Production of bioethanol from corn meal hydrolyzates

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					                                                                  Fuel 85 (2006) 1750–1755

                      Production of bioethanol from corn meal hydrolyzates
                      Ljiljana Mojovic *, Svetlana Nikolic, Marica Rakin, Maja Vukasinovic
                                     ´                   ´                               ´
         Faculty of Technology and Metallurgy, Department of Biochemical Engineering and Biotechnology, University of Belgrade, Karnegijeva 4,
                                                   11000 Belgrade, Serbia, Serbia and Montenegro
                                Received 4 October 2005; received in revised form 13 January 2006; accepted 24 January 2006
                                                            Available online 24 February 2006

   The two-step enzymatic hydrolysis of corn meal by commercially available a-amylase and glucoamylase and further ethanol fermentation of
the obtained hydrolyzates by Saccharomyces cerevisiae yeast was studied. The conditions of starch hydrolysis such as substrate and enzyme
concentration and the time required for enzymatic action were optimized taking into account both the effects of hydrolysis and ethanol
fermentation. The corn meal hydrolyzates obtained were good substrates for ethanol fermentation by S. cerevisiae. The yield of ethanol of more
than 80% (w/w) of the theoretical was achieved with a satisfactory volumetric productivity P (g/l h). No shortage of fermentable sugars was
observed during simultaneous hydrolysis and fermentation. In this process, the savings in energy by carrying out the saccharification step at lower
temperature (32 8C) could be realized, as well as a reduction of the process time for 4 h.
q 2006 Elsevier Ltd. All rights reserved.

Keywords: Enzymatic starch hydrolysis; Corn meal; Corn starch; Bioethanol

1. Introduction                                                                 in fermentable carbohydrates, or those locally available that
                                                                                could be converted to yield the fermentable sugars, are used
   In recent years, research and development efforts directed                   worldwide. Feedstocks based on corn, sorghum, Jerusalem
toward commercial production of ethanol as the most                             artichoke, potato and lignocellulosic biomass are of the
promising biofuel from renewable resources have increased.                      greatest interest for ethanol production in Serbia [8].
In many developed countries in Europe and in USA, the use of                       An important issue regarding the bioethanol production is
bioethanol as an alternative fuel or a gasoline supplement in the               weather the process is economical [9–11]. Research efforts are
amounts up to 15% is highly recommended [1–3], or even                          focused to design and improve a process, which would produce
required as an ecologically favourable fuel oxygenates [4].                     a sustainable transportation fuel. A low cost of feedstock is a
Concerning the EU, a new directive was accepted in November                     very important factor in establishing a cost effective
2001 that requires of members states to establish legislation                   technology [12,13]. In order to analyze the cost effectiveness
about utilisation of fuels from renewable resources. In 2005,                   of bioethanol derived from corn grain, estimates of the net
this utilisation should cover 2% of the total fuel consumption.                 energy balance of ethanol production were done. In the study,
This quota is expected to increase to 5.75% in 2010 and                         which was based on data from processing technologies used in
furthermore. Some member states like Finland, Sweden or                         the early 1980s, Pimentel [13] concluded that the energy
Austria have already fulfilled this quota [5].                                   requirement for producing ethanol derived from corn grain was
   Word ethanol production in 2003 was 32 Mm3 [6]. The                          higher than the energy content of ethanol. However recently,
major world producers are Brazil and the US, which together                     Shapouri et al. [14] and Kim and Dale [1] estimated that the
account for about 80% of the world production. Around 60% of                    energy content of ethanol derived from corn grain was higher
the ethanol is produced by fermentation [7]. Main feedstocks                    than the energy required to produce ethanol. It is believed that
                                                                                the improvement in the last decade was primarily due to
for bioethanol production are sugar cane (in Brazil) and corn
                                                                                increasing corn yields, lower agricultural chemical use, and
grain (USA), while many other agricultural raw materials rich
                                                                                improved corn processing technologies.
                                                                                   The hydrolysis of corn starch may be considered as a first and
 * Corresponding author. Tel.: C381 11 3370 423; fax: C381 11 3370 387.         key step in corn processing for bioethanol production. The main
   E-mail address: (L. Mojovic).                          role of this step is to effectively provide the conversion of
0016-2361/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.        two major starch polymer components: amylose, a mostly linear
doi:10.1016/j.fuel.2006.01.018                                                  a-D-(1–4)-glucan and branched amylopectin, a a-D-(1–4)-glucan,
                                                  L. Mojovic et al. / Fuel 85 (2006) 1750–1755                                       1751

which has a-D-(1–6) linkages at the branch points, to fermentable         concentrations of inoculum (1, 1.35 and 2% w/w) were used
sugars that could subsequently be converted to ethanol by yeasts          for the fermentation of corn meal hydrolyzates.
or bacteria. Recent advances in the developing of termostable
a-amylases, the starch liquefying enzymes which catalyze the
                                                                          2.3. Hydrolysis experiments
hydrolysis of internal a-D-(1–4)-glucosidic linkages in starch in a
random manner [15–18] and effective glucoamylases [19,20], the
                                                                             Corn meal, 100 g was mixed with water in various weight
starch saccharifying enzymes which catalyze the hydrolysis of a-
                                                                          ratios (1:1.25, 1:3, 1:4, 1:5) and 60 ppm of Ca2C (as CaCl2)
D-(1–4) and a-D-(1–6)-glucosidic bonds of starch from the non-
                                                                          ions was added. The mixture was treated with enzymes in two
reducing ends giving glucose as the final product, have led to
                                                                          steps. The first step, liquefaction, was performed at 85 8C and
commercial establishment of the so called ‘two enzyme cold
                                                                          pH 6.0 with various concentrations of Termamyl 120L, and the
process’ [7]. The main advantages of this process are lower
                                                                          second step, saccharification, was performed at 55 8C and pH
energy consumption and a lower content of non-glucosidic
impurities, and thus much better suitability for ethanol                  5.0 with various concentrations of Supersan 240 L. The
production.                                                               liquefaction with Termamyl 120L was usually done in 1 h,
   The aim of this study was to investigate the two-step                  while the saccharification with Supersan 240 L was usually
enzymatic hydrolysis of corn meal by commercially available               operated in 4 h, unless otherwise state. The hydrolysis was
a-amylase and glucoamylase and ethanol fermentation of the                performed in flasks in thermostated water bath with shaking
obtained hydrolyzates by various inoculums concentrations of              (150 rpm).
Saccharomyces cerevisiae yeasts. The conditions of starch
hydrolysis such as the substrate and enzyme concentration and             2.4. Ethanol fermentation of starch hydrolyzates
the time required for the enzymatic action were optimized
taking into account both the effects of hydrolysis and the                   Starch hydrolyzates obtained by the two-step hydrolysis
ethanol fermentation.                                                     of the corn starch meal were subjected to ethanol
                                                                          fermentation by S. cerevisiae under anaerobic conditions
2. Materials and methods                                                  (pH 5.0; 32 8C; mixing rate: 100 rpm). It was considered
                                                                          that the pasteurization of the substrate achieved during the
2.1. Starch                                                               enzymatic liquefaction (85 8C for 1 h) was sufficient thermal
                                                                          treatment, and thus no additional sterilization prior to
   Corn meal obtained by dry milling process was a gift form              fermentation was performed. The mashes containing various
starch industry ‘Jabuka-Pancevo’, Serbia. The corn meal                   initial starch concentrations were fermented by yeast up to
consisted of 90% of particles with average size between 0.5               48 h in flasks in thermostated water bath with shaking. The
and 1 mm and 10% of the particles with a size less then                   effect of initial yeast concentrations was studied by applying
0.5 mm.                                                                   different inoculum concentrations. During the fermentation,
                                                                          the consumption of the substrate was followed as well as
2.2. Enzymes and microorganisms                                           the formation of ethanol. The experiments with simul-
                                                                          taneous fermentation and second step hydrolysis were also
   Termamyl 120L, a heat-stable a-amylase from Bacillus                   performed.
licheniformis was used for corn meal liquefaction. The enzyme
activity was 120 KNU/g (KNU, kilo novo units a-amylases—
the amount of enzyme which breaks down 5.26 g of starch per               2.5. Analytical methods
hour according to Novozyme’s standard method for the
determination of a-amylase). Supersan 240L Aspergillus                        The starch content was determined by Ewers polarimetric
niger glucoamylase, activity 240 AGU/g (AGU is the amount                 method [21]. The water content in the corn meal was
of enzyme which hydrolyses 1 mmol of maltose per minute                   determined by the standard drying method in an oven at
under specified conditions) was used for corn meal sacchar-                105 8C to constant mass. Lipid concentration was determined
ification. The enzymes were gift from Novozymes, Denmark.                  according to the Soxlet method. Protein content was estimated
   S. cerevisiae was used for the fermentation of hydrolyzed              as the total nitrogen by the Kjeldahl method multiplied by 6.25,
corn meal. The culture originated from the collection of BIB-             and the ash content was determined by slow combustion of the
TMF, Belgrade, and was maintained on a malt agar slant. The               sample at 650 8C for 2 h [22]. During the corn meal hydrolysis
agar slant consisted of malt extract (3 g/l), yeast extract (3 g/l),      and fermentation, the content of reducing sugars was
peptone (5 g/l), agar (20 g/l) and distilled water (up to 1 l).           determined by 3, 5-dinitrosalicylic acid [23]. A standard
Before use as an inoculum for the fermentation, the culture was           curve was drawn by measuring the absorbance of known
aerobically propagated in 500 ml flasks in a shaking bath at               concentrations of glucose solutions at 570 nm. The ethanol
32 8C for 48 h and then separated by centrifugation. The liquid           concentration was determined based on the density of alcohol
media consisted of yeast extract (3 g/l), peptone (3.5 g/l),              distillate at 20 8C and expressed in weight % (w/w). At least
KH 2PO 4 (2.0 g/l), MgSO 4!7H 2O (1.0 g/l), (NH 2) 2SO 4                  three measurements were made for each condition and the data
(1.0 g/l), glucose (10 g/l) and distilled water. Various                  given were averages.
1752                                                     ´
                                                L. Mojovic et al. / Fuel 85 (2006) 1750–1755

3. Results and discussion

3.1. Enzymatic hydrolysis

   The corn meal used for the experiments was dry milled
without previous separation of the germ. The content of the
main components was determined by chemical analysis and
presented in Table 1.
   The degree of hydrolysis of native starch from the corn meal
depends on the factors such as the substrate concentration, type
and concentration of the enzyme used, and on the applied
process conditions such as pH, temperature and the mixing
rate. The first set of experiments was conducted in order to
determine the concentration of a-amylase (Termamyl) and the
time needed for an appropriate liquefaction of the corn meal.
The substrate concentration represented by the ratio of
substrate to water (hidromodulus) was fixed at 1:3, which                Fig. 1. Effect of a-amylase (Termamyl) activity on dextrose equivalent of the corn
                                                                        meal suspension. Process conditions: Hidromodulus: 1:3; 85 8C; pH 6.0; 1 h.
corresponded to an initial concentration of starch of 17.5%.
   The effect of the concentration of Termamyl (represented as          hydrolyzate treated for an hour with 12 KNU of Termamyl (up
activity units) on the dextrose equivalent of corn meal is              to DEZ16) was subsequently treated with various amounts of
presented in Fig. 1. The dextrose equivalent (DE) of the corn           Supersan glucoamylase. Fig. 3 presents the DE values
meal hydrolyzate was directly proportional to the activity of           obtained. It may be seen that the highest conversion of corn
the enzyme in a given time of enzyme action. It may be seen             meal starch (DEZ93.8%) was achieved with the combined
from Fig. 1 that DE value of 16 is reached after 1 h of action of
                                                                        action of 12 KNU of Termamyl and 48 AGU of Supersan after
12 KNU of Termamyl, while a DE value of 19 could be
                                                                        48 h. However, a similar and rather high conversion (DEZ
obtained with 14 KNU. Obviously, the same conversions could
                                                                        92.1%) was obtained in a two-step enzymatic hydrolysis with
be achieved with lower enzyme concentrations in a longer
                                                                        36 AGU of Supersan, thus indicating that the lower amount of
period of time. However, to our opinion, extension of the
                                                                        enzyme is sufficient for the effective conversion of the
enzyme reaction in the first step is not economically justified
                                                                        substrate. In all experiments within 24 h more than 90% of
because of the high temperature, which is employed (85 8C). In
                                                                        the total conversion performed within 48 h was attained.
addition, the longer exposure of the enzyme to high
                                                                           The two enzymes used in our study exhibited a high
temperatures, which are needed for gelatinization of the starch
granules and for achieving a good susceptibility to enzyme              efficiency in the conversion of corn starch, which was
action, could lead to slight enzyme deactivation.                       comparable with the results obtained by many other
   A further objective was to determine the optimal degree of           researchers. Arasaratnam et al. [25] reported a lower glucose
liquefaction of the corn meal suspension needed for adequate            yield of 76.0% on corn flour by using a similar combination of
saccharification in the subsequent step. For this purpose,               amylase and glucoamylase. Some differences in the results may
liquefied corn meal suspensions with various degrees of                  originate from different compositions of the initial substrate,
liquefaction (e.g. with different DE values) were subjected to
the action of the saccharifying enzyme Supersan glucoamylase
(24 AGU). The results are presented in Fig. 2. The highest DE
value after treatment with both liquefying and saccharifying
enzymes was obtained when the DE after the treatment with
Termamyl was around 16 (Fig. 2). A similar optimal DE after
liquefaction was reported earlier [7,24].
   In order to determine the optimal concentration of the
glucoamylase, the saccharifying enzyme that is necessary for
complete conversion of starch to glucose, the corn meal
Table 1
Chemical composition of the corn meal

Component                                          (%, w/w)
Starch                                             70.82
Proteins                                           11.80
Lipids                                              7.89                Fig. 2. Effect of the DE value of the hydrolyzate liquefied by Termamyl on the
Ash                                                 5.58                DE value of the hydrolyzate obtained by Supersan. Process conditions for
Water                                               3.91                Termamyl: Hidromodulus: 1:3; 85 8C; pH 6.0; 1 h.; Process conditions for
                                                                        Supersan: 55 8C; pH 5.0; AZ24 AGU; 4 h.
                                                          L. Mojovic et al. / Fuel 85 (2006) 1750–1755                                           1753

                                                                                    11.5; 14; 17.5 and 20% (w/w) were liquefied by the
                                                                                    treatment with Termamyl (1 h; 85 8C; pH 6.0, 12 KNU per
                                                                                    100 g of corn meal), then cooled to 55 8C, adjusted to pH 5,
                                                                                    and treated with Supersan (36 AGU per 100 g of corn meal)
                                                                                    for 4 h. After that the temperature was decreased to 32 8C
                                                                                    and the mixture was inoculated with 1% (w/w) of S.
                                                                                    cerevisiae and subjected to ethanol fermentation. The results
                                                                                    are presented in Table 2.
                                                                                       The initial substrate concentration had a pronounced effect
                                                                                    on both the starch hydrolysis and the ethanol fermentation.
                                                                                    Regarding the yields, lower substrate concentrations are more
                                                                                    suitable since substrate inhibition could be thus avoided.
                                                                                    Arasaratnam et al. [25] reported lower effects of hydrolysis of
                                                                                    corn flour starch for higher initial starch concentrations, e.g.
                                                                                    when a 16% suspension of corn flour was hydrolyzed, the
                                                                                    glucose yield was 76%, while when a 40% suspension was
Fig. 3. Influence of the amount of Supersan on DE values of hydrolyzates.
                                                                                    hydrolyzed the yield was only 50.2%. Similar substrate
Process conditions for Termamyl: Hidromodul: 1:3; 85 8C; pH 6.0; AZ12 KNU;          inhibition was also noticed for ethanol fermentation [9,30].
1 h. Process conditions for Supersan: 55 8C; pH 5.0; AZ24, 36 or 48 AGU; 24         The inhibition of fermentation becomes significant with
and 48 h.                                                                           increase of the initial substrate concentration above 15%
                                                                                    [24]. According to the results presented in Table 2, the
the substrate concentrations and different experimental                             maximum ethanol concentration (% w/w) and the maximum
conditions applied. Dettori-Campus et al. [26] hydrolyzed                           value of product yield on substrate (YP/S) were achieved for the
starch granules from various cereals by amylase from Bacillus                       lowest initial corn starch concentration in the mixture (e.g.
stearothermophilus and obtained up to 80% of conversion for                         11.5% w/w).
barley, corn and rice starches, while the conversion of potato                         However, from the economic viewpoint, it is desired to
starch was less efficient. Similar yields, lower than 80%, were                      attain as high ethanol concentrations as possible in order to
obtained in one step enzymatic hydrolysis with glucoamylase                         decrease the costs of ethanol distillation, which are consider-
performed by Kimura and Robyt [27]. Leach and Schoch [28]                           able in the economical evaluation of the overall process [2,30].
reported that raw corn starch is more susceptible to a-amylase                      In addition, the use of higher initial substrate concentrations is
action than high amylose corn starch. A very efficient                               economically more favourable, since it could decrease the
conversion of corn starch of more than 96% after 24 h was
                                                                                    reactor volumes [7]. Taking into account the ethanol
reported by Karakastsanis and Liakopoulu-Kyriakides [29]
                                                                                    concentrations achieved, the yields of products per substrate
using the simultaneous action of a-amylase and glucoamylase.
                                                                                    (YP/S) and the volumetric productivities of ethanol (P)
Generally considered, the two-step enzymatic process was
                                                                                    presented in Table 2, and related economic points, we can
shown to be more efficient than one-step enzymatic action.
                                                                                    suggest utilization an initial concentration of 17.5% (w/w)
                                                                                    which corresponds to the hidromodulus of 1:3.
4. Ethanol fermentation of the corn meal hydrolyzates
                                                                                    4.2. Effect of inoculum concentration
4.1. Effect of the initial substrate concentration
                                                                                       The time course of the ethanol fermentation of corn starch
   In the first set of experiments, the effect of the initial corn                   hydrolyzate was performed with various inoculum concen-
starch concentrations on ethanol fermentation was assessed.                         trations of S. cerevisiae and the results are presented in Fig. 4.
Mixtures of various concentrations of corn meal and water                           The increase of inoculum concentration did not have
which corresponded to initial starch concentrations C0Z                             pronounced effect on the final ethanol concentration. The final
Table 2
Results of the batch fermentation of corn meal hydrolyzates with different initial starch content

Hidromodulus           Initial substrate       DE after two-step       Maximum % (w/w)         % of theoretical   YP/S (g/g)         P (g/l h)
                       concentration, C0       enzymatic actiona       of ethanolb             yield of ethanol
                       (%, w/w)
1:5                    11.5                    75.5                    5.7                     89.2               0.50               1.21
1:4                    14.0                    72.2                    6.8                     87.5               0.48               1.41
1:3                    17.5                    68.1                    7.7                     78.5               0.44               1.60
1:2.5                  20                      58.4                    6.9                     62.4               0.34               1.43
     Enzymatic treatment: 12 KNU of Termamyl at 85 8C; pH 6.0;1 h; 36 AGU of Supersan at 55 8C; pH 5.0; 4 h.
     Ethanol fermentation: 32 8C; pH 5.0; 48 h; 1% w/w of Saccharomyces cerevisiae.
1754                                                            ´
                                                       L. Mojovic et al. / Fuel 85 (2006) 1750–1755

                                                                               Fig. 5. Time course of simultaneous hydrolysis and fermentation. Enzyme
                                                                               treatment first step: Hidromodulus: 1:3; 12 KNU of Termamyl; 85 8C; pH 6.0;
Fig. 4. Effect of the inoculum concentration of Saccharomyces cerevisiae on    1 h. Ethanol fermentation: pH 5.0; 32 8C; 100 rpm; 1.35% w/w of
the fermentation of starch hydrolyzate. Process conditions for enzyme          Saccharomyces cerevisiae. The second step enzyme treatment with 36 AGU
treatment: Hidromodulus: 1:3; 12 KNU of Termamyl at 85 8C; pH 6.0; 1 h;        of Supersan was performed concurrently with fermentation.
36 AGU of Supersan at 55 8C; pH 5.0; 4 h. Process conditions for ethanol
fermentation: pH 5.0; 32 8C; mixing rate: 100 rpm with various inoculum        the degree of hydrolysis by a-amylase. This incentive effect
concentrations of Saccharomyces cerevisiae.                                    was exhibited in the presence of starch as a substrate, while in
                                                                               the absence of starch the residual enzyme activity was
ethanol yields obtained by fermentation with 1, 1.35 and 2% (w/
                                                                               decreased. As presented in Fig. 5, concentration of glucose
w) of inoculum were 78.5, 80.1 and 81.6% of the theoretical
                                                                               was constantly maintained low during the fermentation, since
yield, respectively. However, the duration of fermentation
                                                                               the produced glucose was simultaneously consumed by the
decreased with the increase of the inoculum concentration.
                                                                               yeast and converted to ethanol. The time course of the glucose
Thus, fermentation with 1% (w/w) of yeast lasted 48 h, with
                                                                               concentration during the fermentation (Fig. 5) suggested that
1.35% (w/w) of yeast 36 h, while with 2% (w/w) of yeast the
                                                                               the production of sugar from starch was in accordance with its
fermentation was accomplished in 32 h (Fig. 4).
                                                                               consumption by yeasts. The progress of ethanol production
                                                                               showed that there was not a shortage of fermentable sugars
4.3. Simultaneous hydrolysis and fermentation                                  during the process.

   In order to reduce the time of the complete process and
make beneficial energy savings, we further performed a                          5. Conclusion
simultaneous process of the second hydrolysis step and
fermentation of corn meal at 32 8C, a temperature which was                       The two-step enzymatic hydrolysis of corn meal by
optimal for the fermentation by yeast. For that purpose, a                     commercially available a-amylase and glucoamylase and the
mixture of corn meal with water (17.5% w/w of starch) was                      subsequent or simultaneous ethanol fermentation of the
first liquefied by treatment with 12 KNU of Termamyl per                         hydrolyzates by S. cerevisiae were studied. The conditions of
100 g of corn meal at 85 8C, pH 6.0 for 1 h. The mixture was                   starch hydrolysis such as substrate and enzyme concentration
subsequently cooled to 32 8C, and pH was adjusted to 5.0. Then                 and the time required for the enzymatic action were optimized
the saccharyfying enzyme, e.g. Supersan glucoamylase was                       taking into account both the effects of hydrolysis and the
added in the amount of 36 AGU per 100 g of corn meal. The                      ethanol fermentation.
mixture was well homogenized and immediately after that                           The optimal DE which should be reached in the first
inoculated with 1.35% w/w of S. cerevisiae. In this way, the                   enzymatic liquefaction step with 12 KNU of Termamyl per
second step hydrolysis, e.g. saccharification, and fermentation                 hundred grams of corn meal was found around 16. Further
were simultaneously carried out at 32 8C. The kinetics of the                  sacharification and fermentation steps were effectively
process is presented in Fig. 5.                                                performed by using 36 AGU of Supersan glucoamylase per
   The time course of the ethanol fermentation and the final                    hundred grams of corn meal and choosing an initial substrate
ethanol concentration achieved (Fig. 5) were very similar to                   concentration of 17.5% (w/w) of starch (1:3 hidromodulus). In
those obtained by fermentation which followed two-step                         this way, an ethanol yield of more than 80% (w/w) of the
enzymatic actions. These data suggested that the ethanol                       theoretical was attained.
produced during the fermentation did not have an inhibitory                       By increasing the inoculum’s concentration from 1 to 2%
effect on the enzymatic hydrolysis. In the literature, there are               w/w of S. cerevisiae, the fermentation time could be reduced
very few recent reports on stimulation of enzyme action by                     from 48 to 32 h, respectively.
ethanol [31]. Apar and Ozbeck, 2005 [31] found that the                           Fermentation and second step hydrolysis can be concur-
addition of ethanol in the range of 5–15% could increase                       rently performed, and no shortage of fermentable sugars was
                                                           L. Mojovic et al. / Fuel 85 (2006) 1750–1755                                                       1755

observed, suggesting that these two processes were in                              [13] Pimentel D. Ethanol fuels energy security, economics, and the
accordance. In this way, the time of the overall process may                            environment. J Agric Environ Ethics 1991;4:1–13.
                                                                                   [14] Shapouri H, Duffield JA, Wang M. The energy balance of corn ethanol: an
be reduced for 4 h (the time required for the second hydrolysis                         update. Agricultural economic report 813, US Department of Agriculture,
step). By concurrent fermentation and hydrolysis, the energy                            Washington, DC; 2002.
savings could be attained since the overall process was                            [15] Konsoula Z, Liakopoulou-Kyriakides M. Hydrolysis of starches by the
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