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Dziugan et al. Biotechnology for Biofuels 2013,
6:158http://www.biotechnologyforbiofuels.com/content/6/1/158
RESEARCH Open Access
Evaluation of the fermentation of high gravitythick sugar beet
juice worts for efficientbioethanol productionPiotr Dziugan1, Maria
Balcerek2*, Katarzyna Pielech-Przybylska2 and Piotr Patelski2
Abstract
Background: Sugar beet and intermediates of sugar beet
processing are considered to be very attractive feedstockfor
ethanol production due to their content of fermentable sugars. In
particular, the processing of the intermediatesinto ethanol is
considerably facilitated because it does not require pretreatment
or enzymatic treatment in contrastto production from starch raw
materials. Moreover, the advantage of thick juice is high solid
substance andsaccharose content which eliminates problems with the
storability of this feedstock.
Results: The objective of this study were to investigate
bioethanol production from thick juice worts and theeffects of
their concentration, the type of mineral supplement, as well as the
dose of yeast inoculum onfermentation dynamics and ethanol
yield.The obtained results show that to ensure efficient ethanolic
fermentation of high gravity thick juice worts, oneneeds to use a
yeast strain with high ethanol tolerance and a large amount of
inoculum. The highest ethanol yield(94.9 2.8% of the theoretical
yield) and sugars intake of 96.5 2.9% were obtained after the
fermentation of wortwith an extract content of 250 g/kg
supplemented with diammonium hydrogen phosphate (0.3 g/L of wort)
andinoculated with 2 g of Ethanol Red dry yeast per L of wort. An
increase in extract content in the fermentationmedium from 250 g/L
to 280 g/kg resulted in decreased efficiency of the process. Also
the distillates originatingfrom worts with an extract content of
250 g/kg were characterized by lower acetaldehyde concentration
thanthose obtained from worts with an extract content of 280
g/kg.
Conclusions: Under the favorable conditions determined in our
experiments, 38.9 1.2 L of 100% (v/v) ethylalcohol can be produced
from 100 kg of thick juice. The obtained results show that the
selection of processconditions and the yeast for the fermentation
of worts with a higher sugar content can improve the
economicperformance of the alcohol-distilling industry due to more
efficient ethanol production, reduced consumption ofcooling water,
and energy for ethanol distillation, as well as a decreased volume
of fermentation stillage.
Keywords: Bioethanol, Fermentation, Thick juice, Sugar beet,
High gravity wort, Yeast
BackgroundBiofuels are defined as solid (biochar), liquid
(bioethanol,biobutanol, biodiesel) and gaseous (biogas,
biosyngas,biohydrogen) fuels that are mainly derived from
biomass.Traditionally, sugar substrates derived from food cropssuch
as sugar cane, corn (maize) and sugar beet have beenthe preferred
feedstock for the production of biofuels [1].
* Correspondence: [email protected] of Spirit
and Yeast Technology, Institute of FermentationTechnology and
Microbiology, Lodz University of Technology, 90-924Wolczanska, Lodz
171/173, PolandFull list of author information is available at the
end of the article
2013 Dziugan et al.; licensee BioMed CentraCommons Attribution
License (http://creativecreproduction in any medium, provided the
or
Bioethanol can be produced from all feedstock thatcontain mono-,
oligo- and polysaccharides (for example,starch and cellulose) [2].
An advantage of raw materialscontaining simple sugars and
disaccharides, such as sac-charose, is the simplified technology of
extraction to thewater medium, followed by fermentation to ethanol
with-out the need of using additional technological
operationsconnected with chemical or enzymatic hydrolysis,
whichcould significantly increase the costs of biosynthesis
[2].From an economic point of view and in comparison withcereals,
sugar beet and beet-processing intermediates con-taining saccharose
are very good raw materials for ethanol
l Ltd. This is an open access article distributed under the
terms of the Creativeommons.org/licenses/by/2.0), which permits
unrestricted use, distribution, andiginal work is properly
cited.
mailto:[email protected]://creativecommons.org/licenses/by/2.0
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Table 1 Chemical composition of raw material
Physicochemical parameters Thick juice
Solid substance (g/kg) 685.2 11.5
pH 7.4 0.2
Reducing sugars as invert sugar (g/kg) 3.1 0.4
Saccharose (g/kg) 598.4 12.5
Total nitrogen (g/kg) 5.6 0.4
Volatile acids as acetic acid (g/kg) 4.4 0.2
Results expressed as mean values standard error (n = 3).
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production due to their content of fermentable
sugars(saccharose) [3,4].The production of ethanol from sugar
beet-processing
intermediates (raw, thin, and thick juices) and from bypro-ducts
(molasses) constitutes an alternative solution forsugar factories
interested in a combined production ofsugar and bioethanol.
Furthermore, the use of inter-mediate products of sugar beet
processing as raw mate-rials for bioethanol production could be
attractive fordistilleries located near the sugar factories, as it
wouldminimize high transportation costs. Cooperation betweenthese
factories could lead to increased production andutilization of the
capacity of both types of facilities.Very high gravity (VHG)
processes are extremely at-
tractive and promising for bioethanol production as theyallow
significant improvements in overall productivity,thus minimizing
production costs thanks to energy sav-ings [5]. On the other hand,
the use of VHG technologyimposes greater stress on yeast cells,
which has beenassociated with the loss of yeast viability during
VHGfermentation, a reduced rate, and incomplete fermenta-tion [6].
Thus, the successful implementation of VHGtechnology in bioethanol
production requires the use ofyeast strains that can efficiently
ferment high sugarconcentrations (>250 g/L) [7]. Such strains
must be resist-ant to the multiple stresses found in the process,
includingthe osmotic stress that results from high sugar
concentra-tion, the ethanol stress at the end of fermentation,
theanaerobic conditions established in large-scale bioreac-tors,
and the cell recycling procedures for the utilizationof the yeast
biomass for several consecutive fermenta-tion cycles [8,9].Balcerek
at al. [10] investigated the effect of various
strains of the yeast Saccharomyces cerevisiae (S. cerevisiae)on
the dynamics and efficiency of alcoholic fermentationof thick juice
worts. The authors tested strains designatedas M1, M2, M3 (from the
Pure Culture Collection of theInstitute of Fermentation Technology
and Microbiology,Lodz University of Technology), commonly used for
thefermentation of molasses worts, as well as strains desig-nated
as Bc-16, D-2, As-4 (purchased from the yeastfactory in Maszewo
Lborskie, Poland), used for the fer-mentation of mashes based on
starch raw materials. Itwas found that S. cerevisiae strains M1 and
M2 dynamic-ally and efficiently (89 to 94% of the theoretical
yield)fermented thick juice worts with an extract concentra-tion of
200 g/kg and 250 g/kg, whereas the strain D-2preferred less dense
worts (extract concentration of200 g/kg). Gumienna et al. [4]
evaluated the efficiencyof alcoholic fermentation of sugar beet and
its processingintermediates using commercial yeast strains such as
Etha-nol Red and Fermiol (Fermentis Division S.I. Lesaffre,France).
Balcerek and Pielech [11] also tested the EthanolRed yeast strain
for the fermentation of triticale starch
mashes with a solid substance concentration of approxi-mately
23%. The obtained results showed high ethanolyields (87.54 0.46% to
88.30 0.46% of the theoreticalyield).According to the declaration
of the producer (Fermentis
Division S.I.), Ethanol Red is a specially selected strainthat
was developed for the ethanol industry. With a highethanol
tolerance, this fast acting strain displays higheralcohol yields
and maintains higher cell viability, especiallyduring VHG
fermentation. Ethanol Red is particularlywell-suited for sugar
substrates (sweet juices, molasses)and also saccharified mashes
[12].The objective of the presented study was to determine
the effect of thick juice worts concentration, the type
ofmineral supplements, and the dose of yeast inoculum onthe
dynamics and efficiency of alcoholic fermentation.
Results and discussionChemical characteristics of thick juiceThe
chemical composition of thick juice applied in thisstudy was
typical of sugar beet processing intermediates(see Table 1). The
high content of saccharose (598.4 g/kg)is advantageous from the
technological point of viewbecause it promotes a high yield of
ethanol from theraw material. Our results are consistent with the
datareported by Rankovi et al. [13] with one exception re-lated to
the total nitrogen content. The thick juice de-scribed by Rankovi
et al. [13] contained four times lesstotal nitrogen (1.4 g/kg) than
the raw material used in ourstudy (5.6 g/kg). The differences in
the content of nitrogencompounds are probably related to the sugar
beet varietiesprocessed in sugar factories in Poland and Serbia
[14],and to different sugar beet cultivation conditions andthe
technology used for processing it into thick juice(Table 1).The
chemical composition of the investigated thick juice
makes this intermediate product of sugar beet processingan
attractive feedstock for alcoholic fermentation. Thickjuice is only
subjected to initial dilution, pH adjustment,and supplementation
with inexpensive mineral sourcesof nitrogen for the yeast (if
needed). This makes theoverall process of bioethanol production
from thick sugarbeet juice relatively simple in comparison to
production
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from starch-based raw materials, which require the lib-eration
of starch (by pressure cooking or by thoroughgrinding) and its
liquefaction and saccharification [11].
The effect of process conditions on fermentationdynamics and the
results of fermentation of high gravitythick juice wortsThe effects
of initial extract content in thick juice worts,the type of mineral
supplement, and the dose of yeastinoculum on the dynamics and
efficiency of alcoholicfermentation were determined. The obtained
results arepresented in Figures 1, 2, and 3.In all the processing
variants, the highest conversion
of saccharose to fermentable sugars (expressed as
reducingsugars) as a result of the activity of the
-fructofuranosidase(EC 3.2.1.26) present in yeast cells, was
observed duringthe first 12 h of fermentation. Due to the prolonged
initialphase of fermentation of worts with an extract content of250
g/kg inoculated with a yeast dose of 1.0 g/L and supple-mented with
diammonium hydrogen phosphate (0.3 g/L ofwort), ethanol production
during the first 6 h of the process
Figure 1 Fermentation dynamics of thick juice worts with an
extract(B) Inoculum content of 1.0 g/L; (NH4)2HPO4 + MgSO4 7 H2O.
(C) Inoculum(NH4)2HPO4. MgSO4 7 H2O.
was very low (close to zero). In the next 6 h of the
process,ethanol concentration increased to 0.8 0.02% (v/v)(Figure
1A). Intensive biosynthesis of ethanol was reportedafter 12 h of
fermentation. After 94 h of fermentation,ethanol concentration in
the wort reached 12.4 0.3%(v/v). On completion of the process, the
real extractconcentration of wort decreased from 250 g/kg to
ap-proximately 50 g/kg (by 80%). Also, the concentrationof residual
reducing sugars was still sufficiently highand amounted to 35.4 g/L
of wort. This indicates thatthe sugar substrates were not fully
utilized (Figure 1A).Yeast cells have specific growth requirements
leading
to an imbalance or limitations resulting in
incompletefermentation. These requirements include specific
levelsof nitrogen, carbon, vitamins, water, oxygen, and metalions.
Metal ions are required for a number of purposes;they include bulk
elements (such as magnesium, calcium,and potassium) and trace
elements (such as zinc, copper,and manganese) needed by yeast cells
[15]. Magnesium isnecessary for the activation of several
glycolytic enzymes,and in practical terms this means that if
industrial media
content of 250 g/kg. (A) Inoculum content of 1.0 g/L;
(NH4)2HPO4.content of 1.5 g/L; (NH4)2HPO4. (D) Inoculum content of
2.0 g/L;
-
Figure 2 Fermentation dynamics of thick juice worts with an
extract of 280 g/kg. (A) Inoculum content of 1.0 g/L; (NH4)2HPO4.
(B) I noculumcontent of 1.0 g/L; (NH4)2HPO4 +MgSO4 7 H2O. (C) I
noculum content of 1.5 g/L; (NH4)2HPO4. (D) Inoculum content of 2.0
g/L; (NH4)2HPO4.
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are magnesium-limited, the conversion of sugar to alcoholmay be
suppressed, leading to slow or incomplete fermen-tation processes
[16].Due to the incomplete fermentation of the wort with
an initial extract content of 250 g/kg supplemented withonly
diammonium hydrogen phosphate and inoculatedwith 1 g yeast per L of
wort, it seemed appropriate toconduct further fermentation
experiments using mixednutrients for yeast in the form of
diammonium hydrogenphosphate (NH4)2HPO4 (0.3 g/L) and magnesium
sulfateheptahydrate (MgSO4 7 H2O) (0.1 g/L) and largeramounts of
yeast inoculum.Supplementation of thick juice wort (with an
extract
content of 250 g/kg) with MgSO4 7 H2O (in addition todiammonium
hydrogen phosphate) did not significantlyimprove the course of the
process, so its efficiency wascomparable to the process conducted
in the presence ofonly diammonium hydrogen phosphate. Upon
comple-tion of this process, ethanol concentration in the
wortsupplemented with Mg2+ ions reached 13.0 0.4% (v/v)and was not
statistically higher than that in wort with-out the addition of
MgSO4 7 H2O (12.4 0.2% v/v,0.05 < P
-
Figure 3 Fermentation results of thick juice worts. Different
letters indicate significant differences (P
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ethanol yield under the cited experimental conditions(Figure
3).The obtained results are in accordance with the findings
of Takeshige and Ouchi [17], who reported inhibited yeastgrowth
and reduced ethanol yield in the process of molas-ses wort
fermentation containing sugar at a concentrationof 300 g/kg.
Moreover, Dodi et al. [18], who fermentedthick juice worts,
observed that with an increase of fer-mentable sugars content from
5% to 20% (w/w), ethanolyields also increased for both investigated
raw materials(molasses and thick juice). However, when an initial
sugarscontent of 20% (w/w) was increased to 25% (w/w), theyields
dropped significantly, from 67 to 56%. The lowyields obtained by
Dodi et al. [18] could have beencaused by the fact that
fermentation was carried outusing bakers yeast, which was most
likely not adaptedto high-density worts. Furthermore, the worts
were notsupplemented with mineral nutrients for yeasts.Hinkov and
Bubnk [19], who fermented concentrated
raw sugar beet juice achieved the highest ethanol yield(88.2 to
94.4% of theoretical yield) when the sugar con-centration in the
wort amounted to 200 g/kg. The effi-ciency of fermentation and
ethanol yield decreased withan increase in wort extract. The
distillery yeast strainstested by Hinkov and Bubnk [19] showed an
increasedtolerance to osmotic pressure and provided higher yieldsin
worts with higher initial concentrations of sugar. Athigh sugar
concentrations, it was observed that the yeastexperienced osmotic
pressure, which led to plasmolysisand a lower ethanol yield [20].
Based on the obtained fer-mentation coefficients for the studied
thick juice worts(Figures 1, 2, 3), the quantity of 100% (v/v)
ethanol ob-tained from 100 kg of this raw material was
calculated.The results show that 38.9 1.2 L 100% (v/v) ethyl
alco-
hol could be produced from 100 kg of thick juice underthe
following favorable conditions, established in our ex-periments:
extract content of 250 g/kg, yeast dose of 2 g/Lof wort and
(NH4)2HPO4 addition of 0.3 g/L of wort.
Analysis of the chemical composition of the
obtaineddistillatesThe quality of bioethanol used for fuel purposes
is strictlydefined by the Polish national and industrial norms.
Highconcentrations of fermentation by-products can cause alower
price of the final product. According to some pro-ducers of
dehydrated ethanol, higher concentrations ofpollutants in the raw
spirit (unpurified ethyl alcohol)can cause fast deterioration of
molecular sieves used inthe process of ethanol dehydration [21].
The chemicalcomposition of distillates obtained is shown in Table
2.Methanol concentration in the obtained raw spiritswas low and
ranged from 7.7 0.7 to 9.3 0.9 mg/L100% (v/v) ethyl alcohol (no
statistically significant dif-ferences, 0.05 < P
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Table 2 Chemical composition of distillates obtained from the
fermentation of thick juice worts
Compound(mg/L 100% v/vethyl alcohol)
Parameters of fermentation
Extract content of 250 g/kg Extract content of 280 g/kg
Inoculum contentof 1.0 g/L;(NH4)2HPO4
Inoculum contentof 1.0 g/L;(NH4)2HPO4 +MgSO4 7H2O
Inoculum contentof 1.5 g/L;(NH4)2HPO4
Inoculum contentof 2.0 g/L;(NH4)2HPO4
Inoculum contentof 1.0 g/L; (NH4)2HPO4
Inoculum contentof 1.0 g/L; (NH4)2HPO4 +MgSO4 7H2O
Inoculum contentof 1.5 g/L;(NH4)2HPO4
Inoculum contentof 2.0 g/L;(NH4)2HPO4
Methanol 8.1 0.8a 7.7 0.7a 9.3 0.9a 7.7 0.8a 9.3 0.9a 8.1 0.8a
9.3 0.9a 9.1 0.9a
Acetaldehyde 763.4 4.2a 1626.0 4.1b 2226.0 6.5c 2214.5 6.3c
3214.5 7.5d 3869.8 8.8e 3971.8 9.2f 4172.9 9.8g
Methyl acetate 8.2 0.8a 9.2 0.8a 8.6 0.8a 9.3 0.8a 7.9 0.8a 9.0
0.8a 8.3 0.8a 9.2 0.8a
Ethyl acetate 266.3 2.5b 248.7 2.5a 284.5 2.8d 270.8 2.6c 248.7
2.5a 246.8 2.5a 243.7 2.5a 285.0 2.8d
Isoamyl acetate 0.0a 0.0a 0.0a 1.9 0.2b 2.6 0.2c 4.2 0.3d 7.1
0.5e 8.7 0.5f
Ethyl butyrate 33.5 0.9c 45.3 1.4d 60.1 1.5f 52.1 1.4e 18.0 0.6a
18.6 0.6a 21.9 0.8b 22.6 0.8b
n-propanol 155.9 2.2bc 177.2 2.8e 187.1 2.8f 158.9 1.6c 152.9
1.6ab 150.3 1.5a 206.1 2.7g 165.9 1.5d
2-methyl-1-propanol 314.3 3.2b 307.2 2.8a 368.9 3.5d 381.1 3.5e
357.8 3.5c 402.2 3.8f 355.7 3.5c 355.3 3.5c
n-butanol 7.4 0.5a 7.8 0.5ab 7.5 0.5a 7.5 0.5a 6.8 0.5a 7.6 0.5a
8.9 0.7b 8.1 0.6b
2-methyl-1-butanol 236.3 2.5a 249.4 2.5b 299.7 2.9e 269.6 2.7c
272.1 2.7c 283.2 2.7d 284.5 2.7d 306.7 3.2f
3-methyl-1-butanol 650.7 3.5a 667.7 3.8b 805.8 4.1d 818.2 4.2e
719.4 3.8c 906.8 4.2g 969.0 4.3h 879.7 4.1f
Results expressed as mean values standard error (n = 3). a-hMean
values in lines with different letters are significantly different
(P
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100% (v/v) ethyl alcohol while the content of
2-methyl-1-propanol was higher and ranged from 307.2 2.8 to402.2
3.8 mg/L 100% (v/v) ethyl alcohol. The amountsof n-butanol in all
the tested distillates were relativelysmall (6.8 0.5 to 8.9 0.7
mg/L 100% v/v ethyl alcohol).The most abundant isoamyl alcohol
detected in the dis-tillates was 3-methyl-1-butanol (650.7 3.5 to
969.0 4.3 mg/L 100% v/v ethyl alcohol), whereas the contentof
2-methyl-1-butanol ranged from 236.3 2.5 to 306.7 3.2 mg/L 100%
(v/v) ethyl alcohol (Table 2). Apart fromthe significantly higher
levels of acetaldehyde in the dis-tillates derived from worts with
a density of 280 g/kg,there was no correlation between the
concentrations ofthe identified byproducts and the fermentation
condi-tions (Table 2).The literature provides scant reports on the
chemical
composition of raw spirits originating from the inter-mediate
products of sugar beet processing. Raw spiritsobtained from the
fermentation of thick juice worts werecharacterized by a lower
content of higher alcohols thanthose obtained by Balcerek and
Pielech-Przybylska [11]following the fermentation of starch mashes
(from triti-cale) with the Ethanol Red yeast strain.
ConclusionsThe results of our study prove that the intermediate
prod-ucts of sugar beet processing, such as thick juice, may
beconsidered an attractive raw material for bioethanol pro-duction.
Saccharose is the principal component of its ex-tract, so the only
necessary operations before alcoholicfermentation are dilution, pH
regulation, and additionof mineral nitrogen sources (if needed).
The fermentation ofthick juice worts with an extract content of 250
g/kg using2 g of the dry distillery yeast Ethanol Red (S.
cerevisiae)per 1 L of wort supplemented with (NH4)2HPO4 as
anutrient for yeast was determined to be favorable, as itenabled a
high ethanol yield (38.9 1.2 L 100% v/v ethylalcohol from 100 kg of
thick juice).Due to limitations on sugar manufacturing in EU
coun-
tries, the capacity of sugar factories is not fully utilizedand
they are ready to increase the processing of sugar beetinto
intermediates, which could serve as feedstock forbioethanol
factories. This would be an alternative to starchprocessing,
especially in the years of crop failures. Anothercrucial issue is
also the ability of biofuels to reduce green-house gas (GHG)
emissions. GHG emissions in the lifecycle of bioethanol depend,
among others, on the rawmaterial and technology of production. The
productionof ethanol from sugar beet intermediate products is
veryfavorable in that it lowers GHG emissions. The resultsof the
study presented in this manuscript are aimed toimprove the
production process leading to measurableeffects in terms of higher
reduction of GHG emissions.
MethodsRaw material and microorganismsThick sugar beet juice was
obtained from Dobrzelin SugarFactory (Dobrzelin, Poland).
Fermentation was carriedout using a preparation of Ethanol Red dry
distilleryyeast (S. cerevisiae), (Fermentis Division S.I.)
designedfor the production of alcohol up to 18% (v/v) at
hightemperature (35C). The number of living cells at packingwas
>2.0 1010 per g, as declared by the manufacturer.
Preparation of fermentation wortsFermentation worts were
prepared by diluting thick juicewith distilled water, initially at
a ratio of 1:1 w/w, and thenobtaining solutions with an extract
content of either 250or 280 g/kg. The worts were acidified with 25%
(w/w) sul-furic acid to pH 4.8 and supplemented with (NH4)2HPO4(0.3
g/L) only or with (NH4)2HPO4 (0.3 g/L) and MgSO4 7H2O (0.1 g/L) as
nutrients for yeast.
Fermentation variantsThe fermentation variants were as
follows:
I. Extract content of 250 g/kg; inoculum content of1.0 g/L;
(NH4)2HPO4
II. Extract content of 250 g/kg; inoculum content of1.0 g/L;
(NH4)2HPO4 +MgSO4 7 H2O
III. Extract content of 250 g/kg; inoculum content of1.5 g/L;
(NH4)2HPO4
IV. Extract content of 250 g/kg; inoculum content of2.0 g/L;
(NH4)2HPO4
V. Extract content of 280 g/kg; inoculum content of1.0 g/L;
(NH4)2HPO4
VI. Extract content of 280 g/kg; inoculum content of1.0 g/L;
(NH4)2HPO4 +MgSO4 7 H2O
VII. Extract content of 280 g/kg; inoculum content of1.5 g/L;
(NH4)2HPO4
VIII. Extract content of 280 g/kg, inoculum content of2.0 g/L;
(NH4)2HPO4
Fermentation experiments were carried out in 6-L glassflasks,
each containing approximately 3 L of wort. Afterinoculation with
yeast, which was preliminarily rehydrated,the flasks were closed
with stoppers equipped with fermen-tation pipes filled with
glycerol and kept in a thermostat-controlled room at 35C. The
process was carried out over4 days (96 h). During the fermentation,
samples for analysiswere collected and the concentration of
ethanol, realextract (after ethanol distillation), reducing sugars,
andsaccharose was measured, allowing us to compare the dy-namics
and biotechnological factors of the entire process.
DistillationWhen fermentation was complete, all ethanol was
distilledfrom worts using a laboratory distillation unit
consisting
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of a distillation flask, a Liebig cooler, a flask for
collectingethanol, and a thermometer. Raw spirits containing 20
to23% (v/v) ethanol were refined to approximately 43% (v/v)in
distillation apparatus equipped with a bi-rectifier
unit(dephlegmator according to Golodetz), and subjected tochemical
analysis.
Analytical methodsThick juice was analyzed by the methods
recommendedfor the sugar industry [24]. Solid substance (total
extract)was measured by using a hydrometer, which indicates
theconcentration of dissolved solids, mostly sugars, calibratedin g
of saccharose per kg of water solution. Total nitrogenwas
determined by the Kjeldahl method. Volatile acids(expressed as
acetic acid) were assayed using steam dis-tillation. Reducing
sugars and total sugars (after inversionwith hydrochloric acid)
were estimated by the Lane-Eynonmethod. Both were expressed in g of
invert sugar per kg ofthick juice. Saccharose concentration was
calculated as thedifference between total sugars and reducing
sugars (takinginto consideration a conversion coefficient of 0.95).
AlsopH was measured (with a digital pH-meter).Worts were analyzed
before and after fermentation using
methods recommended for distilleries. Prior to fermenta-tion,
the worts were analyzed for pH, total extract, and re-ducing sugars
(expressed as invert sugar) and saccharosecontent. On completion of
fermentation, the worts wereanalyzed for real extract (after
ethanol distillation), etha-nol concentration in wort (using a
hydrometer with ascale in % v/v of ethanol) and sugars
content.Distillates were analyzed using the Agillent 6890 N gas
chromatograph (USA, Wilmington) equipped with a flame-ionization
detector (FID), a split/splitless injector and anHP-Innowax
capillary column (60 m 32 mm 0.5 m).The temperature at the injector
(split 1:45) and FID waskept at 250C. The temperature program was
as follows:40C (6 minutes), an increase to 83C (2C/minutes) andthen
to 190C (5C/minutes) (2 minutes). The flowrate of the carrier gas
(helium) through the column was2 mL/minute.
Fermentation evaluationThe intake of total sugars (the
percentage yield of sugarconsumption during fermentation) was
calculated as a ra-tio of sugars used during the fermentation to
their contentin the wort prior to this process, and expressed in
percent.The yield of ethanol was calculated according to the
stoi-chiometric Gay-Lussac equation in relation to total sugarsand
expressed as a percentage of the theoretical yield.
Statistical analysisAll samples were prepared and analyzed in
triplicate.Statistical analysis was carried out using the
MicromalOrigin ver. 6.0 software (Northampton, USA).
Abbreviations(NH4)2HPO4: Diammonium hydrogen phosphate; MgSO4 7
H2O: Magnesiumsulfate heptahydrate; FID: Flame-ionization detector;
GHG: Greenhouse gas;VHG: Very high gravity.
Competing interestsThe authors declare that they have no
competing interests.
Authors contributionsPD and MB designed the experiments. MB,
KP-P and PP performed theexperiments. MB and KP-P wrote the paper.
PD and PP were involved inthe evaluation of results and review of
the paper. All authors read andapproved the final manuscript.
Author details1Department of Fermentation Technology, Institute
of FermentationTechnology and Microbiology, Lodz University of
Technology, 90-924Wolczanska, Lodz 171/173, Poland. 2Department of
Spirit and YeastTechnology, Institute of Fermentation Technology
and Microbiology, LodzUniversity of Technology, 90-924 Wolczanska,
Lodz 171/173, Poland.
Received: 3 July 2013 Accepted: 30 October 2013Published: 8
November 2013
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doi:10.1186/1754-6834-6-158Cite this article as: Dziugan et al.:
Evaluation of the fermentation of highgravity thick sugar beet
juice worts for efficient bioethanol production.Biotechnology for
Biofuels 2013 6:158.
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AbstractBackgroundResultsConclusions
BackgroundResults and discussionChemical characteristics of
thick juiceThe effect of process conditions on fermentation
dynamics and the results of fermentation of high gravity thick
juice wortsAnalysis of the chemical composition of the obtained
distillates
ConclusionsMethodsRaw material and microorganismsPreparation of
fermentation wortsFermentation variantsDistillationAnalytical
methodsFermentation evaluationStatistical analysisAbbreviations
Competing interestsAuthors contributionsAuthor
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