ± 0.6a 2
Fructose (g/L) 19.3 ± 0.8a 18.0 ± 0.7a −5
Total Sugars (S + G + F) (g/L) 188.2 ± 4.9a 182.3 ± 2.4a −19
Titratable reducing sugars (g/L) 58.4 ± 3.0a 54.5 ± 1.1a −5
*
Different lowercase letters represent significant differences between the raw and clarified juices at the 5% probability level. Those parameters that were significant
different are shaded.
†
Below detectable limits.
§
Starch concentrations are a function of the starch method used. Recent results in our laboratory indicate that the method used only measures a portion of the soluble
starch and is an underestimation.

Table 3
Pilot plant clarification of M81-E sweet sorghum juice on November 8, 2011. The error is the standard deviation of sample measurements.*

Parameter Sweet sorghum M-81E raw juice Sweet sorghum M-81E CJ juice % change across clarification

pH 5.50 ± 0.03b 6.44 ± 0.04a 17

Brix 15.18 ± 0.27b 16.39 ± 0.45a 8
Turbidity (NTU) 14,067 ± 24a 230 ± 9b −98
Color (ICU) 3918 ± 9b 6062 ± 96a 54
Phosphate (mg/L) 102.2 ± 0.3a 92.1 ± 0.2b −10
Total protein (mg/L) <20a† <20a† 0
Calcium (mg/L) 4.07 ± 0.02b 8.97 ± 0.02a 120
Starch§ (mg/L) 2163 ± 27a 833 ± 53b −61
Sucrose (g/L) 126.0 ± 0.6b 128.7 ± 0.4a 2
Glucose (g/L) 39.5 ± 0.2b 42.0 ± 0.6a 6
Fructose (g/L) 16.9 ± 0.4b 18.6 ± 0.4a 10
Total Sugars (S + G + F) (g/L) 182.5 ± 1.2b 189.3 ± 1.4a 4
Titratable reducing sugars (g/L) 63.5 ± 1.5a 67.4 ± 4.9a 4
*
Different lowercase letters represent significant differences between the raw and clarified juices at the 5% probability level. Those parameters that were significant
different are shaded.
†
Below detectable limits.
§
Starch concentrations are a function of the starch method used. Recent results in our laboratory indicate that the method used only measures a portion of the soluble
starch and is an underestimation.

et al., 2003). Within experimental error, phosphate and total fer-
mentable sugars did not change with increased maturity in the
raw M81E juice (Tables 2 and 3). Across clarification, there was
no significant change in total sugars across clarification, although
the slight increase in glucose and fructose indicated that some acid
degradation of sucrose had occurred (Table 3). However, like the
performance of M81E juice in October (Table 2), when the sugars
were calculated on a % Brix basis, a slight decrease in total sug-
ars occurred from 1201 to 1155 g/L/Brix. Similar to the pilot plant
clarification of M81E juice in October, the pH of the CJ was 6.44 at
room temperature which was evidence that the target pH of 6.3 at
80 ◦ C was also not exceeded in November (Table 3). Thus, the Nov
results further suggest that the target pH of 6.3 may be slightly low
for pilot plant clarification and a target pH of 6.4–6.5 would better
preserve fermentable sugars across clarification as well as during
the subsequent evaporation process.
Starch levels, as expected, were higher in the M81E raw juice
in November compared to October (Tables 2 and 3) because starch
increases with sweet sorghum maturity (Broadhead, 1972; Zhao
et al., 2009). Starch has previously been reported to interfere with
the processing of sweet sorghum into 88 Brix syrups for raw sugar
manufacture because of its associated increased viscosity and gela-
tion (Ventre, 1940; Smith, 1969). However, this will depend on the
initial concentration of the starch in the juice and the new biopro-
cessing industries are requesting syrups of no greater than 80 Brix
for acceptable flow and handling characteristics. Moreover, starch
could also be a processing opportunity if it can be hydrolyzed into
fermentable glucose with selected ␣-amylases and glucoamylases
that are capable of producing glucose rather than dextrins and
malto-dextrins. The seed heads, not utilized in this study, would
be an additional source of starch that could be processed with the
sweet sorghum stalks into juice or in a separate industrial process
(Zhao et al., 2009). It may also be possible to retrieve the starch
precipitated out in the mud during clarification by re-circulating
it into the downstream fermentation vessel or through a separate
process. Research into this possibility is now warranted especially
as we noted numerous starch granules in the mud in this study
(results not shown) and some other precipitated impurities in the
mud may enhance fermentation.
3.2. Raw and clarified juice evaporation
Generally, during evaporation of sweet sorghum raw juice a vis-
cous, dark green foam formed on the top of the evaporating juice
(Table 4). This foam layer tended to decrease by the end of the
evaporation process. In contrast, the foam produced on evapora-
tion of the sweet sorghum CJ was less viscous and off-white in color.
Moreover, the CJ foam did not persist to the end of the evaporation
process (Table 4). No differences in evaporation rates between the
raw and clarified sweet sorghum juice were noted.
It is well known that removal of certain impurities in sug-
arcane juice during clarification affect heat transfer coefficients
654 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658

Table 4
The quality of raw and clarified syrups produced from the pilot plant evaporation of hybrid-A and M81E raw and clarified sweet sorghum juices, respectively after dilution
to 18.0 Brix. The error is the standard deviation of sample measurements.*

Parameter Hybrid-A; September 14 M81E; October 20 M81E; November 8

Raw syrup Clarified syrup Raw syrup Clarified syrup Raw syrup Clarified syrup

pH 5.0 ± 0.1b 6.1 ± 0.1a 5.3 ± 0.2b 5.7 ± 0.1a 5.1 ± 0.1b 5.70 ± 0.05a
Turbidity (NTU) 6104 ± 47a 253 ± 1b 7905 ± 30a 454 ± 2b 5231 ± 30a 243 ± 1b

Appearance Opaque dark Semi-transparent Opaque dark Semi- Opaque dark Semi-
green brown green transparent green transparent
brown brown
Color (ICU) 5274 ± 311b 6316 ± 149a 5774 ± 98b 6947 ± 97a 6070 ± 201b 8215 ± 114a
Dynamic viscosity (Pa* s) at 4.3 ± 0.2a 1.4 ± 0.2b 5.6 ± 0.2a 0.39 ± 0.06b 621 ± 12a 513 ± 9b
2.5 rad/s at 68.8 Brix
Starch† (mg/L) 1518 ± 57a 1248 ± 15b 1245 ± 32a 797 ± 8b 2934 ± 33a 1143 ± 15b
Sucrose (g/L) 116.7 ± 0.2a 119.2 ± 1.3a 142.8 ± 4.4a 141.9 ± 4.8a 155.6 ± 0.8a 151.8 ± 1.1a
Glucose (g/L) 42.7 ± 0.6a 43.6 ± 0.5a 42.0 ± 0.4a 42.8 ± 1.8a 35.8 ± 0.2a 34.6 ± 0.7a
Fructose (g/L) 18.0 ± 0.7a 18.4 ± 0.3a 18.2 ± 0.1a 19.0 ± 1.2a 12.5 ± 0.1a 11.9 ± 0.2a
Total Sugars (S + G + F) (g/L) 177.4 ± 1.6b 181.2 ± 2.1a 203.0 ± 4.9a 203.7 ± 7.8a 203.9 ± 1.1a 198.3 ± 2.0b
Titratable reducing sugars (g/L) 67.2 ± 2.3a 69.3 ± 1.9a 65.0 ± 2.0a 66.1 ± 3.4a 58.4 ± 1.5a 58.7 ± 1.0a
*
Different lowercase letters represent significant differences between the raw and clarified syrups studied at the 5% probability level, for each sampling date only. Those
parameters that were significant different are shaded.
†
Starch concentrations are a function of the starch method used. Recent results in our laboratory indicate that the method used only measures a portion of the soluble
starch and is an underestimation.

(HTCs) because less scaling occurs on the evaporator calandria
tubes (Allhands, 2007; Doherty, 2011). In this study, within exper-
imental error there was no difference between the HTC before and
after evaporation of raw or clarified juice (results not shown). The
effect of clarification in sweet sorghum juices on the HTC is likely
to be more dramatic after continual usage, when any precipitated
scale can gradually build up on the calandria tubes. A decrease in
HTCs in Robert’s type evaporators in sugarcane factories, particu-
larly the latter evaporators in an evaporation station, can be readily
observed after 8 days of continuous sugarcane juice evaporation
(Eggleston and Monge, 2007).
The increase in Brix for M81E juice between October and
November (Tables 2 and 3) represented an energy savings in
November from not having to evaporate 29 mL of water per liter of
juice to obtain the 70 or 80 Brix syrup (Table 4). The energy savings
would increase the energy return on energy invested for biofuel
production.
(2012b) recently reported that turbidity of sugarcane juices is gov-
erned more by the smaller particles.
Across clarification, the diluted syrups produced from hybrid-A
on September 14 dramatically decreased in the content of smaller
particles (<30 ␮m) leaving a population of larger particles (Fig-
ure. 2a). Furthermore, the mean particle size increased from 36
to 114 ␮m for the hybrid-A raw and clarified syrups, respectively.
The near complete removal of small particulates may be because
of the higher target pH in Sept causing more calcium flocs to form
or it may be because of a cultivar effect, and this warrants further
investigation. However, a similar reduction on clarification for both
smaller (<30 ␮m) and mean particle size on clarification was not
observed in the M81E diluted syrups (Figure 2b and c), although
turbidity removal was still high at 95–98%. Thus, pilot plant clarifi-
cation was not always associated with a reduced mean particle size
in the syrups, and a cultivar effect was noted. The team previously
observed that there was a strong cultivar effect on raw juice quality
and juice clarification performance (Andrzejewski et al., 2013).

3.3. Particle size analysis of diluted raw and clarified syrups

Stored syrups will have to be diluted for fermentation. There-
fore, the team analyzed diluted syrups. Not surprisingly, the diluted
clarified syrups had 96%, 94%, 95% less turbidity levels than the
diluted raw syrups for September 14, October 20, and November
11, respectively (Table 4). There were slight differences between
the turbidities of the sweet sorghum syrups and juices (Tables 1–4).
The turbidities of the clarified syrups tended to be lower than the
corresponding clarified juices which could have been due to the
increased temperature during evaporation acting as a type of clar-
ification, i.e., more turbid particles were precipitated. The team
previously observed that heat alone clarified sweet sorghum juice
but was not considered acceptable clarification (Andrzejewski et al.,
2013). In contrast, there was no clear trend for the turbidities of raw
juices and syrups, but there were indications that turbid particles
were sometimes precipitated in the evaporator.
We compared the particle size analysis distributions for the
syrups in order to assess if specific sizes of the particles were selec-
tively removed by clarification. Both the diluted raw and clarified
syrups exhibited a bimodal distribution for particle size, which is
illustrated in Figure 2. The effectiveness of clarification is often
quantified by removal of the smaller, turbid particles that scatter
visible light. Turbidity, measured in a nephelometer, is influenced
greatest by particles < 5 ␮m in size (Allhands, 2007). Eggleston et al.
3.4. Quality of diluted raw and clarified syrups
Quality parameters for raw and clarified syrups produced in this
study are also listed in Table 4. The viscosity of the syrups were
always higher (P < 0.05) in the raw than clarified syrups. The viscosi-
ties of the raw and clarified syrups produced from the more mature
M81E sweet sorghum in November were dramatically higher than
for the less mature M81E sweet sorghum in October and hybrid-A
in September. This was mostly attributed to the starch concentra-
tions as high starch concentrations can cause gelation to occur and,
therefore, much higher viscosities. However, no significant cor-
relations were measured between the syrup viscosities and final
ethanol yields from the syrups. On observation of the syrups under
digital microscopy we observed more and relatively larger gran-
ules in the raw M81E syrup from November compared to October
(Fig. 3), which explains the much higher viscosity values (Table 4).
There were less large starch granules in the clarified than raw syrup
from November (Fig. 3), thus many of the larger granules must have
precipitated into the mud during clarification. This further high-
lights the need to retrieve starch from the mud or through another
process.
The pH of the syrups were highly correlated (R2 = 0.999;
y = 0.2857x2 − 2.7743x + 11.718) with the corresponding juice pHs.
The syrup pHs, however, were consistently lower than the juice
 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658 655

Fig. 3. Digital micrographs of raw and clarified M81E sweet sorghum syrups, with and without iodine staining for starch, in October and November. The average size of the
starch granules in the November raw syrup was 12 ␮m, ranging from 3 to 33 ␮m and exhibited a CV of 59%. In contrast, the average size of the starch granules in the October
raw syrup from less mature M81E sweet sorghum was 5 ␮m, ranging from 3 to 8 ␮m and exhibited a CV of 33%. Arrows on the micrographs point to starch granules.

pHs because of: (i) the higher Brix of the syrups served to con-
centrate acids, (ii) the formation of acids from the thermal, acid
degradation of sugars during evaporation, and (iii) additionally for
the clarified syrups, the precipitation of alkaline compounds in the
evaporator.
Syrup turbidities had no effect on the ethanol yields under
non-sterile conditions, whereas there was a weak correlation
(R2 = 0.554; y = 1E−05x + 7.655) under the sterile conditions. The
colors of the syrups are also listed in Table 4. At each harvest
date, the color of the clarified syrup was slightly but significantly
(P < 0.05) higher than the raw syrups, which is most likely because
of the acid degradation of sugars (Clarke et al., 1997). When the
total sugars were calculated on a Brix basis it was possible to elu-
cidate the change in sugars from juices to syrups, e.g., during the
evaporation process. In October, 21.0% of the total sugars were lost
to thermal degradation reactions in the raw syrups, whereas 16.1%
was lost in clarified syrup production. In November, 5.7% total sug-
ars were lost during production of raw syrups compared to only
2.7% for clarified syrups. Thus, clarification of the juices reduced
the loss of fermentable sugars during the evaporation stage, and
allowed for better storage (Eggleston et al., 2013).
3.5. Fermentation of diluted raw and clarified syrups
The effects of clarification on fermentation of the diluted syrups
created from thawed frozen syrups were included in this study.
Fermentations were based on industrial methods for sugarcane
juice in the Brazilian bioethanol industry (Basso et al., 2011), i.e.,
10% (w/w) yeast to ensure fast fermentation times and no loss
of ethanol to evaporation. No attempt was made to optimize the
fermentation and growth conditions of the Distiller’s yeast and
the pH of the diluted syrups was not adjusted. This was to eval-
uate the direct effects of the clarification process on fermentation.
Bridgers et al. (2011) reported that sweet sorghum juice is capable
of yeast fermentation without initial input, although Bulawayo et al.
(1996) supplemented sorghum juice with urea and diammonium
phosphate and adjusted the pH to 4.5 before fermentation. We
intend to conduct future investigations on optimized fermentation
 656 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658

10.0 a Under sterile conditions, no significant differences were found

9.0
a a A) between the ethanol yields from the raw and clarified syrups, at
a a each of the three sampling dates (Fig. 4b). These results are not in
8.0
a agreement with those of Bridgers et al. (2011), who reported that
7.0
a 60 Brix sweet sorghum syrup produced after evaporation in an
% ethanol (v/v)

6.0 open vat (previously used to produce molasses) with direct heat
5.0 underneath and constant stirring, that was diluted to 25, 20 and
4.0 15 Brix, showed no signs of fermentation over a 5-day period by
3.0
yeast. Furthermore, “additional fermentation time and sterilization
did not improve conversion” into ethanol (Bridgers et al., 2011).
2.0
Under such conditions juice/syrup temperature can reach 100 ◦ C,
1.0 causing the fermentable sugars to be degraded into unfermentable,
0.0 organic acids. Although it does not matter if, at elevated tem-
9-14-2011 9-14-2011 10-20-2011 10-20-2011 11-08-2011 11-08-2011 peratures, sucrose is acid degraded into glucose and fructose, the
raw CJ raw CJ raw CJ thermal decomposition of glucose and fructose into unfermentable
10.0 compounds does and unfortunately the acid degradation of glucose
9.0 a
a B) and fructose, particularly fructose, is much faster than for sucrose
a a (Clarke et al., 1997; Lee et al., 2011). Another explanation could
a a
8.0
be that the sugar structures changed on thermal degradation, e.g.,
7.0 into furfural, hydroxymethyl furfural, and acetic acid, and inhibited
% ethanol (v/v)

6.0 the fermentation (Palmqvist et al., 1999; Wikandari et al., 2010). In

5.0
4.0
3.0
2.0
1.0
0.0
9-14-2011 9-14-2011 10-20-2011 10-20-2011 11-08-2011 11-08-2011
raw CJ raw CJ raw CJ
Fig. 4. Ethanol yields under (A) non-sterile and (B) sterile fermentation conditions
for raw and clarified diluted syrups (18 Brix), across three sampling dates in 2011.
Different lowercase letters represent significant differences at the 5% probability
level between the raw and clarified juice produced on the same date.
conditions to examine the cost benefits analysis from adding chem-
icals to optimize the pH for fermentation.
In this study, fermentations of the raw and clarified syrups (after
dilution to 18 Brix with deionized water) were investigated under
non-sterile and sterile conditions. Generally, under the non-sterile
conditions, fermentation of the raw and clarified syrups produced
excessive foam that not only clung to the sides of the Erlenmeyer
flask but also to the bottom of the foam stopper. Excess foaming
under commercial conditions in the Brazil sugarcane bioethanol
industry is known to markedly reduce fermentation yields by up
to 5 g/L (Mario Lopes, Fermentec Ltda., personal communication)
and requires the use of expensive antifoam chemicals (Basso et al.,
2011). In comparison, under sterile conditions both the raw and
clarified diluted syrups had less foam formation than the non-
sterile samples and were less viscous. Furthermore, under sterile
conditions, the raw syrup foam was more viscous and occurred
in greater amounts than the foam from fermentation of clarified
syrup.
In general, ethanol yields were lower and more variable under
the non-sterile than sterile conditions because of their susceptibil-
ity to contamination (Fig. 4). Bryan (1990) reported that Leuconostoc
lactic acid bacteria and wild yeasts were among the natural
microflora that readily ferment unsterilized sweet sorghum juices.
In this study, the ethanol yields were kept from being even lower
in the non-sterile conditions, because (i) the high yeast inocula-
tion will have competed with native microflora (Bryan, 1990), and
(ii) the pre-freezing of the juices will have partially sterilized the
juices (Bridgers et al., 2011). Ethanol yields in the sterile fermenta-
tions of the different raw and clarified diluted syrups are illustrated
in Fig. 4b.
this study, evaporation of juices occurred under vacuum to keep
temperatures below 65 ◦ C and, concomitantly, preservation of fer-
mentable sugars. If large-scale production of syrups is to become a
commercial venture, vacuum pan evaporation that minimizes ther-
mal degradation of sugars will be necessary as in the sugarcane,
sugar beet, and fruit juice industries.
Inorganic phosphate in sweet sorghum juice is important for
both acceptable clarification and fermentation. The phosphate
retention in the clarified juice varied dramatically between the
hybrid-A and M81E sweet sorghums examined, and decreased by
96%, 8%, and 10% for September 14, October 20, and November
11, respectively. However, no significant correlations were found
between the phosphate levels in the raw and clarified juices and
the ethanol yields of their respective syrups, under both non-sterile
and sterile conditions. Under non-sterile conditions, the very low
phosphate levels in the hybrid-A clarified juice and syrup (Table 1)
may have caused lower fermentations yields than the raw syrup
(Fig. 4a), but this was not a problem under sterile conditions. For
M81E, the phosphate levels were still high in the clarified syrup,
and this seemed not to affect fermentation under the non-sterile
conditions.
Yeast requires millimolar amounts of phosphate to synthe-
size nucleic acids, phospholipids, and cellular metabolites (Wykoff
and O’Shea, 2001). A standard synthetic yeast medium contains
253 mg/L of inorganic phosphate (Abelovska et al., 2007). Although,
yeast has the potential to metabolize sugars in sweet sorghum juice
under limited phosphate conditions, these are not the optimal con-
ditions for a maximum fermentation rate. A sluggish fermentation
is one that exhibits a rate decrease near the end of fermentation,
and this can be dramatically improved by the addition of diammo-
nium phosphate (400 mg/L) as well as dissolved oxygen (7 mg/L)
(Blateyron and Sablayrolles, 2001). A “stuck” fermentation is one
that leaves residual sugars at the end of fermentation due to lack
of inorganic phosphate (Blateyron and Sablayrolles, 2001). In this
study, sucrose, glucose, and fructose were either not detected or
only detected in trace amounts in most of the wines produced, sug-
gesting nearly complete fermentation. Thus, the results indicated
that clarification and the removal of phosphate did not result in a
“stuck” fermentation. However, it is not known how the phosphate
levels in the raw and clarified syrups affected the fermentation rate
under these non-optimized fermentation conditions, and this will
be investigated in future studies.
The pHs of the syrups were not adjusted in this study.
Under non-sterile conditions there was no significant relationship
between the syrup pH and ethanol yields, which further suggests
 B. Andrzejewski et al. / Industrial Crops and Products 49 (2013) 648–658
that another factor such as contamination was responsible. In com-
parison, under sterile conditions there was a very weak, negative
correlation (R2 = 0.360; y = −90.333x + 12.582) between the ethanol
yields (x axis) and syrup pH (y axis). This suggests that pH had a par-
tial effect on the ethanol yields, and that optimizing the pH to 4.5
would invariably improve yields under the sterile conditions.
Certain, but not all, yeast strains are susceptible to preemptive
flocculation in the presence of excessive amounts of calcium and
settle to the bottom of the vessel decreasing their effectiveness
in yielding a value-added product. Under non-sterile conditions
there was no significant relationship between the clarified juice
calcium concentrations and ethanol yields. Under sterile con-
ditions there was a negative but weak correlation (R2 = 0.430;
y = −0.0974x + 8.5733) between the calcium levels (x axis) and
ethanol yields (y axis). The team previously reported that calcium
ion concentrations in the CJ increased with limed juice pHs from 5.5
to 6.4 but stabilized thereafter (Andrzejewski et al., 2013). Yeast can
be deflocculated by the addition of EDTA or other chelating agents
but at additional cost (Stradford, 1989).
High soluble aluminum concentrations are known to reduce
yeast growth by inhibition of Mg-dependent hexokinase respon-
sible for phosphorylation of six carbon sugars (Trapp, 1980). At the
laboratory scale, we observed no significant correlation between
the aluminum concentration in clarified juices and turbidity
removal across clarification, and sometimes aluminum concen-
trations did not decrease across clarification (Andrzejewski et al.,
2013). Similarly, at the pilot plant scale reported herein there was
no trend in aluminum concentration before and after pilot plant
clarification, with aluminum concentrations ranging from only 1 to
4 ppm in both the raw and clarified juices produced on September
14, October 20, and November 8. Potassium and zinc are also nec-
essary micronutrients for microorganisms (Brock and Brock, 1973).
Like for aluminum there was no trend in potassium concentrations
before and after pilot plant clarification. The average potassium
levels in both the raw and clarified juice were 3433, 2449, and
2645 ppm for September 14, October 10, and November 8, respec-
tively. The amount of potassium containing fertilizer applied to the
field of growing sorghum may influence potassium levels in the
juice. Like the other metals, there was no trend in zinc levels before
and after pilot plant clarification. The average zinc concentrations in
both the raw and clarified juices were 4, 4, and 3 ppm for September
14, October 10, and November 8, respectively.
Overall, these results reported here are important as they
indicate that no significant fermentation yield was lost after clar-
ification while substantial gains are obtained with respect to the
short-term stabilization of juices and long-term storage of 80 Brix
syrups (Eggleston et al., 2013). Furthermore, reduction of the
foam layer during fermentation by pre-clarification of the sweet
sorghum juice had another benefit of higher ethanol yields which
should lead to higher economic gains.
4. Conclusions
At the pilot plant scale, 95–98% turbidity removal across clarifi-
cation (80 ◦ C; target limed juice 6.3; 5 ppm polyanionic flocculant)
was obtained after only 30–50 min of clarification. This high per-
centage of turbidity removal may not be necessary (Steindl, 2011)
for extended syrup storage and downstream fermentations and
turbidity removal requirements will most likely vary with the fer-
mentation end-product. The team intends to investigate the effect
of different syrup turbidities on fermentation yields.
Similar to laboratory-scale clarification (Andrzejewski et al.,
2013), a strong cultivar effect was noted on clarification perfor-
mance at the pilot plant scale. Storage experiments on the syrups
produced in this and another study were undertaken (Eggleston
657
et al., 2013) with storage of 80 Brix syrups considerably more opti-
mal than 70 Brix syrups.
A small amount of fermentable sugars were lost during clar-
ification when the target pH was obtained, but when it was
inadvertently exceeded in September no significant loss of total
fermentable sugars occurred to acid degradation. However, if no
pH adjustment occurred, as in the raw juices, then markedly more
sugars were lost to thermal acid degradation reactions in the
evaporator. Overall, a slightly higher target limed pH of ∼6.5 is rec-
ommended to preserve sugars during clarification and downstream
thermal evaporation. Furthermore, results presented in this paper
have highlighted the need for vacuum pan evaporation in the large-
scale manufacture of sweet sorghum syrups to minimize thermal
degradation of sugars.
Clarification of sweet sorghum juice and evaporation into clar-
ified syrups is only necessary if fermentation capacity is not
sufficient at the processing plant, or if efficient transportation and
long-term storage is required to ensure a year-round supply of feed-
stocks at commercial facilities. This is especially true for facilities
that cannot easily or economically obtain other feedstocks. Overall,
these results reported here are important as they indicate that clar-
ification of the juice reduced losses of fermentable sugars during
the production of syrups and did not significantly affect down-
stream fermentation yields, while substantial gains are obtained
with respect to the long-term storage of 80 Brix syrups (Eggleston
et al., 2013).
Acknowledgements
The authors wish to thank Ms. Jessica Gober and Mr. Eldwin St.
Cyr for their technical assistance. Special thanks are extended to
Dr. Eduardo Burges of Fermentec (Piracicaba, Sao Paulo, Brazil) for
useful discussions on laboratory fermentation techniques and pro-
cesses used in Brazilian ethanol industries. Thanks are also made to
Lynda Wartelle (USDA-ARS-SRRC) for the metal ion determinations
and Dr. Isabel Lima (USDA-ARS-SRRC) for use of the microwave
digestion system. Mention of trade names or commercial products
in this article is solely for the purpose of providing specific infor-
mation and does not imply recommendation or endorsement by
the U.S. Department of Agriculture. USDA is an equal opportunity
provider and employer.
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