Industrial Crops & Products 163 (2021) 113330

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Industrial Crops & Products

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Bioethanol fermentation of clarified sweet sorghum (Sorghum bicolor (L.)

Moench) syrups sealed and stored under vegetable oil
K. Thomas Klasson *, Stephanie A. Boone
USDA, Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd, New Orleans, LA 70124, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Sweet sorghum represents a readily available renewable carbohydrate feedstock for biofuel production.
Sweet sorghum syrup Generally, the sweet sorghum growing season and window for processing is short and it is often desirable to store
Bioethanol fermentation a sugar-rich material for future processing. Therefore, we investigated the possibility of storing three different
Sugar degradation
sweet sorghum sugar syrups under a layer of soybean oil; then we investigated subsequent yeast bioethanol
Soybean oil
Organic acids
fermentation.
Ethanol A layer of oil on the surface of the syrup reduced sugar loss in 30 and 40◦ Bx syrups, but the loss was still 21–36
%. Storage of 50◦ Bx syrup resulted in an 11–18 % loss of sugars with or without a surface layer of oil. Elevated
levels of lactic and acetic acids, caused by bacterial contamination, almost completely inhibited yeast fermen­
tation; however, when ethanol-producing microorganisms were part of the contamination, stored syrups pro­
duced a bioethanol at a yield of 83–91 %, compared to theoretical.

1. Introduction spoiled within five hours at normal temperatures. Over three days at 25
◦
C, 12–30 % fermentable sugars can be lost and 40–50 % can be lost in
For some time, emphasis in the U.S. is to produce biofuels and bio­ one week (Wu et al., 2010). Concurrently, predominantly lactic acid
products from lignocellulosic biomass or sugar-rich crops, such as sweet bacteria increased 30- to 300-fold. Refrigeration (4 ◦ C), reduced the loss
sorghum (Sorghum bicolor (L.) Moench) and sugar (or energy) beets (Beta to less than 1 and 3 % after 1 and 2 weeks, respectively. In a similar
vulgaris). Sugar crops are advantageous as renewable feedstocks because study with fresh sweet sorghum juice, investigators determined that the
they are already available, and the plants have high yields of sugar and most dominant bacteria found in the juice were species of Lactobacillus,
other carbohydrates per unit growth area. Worldwide, approximately 50 followed by species of Acinetobacter, Enterobacter, Erwinia, and Pseudo­
% of fuel ethanol production comes from sugar crops (by sugarcane juice monas (Daeschel et al., 1981). Thus, to limit sugar loss during storage
or molasses fermentation in Brazil and India). The remaining 50 % and improve overall fermentation yields after storage, technologies for
originate from grains (e.g., corn in the U.S.). Sugar crops have an stabilization and preservation of sugar juices are needed.
advantage over grain crops as their juices are directly fermentable Stabilization and preservation processes for foods and beverages
without much processing. For decades, research has been focused on mostly consist of chemical, physical, or biological processes (i.e.,
lignocellulosic conversion technologies for second generation biofuels, fermentation) (Desrosier and Singh, 2018). Except for the biological
but there is also an interest in developing sustainable sugar feedstocks in processes, these methods control the growth of microorganisms. The
combination with flexible biorefineries with lower cost, reduced sugar biological processes promote growth of some microorganisms while
loss during temporary storage, and improved feedstock quality limiting others, often by pH reduction. Ultraviolet light irradiation has
(Koninckx, 2013). been used to control several types of microorganisms and has been
Juices from sweet sorghum and energy beet are very susceptible to tested for preservation of maple sap (Morselli and Whalen, 1983);
microbial spoilage during storage due to their high water activity and however, Eggleston et al. (2015b) reported that UV germicidal light was
sugar contents (Wu et al., 2010). To a much lesser extent, the juice is also incapable of stabilizing sweet sorghum juices because of the latter’s
sensitive to enzymatic and acid deterioration reactions (Eggleston, relatively high sugar content, turbidity, and color values which all
2002; Singh et al., 2006). Daeschel et al. (1981) noted that fresh juice interfered with the UV light irradiation. Juice clarification (a

* Corresponding author.
E-mail address: [email protected] (K.T. Klasson).

https://doi.org/10.1016/j.indcrop.2021.113330
Received 27 October 2020; Received in revised form 25 January 2021; Accepted 5 February 2021
Available online 17 February 2021
0926-6690/Published by Elsevier B.V.
K.T. Klasson and S.A. Boone Industrial Crops & Products 163 (2021) 113330

combination of thermal, chemical, and precipitation processes) stabi­ sugar levels in the received syrups (Dale and M-81E by P.A. and Dale by
lized sweet sorghum juice and allowed storage at 25 ◦ C for at least two P.B.) were 65–75 ◦ Bx.
days (Eggleston et al., 2015b). Clarified juices can be further stabilized
by evaporation of water, concentrating the juice into syrup for efficient 2.2. Storage experiments
transport and storage. Clarification of juice, prior to evaporation, (1)
removes suspended and turbid particles (including microorganisms) Glass beverage containers (3.76 L) were cleaned by soaking them in a
that reduce scaling in downstream evaporators, (2) reduces microbial diluted (10 % v/v) commercial bleach solution (Clorox Company,
deterioration, (3) adjusts the pH to reduce unwanted acid degradation of Oakland, CA) for 15 min, followed by rinsing with a 2 % (w/v) sodium
glucose and fructose during evaporation (Clarke et al., 1997), and (4) thiosulfate solution. Each clean glass container was then turned upside
improves end product yields and quality (Andrzejewski et al., 2013; down, air dried on a sterile cloth, and stored sealed. Autoclaved sterile
Eggleston et al., 2015a). stainless steel containers or glassware was used to handle all syrup
Commercial-scale storage of sweet sorghum and sugarcane syrups is during sample preparation. The syrups were diluted with sterile auto­
limited. However, commercial storage of sugar beet syrup is common claved water until 30, 40, and 50◦ Bx were obtained. Homogeneous al­
among European and U.S. sugar beet factories to extend the processing iquots of 1.5 L were distributed into each glass container and a 1.7-cm
season. Efforts to improve the storage conditions of beet syrups have also layer of Crisco Pure Vegetable Oil (J.M. Smuckers Co., Orrville, OH)
been published (Blowers et al., 2010; Hatch et al., 2013). On a labora­ made from soybean oil (containing high polyunsaturated fatty acids)
tory scale, storage of sweet sorghum and sugarcane syrups have been (Eggleston et al., 2018) was added to the syrup using sterile pipettes.
researched. Polyethylene glycolate (a mixture of polyethylene glycol Containers were then stored indoors at ambient temperatures (11− 22
and caustic soda) was used in studies with 68◦ Bx sugarcane syrup with ◦
C), which fluctuated during the season. Fifty milliliter samples were
some success, protecting the surface and raising the pH of the syrup to taken for 10 weeks from the bottom of the container (via spigots,
control microbial contamination (Linares and Ehrenhauser, 2016). In cleaned with ethanol and purged) and stored frozen in sterile tubes until
other laboratory studies, Zhang et al. (2018) extracted and evaporated analysis. Duplicate storage experiments (i.e., duplicate containers) were
sweet sorghum juice and found that methyl paraben could be used as a used with soybean oil sealed syrups. Only single experiments were done
preservative while storing 40 % (w/v) syrup without much sugar loss. when syrups were not stored under an oil layer. After storage, the ◦ Bx
Natural sugar syrups are not free of microorganisms. Moroz (1963) level was determined and the syrups were diluted with distilled water to
reported finding yeasts in sugar syrups such as Saccharomyces cerevisiae 22◦ Bx before yeast inoculation and fermentation.
in 67◦ Bx sugarcane refinery sucrose syrup, Saccharomyces rouxii in
72–76 ◦ Bx partial invert syrups, Rhodotorula and Candida in 55◦ Bx su­ 2.3. Fermentation protocol
crose and partial invert syrups, and Saccharomyces mellis in 68–70 ◦ Bx
sucrose-corn syrup blends. However, there was no examination of syrup Fermentation of stored sweet sorghum syrups was performed in
quality, sugar loss, and sugar profiles after storage. ANKOM (Macedon, NY) fermentation bottles (250-mL) equipped with
Eggleston et al. (2015a) reported that, due to condensation, the pressure control and monitoring for CO2 production quantification. The
surface of sweet sorghum syrups showed fungal growth during storage. materials and containers were clean but not sterilized to mimic antici­
Covering the surface with a thin layer of soybean oil or Candelilla wax pated industrial conditions. Dry yeast cells (Red Star Distiller’s Active
showed promise as surface sealant for storage of 70–80 ◦ Bx sweet sor­ Dry Yeast, BSG Handcraft, Shakopee, MN) were activated by adding 1 g
ghum syrups for at least 80 days. In a follow-up study, the storage of less of yeast per 19 mL of activation broth containing 5.2 g/L of (NH4)2HPO4
concentrated syrups (i.e., 28 and 65◦ Bx) was performed and found that in distilled water and gently shaking at 32 ◦ C for 30–40 min. The acti­
the 65◦ Bx syrups could be stored under a vegetable-oil surface sealant vated culture was added to each of the fermentation bottles with sweet
for up to a year without major deterioration, while the 28◦ Bx syrup sorghum syrups [20 g activation broth per 100 g of diluted syrup
degraded in less than a week (Eggleston et al., 2018). In the current (22◦ Bx)]. An initial liquid sample was collected, the headspace was
studies, we investigated if partially concentrated syrups (i.e., less than flushed with nitrogen gas and the fermentation was allowed to progress
65◦ Bx) could be stored for intermediate durations (greater than a week until CO2 production had ceased. All fermentation bottles contained a
but for less than 80 days) prior to fermentation to bioethanol. Syrups magnetic stir bar and were agitated (100 rpm) in a controlled temper­
stored at lower ᶛBx concentrations save time, energy and money thereby ature environment (30 ◦ C). Fermentations were conducted twice (at
becoming more efficient for syrup producers and saving water during different dates) for the 30◦ Bx stored syrup, while only conducted once
fermentation rehydration. As previous work by us (and others) has not for the 40 and 50◦ Bx syrups.
focused on intermediate ◦ Bx levels and intermediate storage durations,
the work is novel. There is also a lack of research in the literature on 2.4. Measurements techniques
storage of commercially generated sweet sorghum syrups.
Sugar ◦ Bx values were measured with a Model DR6000-T Kruss
Optronic (Hamburg, Germany) Refractometer. Water activity (Aw) was
2. Materials and methods quantified with a dew point and a hygroscopic polymer sensor that
measures volatile ingredients (Aqualab TEV4, Sydney, Australia). Syrup
2.1. Syrups used pH was evaluated with an Oakton pH meter 510 series, Oakton in­
struments (Vernon Hills, Illinois, USA). Chemical analyses of sugars and
Sweet sorghum syrups were received from two commercial pro­ products were carried out by high performance liquid chromatography
ducers of syrups, Partner A (P.A.) and Partner B (P.B.). In both cases, the (HPLC) with a refractive index detector (Series 1100, Agilent, Santa
top panicles of the sweet sorghum stalks were removed during har­ Clara, CA, held at 30 ◦ C). The mobile phase was 5 mM H2SO4, which was
vesting. The harvested sweet sorghum stalks were crushed in two tan­ pumped at 0.6 mL/min through an Aminex HPX-87H column (Chinnici
dem roller mills. In the case of Partner A, the cultivars Dale and M-81E et al., 2005; Duarte-Delgado et al., 2015) with a Cation H guard column
were harvested and processed separately, and the juice was passed (Bio-Rad, Hercules, CA). Samples were injected (2–10 uL) twice; once
through a 2-mm screen before clarification as previously described for sugar analysis with the column held at 20 ◦ C to prevent hydrolysis of
(Eggleston et al., 2016; Heckemeyer et al., 2016). In the case of Partner sucrose (Tomlins et al., 1990) and secondly with the column held at 50
B, only cultivar Dale was harvested and the juice was filtered through a ◦
C for analysis of all other components. One standard stock solution was
0.6 mm pore size screen before a proprietary clarification process. In all made with 25 g/L of each of the sugars. The other standard stock solu­
cases, concentration to syrup was done in open pan evaporators. The tion contained succinic acid (10 g/L), L-lactic acid (12 g/L), acetic acid

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K.T. Klasson and S.A. Boone Industrial Crops & Products 163 (2021) 113330

(12 g/L), trans-aconitic acid (5 g/L), glycerol (8 g/L), and ethanol (15
g/L). All calibrations were found to be linear with very good correlations
(r2>0.99).

2.5. Statistics

Pooled standard deviations, from the duplicate experiments, were

calculated for total sugars, total degradation products, and ethanol for
each ◦ Bx storage condition. This pooled standard deviation was used to
calculate standard error of the mean from two experiments (i.e., SDp/
√2). This standard error was assumed constant within each ◦ Bx storage
condition. Two-way ANOVA evaluations were performed using Sigma­
Plot for Windows, Version 11.0 (Systat Software, San Jose, CA, USA),
which uses Type III Sums of Squares (Shaw and Mitchell-Olds, 1993).
Comparisons between means were performed using the Holm-Sidak
method (Holm, 1979; Sidak, 1967) at the **P = 0.05 and *P = 0.10
levels.

3. Results and discussion

During storage of sugar solutions, microbial degradation is a major

cause of fermentable sugar loss in syrups with water activities above
0.900 and the quantity of fermentable sugar loss is affected by microbial
quantity, species or genus variations in microbe community, pH, water
activity, storage temperature, and osmotic pressure (Lievens et al.,
2015). Yeast and bacteria rarely grow in food products below water
activities of 0.900− 0.910, while fungi can tolerate lower water activities
(Brown, 1976; Desrosier and Singh, 2018). The water activity (Aw) of the
syrup samples ranged from 0.960 to 0.972 for the 30◦ Bx syrups,
0.939− 0.948 for the 40◦ Bx syrups, and 0.908− 0.926 for the 50◦ Bx
syrups. Measurements and theoretical models show that water activities
of 30, 40, 50, and 60◦ Bx of pure sucrose solutions are 0.973, 0.957,
0.931, and 0.890 (Sereno et al., 2001). Thus, the syrups used here had
slightly lower water activities than similar pure sucrose solutions of the
same ◦ Bx. During the 10-week storage, the ◦ Bx levels dropped and the
water activity increased (Table 1). Acidity, as measured by pH, was an
indicator of degradation of sugars and production of organic acids. In
the most extreme cases (30◦ Bx syrups), the pH dropped as much as 0.81
pH units (data not shown).
The loss of sugars (sucrose, glucose, and fructose) during the storage
can be seen in Fig. 1. As is noted, 30, 40 and 50◦ Bx syrups that had been
stored for 10 weeks lost sugar during the storage, regardless of the
cultivar, the producer, or a protective oil layer. Without an oil layer,
there was a statistical difference (P < 0.05) between how much sugar (as
glucose equivalents) was lost in the 30, 40 and 50◦ Bx syrups. The same
was true with a layer of oil. Thus, the sugar level (and thus the water
activity) clearly impacts the degradation of sugar during syrup storage. Fig. 1. Sugar concentration before and after storage with and without a
With higher Brix levels (e.g., 65◦ Bx) sweet sorghum syrups can be stored surface-covering oil layer. (P.A.=Partner A; P.B.=Partner B; A.S.=After Storage;
for up to a year under an oil layer without microbial growth (Eggleston w/=with; w/o = without).
et al., 2018).
This fraction of sugars (i.e., as glucose equivalents) lost during the
10-week storage is shown in Table 2. As is noted, the lower the ◦ Bx value
of the stored syrup, the greater the sugar loss, which was statistically

Table 1
◦
Bx decrease and water activity (Aw) increases during 10-week storage, with or without an oil layer, in syrups of two sweet sorghum cultivars and two producers. (*P <
0.10; **P < 0.05; comparing “Oil” and “No oil”, within the same ◦ Bx).
Dale (P.A.) M-81E (P.A.) Dale (P.B.)

Storage Bx
◦
Surface layer ◦
Bx decrease during storage Aw increase ◦
Bx decrease during storage Aw increase ◦
Bx decrease during storage Aw increase

Oil 14 % 0.6 % 15 % 0.9 % 19 % 0.0 %

30 Bx
◦
No oil 22 %** 1.0 %** 23 %** 0.9 % 20 % 0.1 %
Oil 12 % 1.3 % 12 % 1.4 % 12 % 1.3 %
40◦ Bx
No oil 12 % 1.3 % 12 % 2.0 %** 20 %** 2.2 %**
Oil 9% 1.6 % 5% 1.0 % 11 % 2.5 %
50◦ Bx
No oil 12 % 1.1 % 6% 1.9 %** 10 % 2.2 %

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K.T. Klasson and S.A. Boone Industrial Crops & Products 163 (2021) 113330

Table 2
Sugar loss (i.e., as glucose equivalents) during 10-week storage before fermentation, with or without an oil layer, in syrups of two sweet sorghum cultivars and two
producers. After storage the syrup was diluted to 22◦ Bx and fermented. Ethanol yield was calculated as measured titer of ethanol, after fermentation (24-120 h) divided
by theoretically estimated ethanol titer. (*P < 0.10; **P < 0.05; comparing “Oil” and “No oil”, within the same ◦ Bx).
Dale (P.A.) M-81E (P.A.) Dale (P.B.)

Storage Bx
◦
Surface layer Sugar loss during storage Ethanol yield Sugar loss during storage Ethanol yield Sugar loss during storage Ethanol yield

Oil 32 % 90 % 36 % 85 %** 35 % 90 %**

30 Bx
◦
No oil 48 %* 90 % 56 %* 42 % 39 % 67 %
Oil 21 % 91 % 22 % 84 % 23 % 89 %**
40◦ Bx
No oil 22 % 90 % 22 % 84 % 35 %** 84 %
Oil 13 % 91 % 18 % 83 % 15 % 86 %
50◦ Bx
No oil 17 % 90 % 11 % 84 % 11 % 86 %

true (P < 0.025). In general, storage conditions with an oil layer reduced
the sugar loss, but not always. The greatest loss was noted when 30◦ Bx
syrups were stored without an oil layer.
In the storage study with 30◦ Bx syrups, 32–36 % of the sugars were
lost from the syrup when sealed with a layer of oil, while 39–56 % were
lost when the syrup was stored without an oil layer (Table 2). This dif­
ference between the two storage conditions (with or without an oil
layer) was statistically significant (P < 0.1) for two of the syrups. With a
water activity of >0.960 in the 30◦ Bx syrup, many microbes can survive
and grow (Lievens et al., 2015). Thus, it was not unexpected that sugar
was lost due to microbial degradation. Significant loss of sugar, reduced
pH, and presence of microorganisms were noted in the storage of 28◦ Bx
syrups (Eggleston et al., 2018). While no detailed analysis of microor­
ganisms (responsible for the sugar degradation) was performed, past
research on sweet sorghum syrups suggests that bacteria are more
prevalent than mold, slime mold, and yeast in 28 and 65◦ Bx sweet
sorghum syrups, before and after storage (Eggleston et al., 2018).
In the study with 40◦ Bx syrups, 21–23 % of sugars were lost when the
syrup was stored under a layer of oil, while 22–35 % were lost when the
syrup was stored without an oil layer. The amount of sugar lost was
dependent on the syrup type and storage conditions (i.e., Two-Way
ANOVA interaction was noted, P = 0.007). For example, there was a
clear impact (see Fig. 1, 40◦ Bx and Table 2) of an oil sealant in the case
of syrup from Partner B (Dale) but not in the case from Partner A (Dale or
M-81E). This could indicate the importance of clarification procedures,
which differed between the two partners. It has previously been shown
that clarification has a clear positive impact by reducing microbial
contamination (Eggleston et al., 2015b). In a storage study with similar
levels of sugars, Zhang et al. (2018) noticed an approximate 50 % loss of
total sugars after 10 weeks of sweet sorghum syrup storage without a
chemical preservative; but with a suitable preservative (methyl para­
ben), the fermentable sugar loss was <5 %.
In the case of 50◦ Bx syrups, 13–18 % of sugars were lost when stored
under a layer of oil, while 11–17 % were lost when the syrup was stored
without an oil layer but it was not statistically different between syrup
types/sources or protective layer. The lesser loss of sugars in the 50◦ Bx
syrups (oil-sealed or unsealed), as compared to the 30 and 40◦ Bx syrups,
reflect decreases in microbial metabolic activity resulting from
increased osmotic pressure and lower water activity (0.969− 0.974,
0.953− 0.964, and 0.918− 0.941 after 10 weeks of storage for 30, 40, and
50◦ Bx, respectively). Growth of many non-halophilic and anaerobic
bacteria are inhibited in media (syrup, molasses, etc.) of high osmolar­
ity, a fact that is usually attributed to low water activity (Walter et al.,
1987). Generally, water activity and osmotic pressure are easily related
using the Van’t Hoff equation (Gekas et al., 1998).
The sugar loss only tells part of the story; the products formed during
storage give additional insights (Fig. 2). Syrup from Partner A, of the
M81-E cultivar, showed significant sugar degradation products such as
lactic and acetic acids in the original syrup before storage. This suggests Fig. 2. Sugar degradation products before and after storage with and without a
surface-covering oil layer. (P.A.=Partner A; P.B.=Partner B; A.S.=After Storage;
that degradation had taken place prior to syrup evaporation, perhaps
w/=with; w/o = without.
between harvest and processing (Misra et al., 2017; Solomon, 2009).
During the storage of 30◦ Bx M81-E syrup, additional lactic acid was

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K.T. Klasson and S.A. Boone Industrial Crops & Products 163 (2021) 113330

produced, suggesting sugar deterioration by bacteria. Lactic acid was syrups that had or had not been stored under a layer of oil. However, the
not a major product when 40 and 50◦ Bx syrups were stored. In all cases, results differed in the case of 30◦ Bx syrups stored under oil.
ethanol was the main product, regardless of ◦ Bx level, cultivar, or syrup The fermentation of the 30◦ Bx syrup from the Dale cultivar from
producer. It is possible that the lower water activity in the higher ◦ Bx Partner A (P.A.) proceeded normally, but the fermentation rate varied
syrups may have prevented some bacteria (e.g., lactic acid producers) to slightly from experiment to experiment (see Fig. 4). The high level of
grow (Brown, 1976). This is fortuitous, if the desired final product is lactic acid (13 g/L, measured after dilution of stored syrup to 22◦ Bx and
bioethanol or subsequent products made from ethanol (e.g., vinegar) after yeast inoculation) present in some of the syrups before fermenta­
(Mat Isham et al., 2019). tion most likely impacted the fermentation rate. As is noted in Fig. 4, the
Yeast fermentation results for the stored and then diluted (22◦ Bx) 30◦ Bx sweet sorghum syrup that had been stored without an oil layer,
syrups are shown in Fig. 3. The fermentations progressed over 120 h in a produced less CO2 than the two replicate experiments which had a layer
few cases, but most fermentations were completed within 24 h. In the of oil on top. However, it should not be concluded that less ethanol was
case of 40 and 50◦ Bx syrup storage experiments, the product composi­ present in the control experiment (without oil). All the sugars were
tion and concentrations did not differ between fermentations done with consumed (data not shown) while ethanol and the coproduct, glycerol,
were the major products.
The fermentation of 30◦ Bx syrup from the M-81E cultivar from
Partner A (P.A.) that had been stored without an oil layer failed to
ferment. There was no evidence of CO2 production (data not shown) and
the products that existed before fermentation also existed after the
fermentation (Fig. 3, 30◦ Bx). The lactic acid level after storage and
before fermentation was 18 g/L, which is high. Others have reported
decreased yeast fermentation efficiency in the presence of lactic and
acetic acids (Graves et al., 2006; Narendranath et al., 2001a, b). In the
presence of 30− 40 g/L of lactic acid, reduced ethanol titer from corn
mash was noted by Graves et al. (2006). In a defined medium, 25 g/L of
lactic acid inhibited the growth of two yeast strains (Narendranath et al.,
2001b). Reduced growth, lower sugar consumption, and lower ethanol
production was noted for lower concentrations (2− 6 g/L) of lactic acid.
The negative impact on yeast when both acetic and lactic acids were
present was amplified when compared to the presence of a single acid
(Narendranath et al., 2001b) and it is well known that lactic acid bac­
teria are useful when preserving foods (Singh, 2018). Thus, our
conclusion is that the organic acid concentration inhibited ethanol
fermentation.
To estimate the overall impact of storage on ethanol production via
fermentation, we calculated the theoretical final ethanol titer based on
initial (before storage) sugar and ethanol concentrations and assuming
the following theoretical reactions for conversion of sugars
inversion
1 mol sucrose ̅̅̅̅→ 1 mol glucose
fermentation
+ 1 mol fructose ̅̅̅̅̅̅→ 4 mol ethanol

fermentation
1 mol glucose OR 1 mol fructose ̅̅̅̅̅̅→ 2 mol ethanol
This value was compared with the titer of ethanol in the final fer­
mented product, corrected for any dilutions (i.e., dilution before
fermentation and by inoculum). The ethanol yield was calculated as:

Fig. 3. Concentration of sugar degradation products after storage (with and

without a surface-covering oil layer) and after fermentation of the stored
syrups, diluted to 22◦ Bx. The concentrations after storage have been adjusted Fig. 4. Example of yeast fermentation rates for one cultivar (Dale) from one
for dilution factors. (P.A.=Partner A; P.B.=Partner B; A.S.=After Storage; A.F.= partner (P.A.=Partner A) and three storage containers (two replicates and
After Fermentation; w/=with; w/o = without). one control).

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K.T. Klasson and S.A. Boone Industrial Crops & Products 163 (2021) 113330

ethanolbeforestorage+ethanolpredictedfromsugarfermentation Declaration of Competing Interest

EthanolYield=
ethanolafterstorageandfermentation
The authors report no declarations of interest.
These results are shown in Table 2. The final ethanol yield was not
impacted by storage conditions for any of the ◦ Bx levels of the Dale Acknowledgements
cultivar from Partner A, as the majority of the degradation of sugars
during storage produced the desired ethanol. The yeast fermentation This research was supported by the U.S. Department of Agriculture
that followed the storage, simply increased the ethanol concentration. (USDA), Agricultural Research Service, and addressed goals outlined in
Overall, the ethanol yields are lower for the M-81E cultivar from the strategical research plans. Mention of trade names or commercial
same partner (Partner A), because the sugars degraded to several other products in this publication is solely for the purpose of providing specific
products during storage (see Fig. 2). However, the ethanol yield was not information and does not imply recommendation or endorsement by the
significantly impacted by storage (with or without an oil layer) for any USDA. USDA is an equal opportunity provider and employer.
of the ◦ Bx levels of the M-81E cultivar, except for in one case. As pre­
viously mentioned, fermentation of diluted 30◦ Bx syrup made from the References
M-81E cultivar and stored without an oil layer failed. While fermenta­
tion failed, ethanol was produced during storage, accounting for 42 % of Andrzejewski, B., Eggleston, G., Powell, R., 2013. Pilot plant clarification of sweet
sorghum juice and evaporation of raw and clarified juices. Ind. Crops Prod. 49,
theoretical (Table 2). The ethanol yield for the Dale cultivar from
648–658. https://doi.org/10.1016/j.indcrop.2013.06.027.
Partner B was slightly lower than from the same cultivar from Partner A, Blowers, M., Bowler, G., Goddard, M., Hopwood, S., Parkin, G., Roberts, D., 2010. The
but not significantly. The lower ethanol yield (67 %) noted for 30◦ Bx Storage of Thick Juice. British Sugar, Wissington Sugar Factory, Norfolk, United
Kingdom.
syrup from Partner B stored without oil was the result of incomplete
Brown, A.D., 1976. Microbial water stress. Bacteriol. Rev. 40, 803–846.
fermentation of fructose (data not shown). Busic, A., Mardetko, N., Kundas, S., Morzak, G., Belskaya, H., Santek, M.I., Komes, D.,
No recovery and purification of the produced bioethanol was part of Novak, S., Santek, B., 2018. Bioethanol production from renewable raw materials
this research, but it is assumed that the recovered ethanol would have and its separation and purification: a review. Food Technol. Biotechnol. 56,
289–311. https://doi.org/10.17113/ftb.56.03.18.5546.
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