Biochemical Engineering Journal 121 (2017) 1–12

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Intermittent agitation contributes to uniformity across the bed during

pectinase production by Aspergillus niger grown in solid-state
fermentation in a pilot-scale packed-bed bioreactor
Anelize Terezinha Jung Finkler a , Alessandra Biz b , Luana Oliveira Pitol c ,
Bruna Schweitzer Medina b , Henrique Luithardt b , Luiz Fernando de Lima Luz Jr. a ,
Nadia Krieger d , David Alexander Mitchell b,∗
a
Departamento de Engenharia Química, Universidade Federal do Paraná, Cx. P. 19011 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil
b
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil
c
Departamento de Engenharia Química, Universidade Estadual de Maringá, Av. Colombo 5790, Maringá 87020-900, Paraná, Brazil
d
Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil

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

Article history: Solid-state fermentation could be used to produce low-cost pectinases that could then be used to sac-
Received 9 July 2016 charify pectin in citrus waste biorefineries. Recently, we produced pectinases in a pilot-scale packed-bed
Received in revised form 5 January 2017 bioreactor, growing Aspergillus niger on a substrate mixture consisting of 27 kg of wheat bran and 3 kg
Accepted 21 January 2017
of sugarcane bagasse (dry mass). However, the agglomeration of particles and shrinkage of the bed cre-
Available online 23 January 2017
ated preferential flow paths, leading to overheating within the bed and poor uniformity of pectinase
levels at the end of the fermentation. In the current work, we used intermittent agitation as a strategy
Keywords:
to minimize agglomeration, comparing one agitation (10 h), three agitations (at 8, 10 and 12 h) and five
Solid-state fermentation
Packed-bed bioreactor
agitations (every 2 h from 8 to 16 h). The pectinase activity in the bed was uniform after agitation, but
Aspergillus niger poor uniformity occurred when the bed was left unmixed for more than 10 h. The best regime was that
Intermittent agitation with three agitations: For 15 samples removed from different vertical and horizontal positions of the bed
Pectinases at 20 h, the average pectinase activity was 22 U g−1 , with a sample standard deviation of 2 U g−1 . We con-
Product uniformity clude that the use of intermittently-mixed packed-bed bioreactors is a promising strategy for producing
pectinases in solid-state fermentation.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction For the saccharification of pectin, enzymatic hydrolysis is prefer-

Several authors have recently suggested the possibility of estab-
lishing biorefineries to produce a range of products from citrus
wastes, including limonene, pectin, ethanol and biogas [1,2]. How-
ever, since the amount of pectin contained in citrus wastes is
several-fold greater than the world pectin market [3], it would be
more interesting to saccharify the pectin to liberate D-galacturonic
acid, which constitutes over 75% of the polymer [4]. D-galacturonic
acid could then be converted to mucic acid and L-galactonic acid,
which have several current or potential commercial applications
and can also act as precursors of other products such as ascorbic
acid, bioplastics, mucic acid and L-galactonic acid [5–7].
∗ Corresponding author.
E-mail address: davidmitchell@ufpr.br (D.A. Mitchell).
able to acid hydrolysis since it preserves the d-galacturonic units
that are released. This is unlike acid hydrolysis, for which processes
that are strong enough to hydrolyze the glycosidic bonds between
the d-galacturonic acid residues in pectin also degrade a significant
proportion of the d-galacturonic that is released [8]. However, in
order for enzymatic hydrolysis to be viable, it is essential to have
low-cost pectinases.
One possible strategy for producing low-cost pectinases is to use
solid-state fermentation (SSF) [9]. However, there are no general
rules for guiding scale-up of SSF bioreactors [10] and it is therefore
necessary to scale-up processes on a case-by-case basis. In the case
of pectinases, a scale-up study was recently initiated by Pitol et al.
[9]. Their work represents the largest pilot-scale study of pectinase
production by SSF to date: the filamentous fungus Aspergillus niger
was cultivated on 27 kg of wheat bran, with the addition of 3 kg
of sugarcane bagasse as a bed porosity modifier (dry masses). The
bioreactor was operated as a packed-bed with forced aeration. The

http://dx.doi.org/10.1016/j.bej.2017.01.011
1369-703X/© 2017 Elsevier B.V. All rights reserved.
2 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
bed remained completely static during the fermentations and, as
a result, problems were encountered. For example, in their third
pilot-scale fermentation, the fungus knitted the substrate particles
into agglomerates and the bed shrank, producing gaps between the
bed and the bioreactor wall. When this happened, the air flowed
through these gaps rather than through the bed itself. As a con-
sequence, the bed overheated, with temperatures as high as 43 ◦ C
being measured at the top of the bed [9]. At the end of the fermen-
tation, the pectinase activities measured in different locations of
the bed were quite different, varying from 11 to 28 U per gram of
dry substrate.
The problem of substrate agglomeration by filamentous fungi
in SSF was investigated by Schutyser et al. [11], who studied the
growth of Aspergillus oryzae on wheat grains in a horizontal drum.
In their studies, the bed was initially left static and then later agi-
tated with the intention of breaking the agglomerates apart. They
concluded that, if the bed is left too long before the first agitation
event, large agglomerates will form in which the “mycelial bonds”
between the particles are sufficiently strong to remain intact when
the bed is mixed by rotating the drum. They suggested that the
best strategy to prevent the formation of unbreakable agglomer-
ates is to use an agitation event early in the fermentation to disrupt
the mycelial bonds between the particles before they become too
strong. Although their experimental investigations were limited
to a single preemptive agitation event, they did raise the pos-
sibility that it might be necessary to have subsequent agitation
events to maintain the substrate bed free of agglomeration prob-
lems throughout the whole process.
The recommendations made by Schutyser et al. [11] regarding
the use of preemptive agitation events to prevent substrate agglom-
eration prompted us to investigate whether this strategy would
mitigate the agglomeration problems reported by Pitol et al. [9] for
the cultivation of Aspergillus niger on the mixture of wheat bran and
sugarcane bagasse in the pilot-scale bioreactor.
2. Materials and methods
The system studied in the current work is the same as that
studied by Pitol et al. [9], namely the growth of Aspergillus niger
CH4 (kindly provided by Dr. Jesus Cordova and Prof. Dr. Gustavo
Viniegra-Gonzalez) on a substrate mixture consisting of 27 kg of
wheat bran and 3 kg of sugarcane bagasse (dry mass), in a pilot-
scale packed-bed bioreactor with a bed capacity of 200 L. This
bioreactor can be agitated intermittently by rotation around its cen-
tral axis. Note that moisture contents are reported throughout this
paper as %w/w, on a wet basis. They were determined in an infrared
balance (Gehaka IV 2000, São Paulo, Brazil).
2.1. Source and preparation of raw materials
The sugarcane bagasse was donated by Usina de Álcool Mel-
horamentos (Jussara, Brazil). It was sun-dried in the open air for
several days before use. Otherwise, it was used as received (i.e.
without milling or sieving). The wheat bran was donated by Ana-
conda Industrial e Agrícola de Cereais (Curitiba, Brazil) and used as
received.
2.2. Pilot-scale packed-bed bioreactor
The bioreactor consists of an AISI 306 stainless steel cylinder
with a perforated plate (70 cm × 60 cm) at the bottom, which sup-
ports the substrate bed (Fig. 1 a and b) [9,12]. A wire mesh with
1-mm apertures covers the base plate. The air is injected below the
base plate and flows from the bottom to the top of the bed.
The air preparation and temperature measurements are the
same as for Pitol et al. [9] and Biz et al. [12]. A blower captures air
from outside the building and blows it through a glass wool filter
and into the bottom of a humidification tower. The flow is measured
by a differential pressure flow meter (Kobold, Hofheim, Germany).
Water is pumped from a 1200-L reservoir (kept at 32 ± 2 ◦ C) into the
top of the humidification tower and drains back into the reservoir.
The air exits the top of the humidification column with a relative
humidity of 99% and a temperature close to the water reservoir
temperature. By the time that it enters the bottom of the biore-
actor, it is about 2 ◦ C cooler than when it left the humidification
column. The outlet air passes through a gas washer. Thermocouples
measure the inlet and outlet air temperatures. There are also four
thermocouple arrays within the bioreactor, positioned at heights
of 5, 18, 33 and 46 cm (Fig. 1b, items 2, 3, 4 and 5, respectively).
Each array measures the temperature at four different horizontal
positions. Temperatures reported for each bed height represent the
averages of these four measurements on the horizontal.
2.3. Preparation of inoculum for the pilot-scale bioreactor
The inoculum was prepared in the same manner as that
described by Pitol et al. [9]. Fresh cultures of Aspergillus niger CH4
were grown on potato dextrose agar at 30 ◦ C for 4 days to allow
sporulation. The spores were suspended in sterile distilled water
and the suspension was filtered through sterile gauze to remove
any mycelium. Spores were counted in a Neubauer chamber and
the concentration in the suspension was adjusted to 2 × 107 spores
mL−1 . This preparation is denoted as “pre-inoculum”.
Forty 250-mL Erlenmeyer flasks, each containing 10 g (dry mass)
of a mixture of 30% sugarcane bagasse and 70% wheat bran (w/w),
were autoclaved (121 ◦ C, 15 min). (NH4 )2 SO4 solution was auto-
claved (121 ◦ C, 15 min) and added to each flask to obtain a final
moisture content of 50% (w/w, wet basis) and a final concentration
of (NH4 )2 SO4 of 4.4% (w/w, based on total dry substrate). Pre-
inoculum was added to give 107 spores per gram of dry substrate
and the flasks were incubated at 30 ◦ C for 7 days to allow sporula-
tion. The spores were suspended in sterile distilled water and then
filtered in a funnel with sterile gauze to remove residual substrate.
The spore concentration in the suspension was determined using
a Neubauer chamber. The spore concentration in the suspension
was adjusted to 2 × 107 spores mL−1 . This preparation is denoted
as “pilot-inoculum”.
2.4. Solid-state fermentation in the pilot-scale bioreactor
Except for the fact that the bed was intermittently agitated,
the pilot-scale bioreactor was operated as described by Pitol et al.
[9]. The air flow rate was kept constant in all fermentations at
150 m3 h−1 , resulting in a superficial air velocity of 0.1 m s−1 . All
fermentations were carried out with 27 kg of wheat bran and
3 kg of sugarcane bagasse (dry masses), which resulted in an ini-
tial bed height of 40 cm. The solid substrate, sufficient solution of
(NH4 )2 SO4 to give an initial concentration of 4.4% (w/w, based on
total dry substrate) and the additional water needed to obtain an
initial moisture content of 62% were autoclaved (121 ◦ C, 2 h). After
cooling, sufficient pilot-inoculum was added to give 2 × 107 spores
per gram of dry substrate and then the inoculated solid was added
to the bioreactor, which was rotated for 30 min to homogenize the
bed. After this period, the bed surface was leveled using a rake, the
bioreactor was closed and the air supply system was connected.
The bioreactor was left static for most of the time. For each agita-
tion event, the air inlet, the air outlet and the thermocouple cables
were disconnected and then the bioreactor was rotated around its
central axis at 5 rpm for 2 min. After this, the top of the bed was
again leveled with a rake. The inlet and outlet air lines and cables
were then reconnected.
 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12 3

Fig. 1. The pilot-scale bioreactor. (a) Overview of the bioreactor and the air preparation system. Key: (1) air blower; (2) air filter; (3) humidification tower; (4) water reservoir;
(5) water pump; (6) water entry into the humidification tower; (7) supply of saturated air to bioreactor; (8) return of excess water back to the reservoir; (9) measurement
of inlet air temperature; (10) air inlet (11) measurements of bed temperature; (12) air outlet; (13) measurement of outlet air temperature; (14) oxygen sensor; (15) data
acquisition equipment; (16) to gas washer. (b) Design details of the pilot-scale packed-bed bioreactor; Key: (1) 1-mm aperture wire mesh supported on a perforated base
plate; (2) thermocouple at 5-cm height; (3) thermocouple at 18-cm height; (4) thermocouple at 33-cm height; (5) thermocouple at 46-cm height; (6) electric motor with
variable frequency attached to the central axis; (c) locations where the samples were removed at the end of the fermentation for mapping the bed. Samples from the bottom
were collected at around 2-cm height, from the middle at about 20-cm height and from the top at about 40-cm height. This figure is reproduced from Biz et al. [12] with the
kind permission of Elsevier and is a modified version of the figure originally given in Pitol et al. [9].

During the fermentation, samples for the determination of
pectinase activity were collected only from the top of the bed, in
order to avoid the creation of preferential paths for air flow. At
the end of the fermentations, in order to investigate the uniformity
of the bed, samples were removed from five different horizontal
positions near the base, in the middle and near the top of the bed
(Fig. 1c). This procedure, which is identical to that carried out by
Pitol et al. [9], is referred to as “mapping” of the bed.
2.5. Determination of pectinase activity
To samples of 1 g of dry substrate, 20 mL of acetate buffer (0.2 M,
pH 4.5) was added, with extraction at 180 rpm and 30 ◦ C for 30 min.
The crude extracts were vacuum-filtered through Whatman n◦ 1
filter paper. For the enzyme assays, 0.25 mL of crude extract was
incubated with 0.25 mL of 1% (w/v) citric pectin (Sigma-Aldrich,
Missouri, USA, 75% methylation) in acetate buffer (0.2 M, pH 4.5) for
20 min at 30 ◦ C [13]. The release of reducing sugars was followed
using the DNS method [14]. A standard curve was constructed with
D-galacturonic acid (Sigma-Aldrich, Missouri, USA, ≥98.0% purity).
Pectinase activities are expressed on the basis of the total mass of
dry substrate in the sample (i.e. U g−1 ).
3. Results
Six fermentations were carried out in which Aspergillus niger
was grown on a 90:10 mixture (% m/m) of wheat bran and sugar-
cane bagasse. Each fermentation is denoted by a code showing the
number of mixing events, with the time of harvesting, in hours, as
a subscript. MIX-020 had no mixing events. MIX-320 and MIX-326
had mixing events at 8, 10 and 12 h. MIX-315 had mixing events
at the same times and was terminated at 18 h. However, for this
fermentation, the lag phase was 3 h longer than normal, due to
problems in the temperature control of the water reservoir imme-
4 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
diately after inoculation. As a result, 3 h have been discounted from
all times for plotting the pectinase activity and moisture content
results for this particular fermentation and the harvesting is con-
sidered as though it had occurred at 15 h of a normal fermentation.
MIX-526 had mixing events at 8, 10, 12, 14 and 16 h. The results of
these fermentations were compared with those of two fermenta-
tions reported for the same system by Pitol el al. [9]: MIX-026 is
their third fermentation, which was harvested at 26 h, while MIX-
012 represents the first 12 h of their fourth fermentation (i.e. before
they began to vary the temperature of the inlet air). In the text
below, values given in cm are heights within the substrate bed,
with 33 cm being the highest value at which temperatures in the
bed were measured. The data for moisture contents for MIX-026
and MIX-012 were obtained from Pitol [15].
3.1. Axial temperature profiles in the bed
In all fermentations with intermittent agitation, the axial tem-
perature profiles in the bed were smaller than those of the
fermentations without agitation (Fig. 2a, c, e, g and i). In all cases, the
inlet air temperature was well controlled at around 30 ◦ C (Fig. 2b,
d, f, h and j). In the fermentations without agitation, temperatures
over 40 ◦ C were measured at 33 cm: MIX-026 gave a maximum of
43 ◦ C at 16 h [9] (Fig. 2a), while MIX-020 gave a value of 41 ◦ C at
15 h (Fig. 2c). There is clear evidence that, in the absence of mix-
ing, preferential air paths had formed in the bed by 15 h, with the
temperature at 33 cm being higher than the outlet air temperature
for both MIX-026 (compare Fig. 2a with b) and MIX-020 (compare
Fig. 2c with d).
In the fermentations with mixing events, the axial tempera-
ture profiles measured soon after mixing were considerably smaller
than those observed in the unmixed fermentations. For example,
in MIX-126 , the temperature at 33 cm remained around 35 ◦ C from
17 to 21 h (Fig. 2e). However, with MIX-126 and MIX-326 , the out-
let air temperature fell below the bed temperature at 33 ◦ C late
in the fermentation; this is a clear indication that at this time the
air was flowing through preferential paths rather than through the
bed itself. For example, at 24 h in MIX-126 , the temperature at 33 cm
was 39 ◦ C, compared to an outlet air temperature of 32 ◦ C (compare
Fig. 2e with Fig. 2f). Likewise, at 20 h in MIX-326 , the temperature
at 33 cm was 3 ◦ C higher than that of the outlet air (compare Fig. 2i
with Fig. 2j). In MIX-526 , the additional agitation events prevented
this formation of preferential air paths, as shown by the fact that in
this fermentation the outlet air temperature never fell below the
temperature at 33 cm (compare Fig. 2g with Fig. 2h).
In MIX-526 and MIX-326 , the highest temperatures in the bed
were measured at 5 cm (see Figs. 2g and i). This was due to a local-
ized problem caused by the design of the bioreactor. The sleeve for
the thermocouples at 5 cm is close to the wall of the bioreactor, and
when we removed the bed at the end of these fermentations, we
found an agglomeration of substrate trapped in the narrow space
between the 5-cm thermocouple sleeve and the side and bottom of
the bed. We presume that this localized agglomeration of substrate
was not broken up by the agitation events, and the agglomeration
would have decreased the permeability of this region, leading to
the localized increase in temperature.
3.2. Horizontal uniformity of bed temperatures
Agitation also affected the uniformity of the temperatures mea-
sured by the horizontal thermocouple arrays positioned at different
bed heights. For the first 10 h of the fermentation, all thermocou-
ples within the same horizontal sleeve gave very similar readings
(Fig. 3). In the absence of mixing, when preferential air paths
started to form at around 12–13 h, the readings of the thermocou-
ples within the same horizontal sleeve started to diverge from one
another. This occurred at both the bed heights of 18 cm (see Fig. 3a
and c for MIX-026 and MIX-020 , respectively) and 33 cm (see Fig. 3b
and d for MIX-026 and MIX-020 , respectively).
Mixing improved the horizontal uniformity of temperature
measurements. For MIX-526 , the temperatures measured by the
same horizontal sleeve were quite close to one another through-
out the entire fermentation (see Fig. 3e and f for the bed heights of
18 and 33 cm, respectively). For MIX-320 , the temperatures mea-
sured by the horizontal sleeve at 18-cm bed height were quite
close to one another until 16 h and then diverged slightly (Fig. 3g).
Likewise, the temperatures measured by the horizontal sleeve at
33-cm bed height were close to one another until 16 h and then
started to diverge, but somewhat more noticeably (Fig. 3h). This
divergence in the temperatures after 16 h for MIX-320 is consistent
with the observation in Section 3.1 that preferential air paths were
well established in MIX-326 by 20 h.
3.3. Temporal profiles for pectinase activities and moisture
contents at the top of the bed
With the poor temperature control in the fermentation with-
out agitation (MIX-026 ), the pectinase activity at the top of the
bed reached 23 U g−1 at 14 h and actually decreased after the
temperature at 33 cm reached 43 ◦ C, before recovering when the
temperature fell again (Fig. 4a) [9]. A similar result was obtained in
MIX-020 , with the pectinase activity at 20 h being lower than that
measured at 18 h, which was 22 U g−1 . With one agitation event
(MIX-126 ), the maximum pectinase activity at the top of bed was
slightly lower, 19 U g−1 at 18 h (Fig. 4c). For MIX-526 , the maximum
pectinase activity at the top of the bed, 23 U g−1 , was similar to
the control, but this was achieved at a much longer fermentation
time, 24 h (Fig. 4e). In this case, the five agitation events affected the
production of pectinases deleteriously: at the last agitation event
at 16 h, the pectinase activity at the top of the bed was around
19 U g−1 , but it increased only very slowly after this. On the other
hand, in the fermentations with three agitation events, high pecti-
nase activities, 24 U g−1 for MIX-320 and 22 U g−1 for MIX-326 , were
measured at the top of bed at 18 h, which is only slightly longer than
the 14 h of the fermentation without agitation (Fig. 4g).
In all cases, the moisture content at the top of the bed decreased
during the fermentation. The moisture content decreased by
around 7 percentage points over 26 h when there was no agitation
(MIX-026 ) or only one agitation event (MIX-126 ) (Fig. 4b and 4d,
respectively), and 10–13 percentage points when there were more
agitation events (MIX-526 and MIX-326 ) (Fig. 4f and 4 h, respec-
tively). In no case did the moisture content fall below 50%. In those
cases in which preferential air paths formed, which happened at
about 15 h for MIX-026 and at about 18 h for MIX-126 , the mois-
ture content at 26 h was around 4.3 percentage points higher than
the corresponding values measured for MIX-526 and MIX-326 . This
is understandable: when preferential air paths form, then the air
flows almost entirely around the bed, which reduces the evapora-
tion rate compared to the case where air flows through the bed.
3.4. Uniformity of pectinase activities within the bed at the time
of harvesting
When the fermentations were terminated, samples were
removed from various heights and horizontal positions in the bed
(see Fig. 1c). The average pectinase activities and sample standard
error were calculated for the samples removed at each of the three
different bed heights.
There was notable heterogeneity of pectinase activity with bed
height at the end of MIX-026 , with the average activity measured at
the bottom of the bed being 23 U g−1 , compared to values around
15 U g−1 at middle height and 16 U g−1 at the top of the bed (Fig. 5).
 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12 5

Fig. 2. Effect of the agitation regime on temporal temperature profiles of the inlet and outlet air and at different heights within the bed. The column on the left shows
temperature profiles at three bed heights (5, 18 and 33 cm). The column on the right shows the temperature profiles for the inlet and outlet air. The number after the dash
represents the number of agitation events, while the subscript indicates the time (h) at which the fermentation was terminated. The arrows show the times at which the bed
was agitated. The results for MIX-026 are reproduced from Pitol et al. [9].
6 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12

Fig. 3. Effect of the agitation regime on temperatures measured within the bed by the thermocouples within the same horizontal sleeve. The column on the left shows
the temperatures measured by the individual thermocouples (denoted T4, T5, T6 and T7) at 18 cm while the column on the right shows the temperatures measured by the
individual thermocouples (denoted T8, T9, T10 and T11) at 33 cm.
 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12 7

Fig. 4. Effect of the agitation regime on temporal profiles for pectinase activity and moisture content for samples removed from the top of the bed. The column on the left
shows the pectinase activities while the column on the right shows the moisture contents. The arrows show the times at which the bed was agitated. Replicate fermentations
that were terminated at different times are plotted together. The results for pectinase activity for MIX-026 and MIX-012 are reproduced from Pitol et al. [9] and the results for
moisture content for MIX-026 and MIX-012 are reproduced from Pitol [15].
8 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
Fig. 5. Uniformity of pectinase activities along the vertical axis of the bed. Samples from the bottom were collected at around 2-cm height, from the middle at about 20-cm
height and from the top at about 40-cm height. The bars represent the average pectinase activity for the five samples removed from the same horizontal plane (the locations
of which are indicated in Fig. 1c). The error bars represent the sample standard error for these samples. The results for MIX-026 were calculated from Pitol et al. [9].
Similar heterogeneity at the end of the fermentation occurred for
MIX-126 and MIX-326 . For MIX-126 , the average pectinase activ-
ity was 25 U g−1 at the bottom of the bed and 17 U g−1 at the top
of the bed, while for MIX-326 , the corresponding values were 29
and 22 U g−1 , respectively. MIX-526 had significantly better axial
homogeneity at the end of the fermentation: for all bed heights,
the average pectinase activity was around 22–23 U g−1 .
Agitation also affected the heterogeneity of pectinase activities
in the same horizontal plane, which is reflected in the value of the
sample standard error of the five samples collected from that plane.
For MIX-026 , the samples removed from the same horizontal plane
had sample standard errors ranging from 3 to 5 U g−1 (Fig. 5). For
MIX-126 and MIX-326 , the sample standard errors were only slightly
lower, ranging from 1.7 to 3.2 U g−1 for MIX-126 and from 1.7 to
3.8 U g−1 for MIX-326 . The use of five agitation events did promote
horizontal homogeneity (MIX-526 ), even 10 h after the last agitation
event at 16 h, with all sample standard errors being below 2 U g−1
at 26 h.
With three mixing events, it is possible to see the evolution of
both axial and horizontal heterogeneity of pectinase activities in the
bed over time since the last mixing event at 12 h. At 15 h, the aver-
age pectinase activities at all bed heights were around 16–17 U g−1
and the sample standard errors for the three bed heights were
all below 2 U g−1 . At 20 h, the average pectinase activities at all
bed heights had increased to 21–24 U g−1 and the sample standard
errors for the three bed heights were around 2 U g−1 . Finally, at 26 h,
only the pectinase activity at the bottom of the bed had increased
noticeably in relation to its value at 20 h, reaching 29 U g−1 . Addi-
tionally, the sample standard errors for the three bed heights were
now in the range of 2–4 U g−1 .
3.5. Reproducibility of the fermentations
In both the fermentation without mixing and the fermentation
with three mixing events, three replicate experiments were done,
with the only difference being that the fermentations were termi-
nated and the bed mapped at different times. When the temporal
pectinase activity profiles at the top of the bed for MIX-315 , MIX-
320 and MIX-326 are plotted on the same graph, they are quite
close to one another (Fig. 4g). In comparison, the pectinase activ-
ity profiles for MIX-012 , MIX-020 and MIX-026 show more variation
(Fig. 4a). The temporal moisture content profiles at the top of the
bed show a similar result: the profiles for MIX-315 , MIX-320 and
MIX-326 (Fig. 4h) are closer to one another than are the profiles for
MIX-012 , MIX-020 and MIX-026 (Fig. 4b). Our conclusion is that mix-
ing, even if intermittent, promotes fermentation-to-fermentation
reproducibility.
4. Discussion
Not only does our work show that intermittent agitation is
beneficial for the production of pectinases by SSF in a pilot-scale
packed-bed bioreactor, but it also makes a more general contri-
bution by studying intermittent agitation and its effect on the
product uniformity within the bed. These contributions are dis-
cussed below.
4.1. Pilot-scale production of pectinases by SSF
In the current work, intermittent agitation gave better results for
the pilot-scale production of pectinases by Aspergillus niger in SSF
than those obtained by Pitol et al. [9], who used the same microor-
ganism, bioreactor and substrate (i.e. composed of the same 90:10
mixture of wheat bran and sugarcane bagasse) but did not agitate
the bed. We conclude that the best strategy is to use three agitation
events and to harvest at 20 h. This strategy balanced the positive
and negative effects of agitation: it gave a reasonably uniform bed
at 20 h, while avoiding the deceleration of the process that occurred
with five agitation events.
It is important to base decisions about harvesting times on the
results of the mapping of the bed and not on the results of sam-
ples removed from the top of the bed. Samples are removed from
the top of the bed during periods of static operation in packed-bed
bioreactors in order to avoid disturbing the bed. The removal of
samples from the inner parts of the bed would promote the for-
mation of preferential flow paths and subsequent overheating of
the bed. However, as our results show, the real performance of the
fermentation cannot be properly evaluated on the basis of samples
removed from the top of the bed. Based on such samples, the best
productivity would be calculated for MIX-026 : with harvesting at
14 h, the pectinase activity of 23 U g−1 would suggest a productiv-
ity of 1.64 U g−1 h−1 . Application of the same criterion to MIX-320
 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
would lead to a harvesting time of 18 h, with the pectinase activity
at this time of 24 U g−1 suggesting a productivity of 1.33 U g−1 h−1 .
Our mapping of the bed, which takes the whole bed into account,
gave a different result: the average pectinase activity of 22 U g−1
that was obtained at 20 h in MIX-320 is significantly better (p = 0.05)
than the average pectinase activity of 18 U g−1 obtained at 20 h in
MIX-020 . The corresponding real productivities at the time of map-
ping are 1.1 U g−1 h−1 for MIX-320 and 0.9 U g−1 h−1 for MIX-020 .
The better reproducibility between fermentations is an addi-
tional benefit of using intermittent mixing: from the quality control
viewpoint, in commercial processes, reproducibility between fer-
mentations is as important as the product uniformity within the
bed in individual fermentations.
4.2. Agitation regimes and uniformity in SSF bioreactors
Significant temperature and moisture content gradients can
occur within the bed in static SSF bioreactors [10]. Intermittent agi-
tation has been used to decrease such gradients, with some authors
comparing the performance of the fermentation with different
agitation regimes. However, the effect of different intermittent agi-
tation regimes on product distribution in the bed has not been
studied at scales involving 1 kg of moist substrate or more.
The effect of non-uniform growth conditions on the distribu-
tion of product within the bed was addressed by Ghildyal et al.
[16], who studied the production of amyloglucosidase on 750 g of
wheat bran (dry mass) in a packed-bed bioreactor 34.5 cm high and
15 cm in diameter. They obtained temporal profiles for bed tem-
peratures and amyloglucosidase activities at three different bed
heights. They did this for five air flow rates. In their system, even at
the highest air flow rate, there was a significant axial temperature
profile in the bed: with an inlet air temperature of 30 ◦ C, a value of
37.4 ◦ C was measured at 28-cm bed height. Their key result was
that the amyloglucosidase activity tended to decrease with bed
height, correlating with the fact that the temporal temperature
profiles peaked at higher values at the higher bed heights. How-
ever, although they pointed out that it was necessary to minimize
the temperature gradients in order to maximize amyloglucosidase
levels across the whole bed, they did not investigate the use of
infrequent agitation as a possible strategy for achieving this. Like-
wise, although Pitol et al. [9] demonstrated the lack of uniformity
of pectinase activities by mapping the bed at the end of fermenta-
tions undertaken using the same fungus, substrate and bioreactor
as those used in the current work, they did not study the use of
intermittent agitation.
On the other hand, the studies that have investigated the
potential of intermittent agitation for increasing fermentation per-
formance have not investigated the effect of the agitation regimes
on product uniformity in the bed at the end of the fermentation
(Table 1) [11,17–28].
Some studies of intermittent agitation have been undertaken
with rotating-drum and horizontal stirred-drum bioreactors. For
example, de Reu et al. [17] compared a static bed and discontinuous
agitation (at a frequency of about 1.4 h−1 ) for the production of 1 kg
of soybean tempe in a rotating-drum bioreactor, but the compari-
son was done on the basis of the temperature, measured at a single
point in the bed. In a subsequent study of the same process in a sim-
ilar rotating-drum bioreactor, but at a scale involving 100–140 kg
of cooked soybeans, Han et al. [18] applied intermittent agitation
at a frequency of 17.1 h−1 . However, a single value was given for
each measured variable at each sampling time, so, once again, the
uniformity of the bed was not characterized. The lack of concern
of these authors with product uniformity is understandable: with
frequent intermittent agitation in a rotating-drum bioreactor, the
bed is maintained in a reasonably uniform state. However, even in
9
those cases in which rotating drums are left static for several hours,
product uniformity in the bed has not been investigated [21,23,26].
In intermittently-agitated packed-bed bioreactors, agitation is
typically applied at intervals of several hours. As a result, the con-
ditions within the bed become heterogeneous and remain so for
several hours. Despite this, there have been no attempts to charac-
terize how this lack of uniformity of growth conditions affects the
uniformity of product levels within the bed (Table 1). For example,
although temperature differences of up to 15 ◦ C were noted in the
bed of an intermittently-agitated packed-bed bioreactor of 200-
kg capacity, the consequences of these temperature differences for
product uniformity within the bed were not investigated [20].
When mixing is frequent in intermittently-agitated bioreactors,
there is no problem with the mycelium binding the substrate par-
ticles together: any agglomerates that start to form are quickly
broken up. The situation is different when agitation is applied only
a few times per day. This allows the mycelium to bind the particles
together, and it is this situation that applies in our fermentations
and which was addressed by Schutyser et al. [11]. Our results con-
firm the idea of the Schutyser et al. [11] that, in this case, it is
appropriate to agitate early in the fermentation in order to break
up agglomerates before they become too strong. This is especially
important when the agitation is obtained by rotation of the bioreac-
tor body, such as occurred in the bioreactor of Schutyser et al. [11]
and in our packed-bed bioreactor, as this mode of agitation is less
forceful than that which can be obtained with a mechanical agitator.
We also confirmed their suggestion, which they did not investi-
gate experimentally, that one early agitation event might not be
sufficient: when we applied one agitation event at 10 h (MIX-126 ),
agglomeration problems and subsequent formation of preferential
flow paths occurred by the end of the fermentation, with relatively
poor uniformity of pectinase levels in the bed at 26 h (varying from
17 to 25 U g−1 , see Fig. 5).
5. Conclusions
We have extended the studies of Pitol et al. [9] into the pro-
duction of pectinases by SSF in a pilot-scale packed-bed bioreactor
containing 27 kg of wheat bran and 3 kg of sugarcane bagasse (dry
masses), which is the largest substrate loading yet reported for
pectinase production by SSF. We have demonstrated that agitat-
ing the bed a few times early in the fermentation could minimize
problems with substrate agglomeration and could thereby ensure
uniform pectinase levels in the bed at the time of harvesting. We
conclude that the best regime is a 20-h fermentation, with agitation
events at 8, 10 and 12 h. At this time, the average pectinase activity
in 15 samples removed from different vertical and horizontal posi-
tions of the bed was 22 U g−1 , with a sample standard deviation of
only 2 U g−1 .
Acknowledgements
This research was supported by a “Universal” Grant from CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico),
a Brazilian government agency for the advancement of science
and technology. Research scholarships were granted to Bruna
Schweitzer Medina, Henrique Luithardt, Luiz Fernando de Lima
Luz Jr, Nadia Krieger and David Mitchell by CNPq, to Luana de
Oliveira Pitol by Fundação Araucária, an agency for the support of
science and technology in the State of Paraná, and to Anelize Terez-
inha Jung Finkler and Alessandra Biz by CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior), a Brazilian govern-
ment agency for the development of personnel in higher education.

Table 1
Previous studies of the effect of intermittent agitation in solid-state fermentation.
Organism, substrate and
product
Rhizopus oligosporus on
soybeans producing a
tempe-like product.
Rhizopus microsporus and R.
oligosporus on soybeans
producing a tempe-like
product.
Aspergillus oryzae on wheat
grains.
Aspergillus niger on wheat bran.
Gibberella fujikuroi on extruded
wheat bran granules producing
gibberellic acid.
Aspergillus awamori on white
grape pomace producing
hydrolytic enzymes.
Trichoderma reesei on wet corn
distillers grain producing
cellulase (filter paper activity).
Phanerochaete chrysosporium
on apple pomace producing
ligninolytic enzymes.
Aspergillus niger on sugarcane
bagasse and palm kernel cake
(50% each by mass) producing
cellulase (filter paper activity).
Bioreactor and operation
Discontinuous rotating drum. Mixing events
were triggered by the bed temperature rising
to a set point.
(i) Agitated SSF in a 450-L rotating drum;
rotation at 1.5 rpm for 30 s followed by a
stationary period of 3 min. (ii) Static SSF in
plastic boxes (205 × 90 × 45 mm) with a
regular pattern of 1-mm diameter perforations.
28-L rotating drum. Injection of air under bed
surface at 4 points. Mass of substrate not clear.
Bed mixed initially to distribute inoculum
uniformly, then left static for several hours.
After a single mixing (1 rpm for 5 min), the
whole substrate bed was harvested.
Packed bed (bed of 5.5-cm height).
Temperature of the outlet air monitored and
used in an inference model to estimate bed
moisture content. Intermittent agitation
events in which water mixed into the bed.
Packed bed (bed of 150-cm diameter, 40 cm
height). Capacity for 200 kg of substrate.
30-min long mixing events, at intervals of
4–10 h (depending on bed temperatures and
the need to add water).
250-mL rotating drum (roller bottle).
(i) 50-mL Erlenmeyer flasks, mixed with
vortexer. (ii) 1-cm diameter glass columns
(contents emptied into 100-mL beakers, mixed
with vortexer and transferred back to the
columns). Mixing frequencies of 0, 1, 2, 3, and
6 per day. In all cases, the first mixing event
was at ∼66 h (after the end of the lag phase).
12-L rotating drum (when rotated, rotated at
2 rpm). Three agitation regimes studiedb : (i)
C = continuous agitation during the whole
fermentation. (ii) C/D/C = continuous agitation
during lag phase, discontinuous agitationc
during exponential phase, continuous agitation
during stationary phase. (iii)
D/C/D = discontinuous agitationc during lag
phase, continuous agitation during exponential
phase, discontinuous agitationc during
stationary phase.
“FERMSOSTAT” bioreactor containing from 0.5
to 1 kg of substrate (corresponding to bed
depths of 4 and 6 cm, respectively). Details of
bioreactor and operation not given.
10
Key measurements and findings related to performance Differences from the current Ref.
study
Bed temperature measured at a single position. With static Product formation not studied. [17]
operation, the bed temperature reached 45 ◦ C. With Temperature and moisture
discontinuous rotation, the temperature was controlled gradients in the bed not
close to the desired set point. studied.
Insufficient information given about samplinga . In static Effect of intermittent agitation [18]
SSF, the temperature was measured in the center of the on product uniformity within
bed. R. microsporus tolerated agitation well while R. the bed not studied.
oligosporus was strongly affected. Temperatures in static
SSF increased to about 12 ◦ C above that of the incubator.
The rotating drum with intermittent rotation enabled
adequate temperature control.
Whole bed contents harvested and separated into three Uniformity in the bed not [11]
fractions: single grains; small aggregates (2–20 grains); studied. The focus was on the
and large aggregates (>20 grains). The longer that the bed effect of mixing on the
A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
is left static before the first mixing event, the more formation of aggregates.
strongly the mycelium binds the wheat particles into
agglomerates (i.e. a greater fraction of the particles of the
bed remains agglomerated after the mixing event).
Samples representing the whole bed height cut into three Small bed height chosen to [19]
equal layers (bottom, middle, top). Moisture contents at minimize gradients, so not
different heights in the bed remained close to one another useful for investigating
even between mixing events. heterogeneity.
Bed temperature measured at 4 positions (heights of both Effect of temperature [20]
5 and 20 cm, radial positions of both 10 cm from the wall differences on product
and 10 cm from the center). Temperatures at the different uniformity not characterized.
positions were different by up to 15 ◦ C.
Insufficient information given about samplinga . Highest Effect of intermittent agitation [21]
enzyme activities for static culture or with a single daily on product uniformity within
agitation of 1 min. the bed not studied.
Insufficient information given about samplinga . CO2 Effect of intermittent agitation [22]
monitored in off-gases of glass columns. Mixing caused on product uniformity within
about a tenfold increase in spore production compared to the bed not studied.
static fermentation. Highest final cellulase level for static
fermentation. Mixing reduced the level by 5–10%.
Insufficient information given about samplinga . C/D/C gave Effect of intermittent agitation [23]
highest production of manganese peroxidase, lignin on product uniformity within
peroxidase and laccase. the bed not studied.
Every 24 h, substrate mixed for 5 min then 10 g of substrate Effect of intermittent agitation [24]
removed from each sampling port (locations not specified). on product uniformity within
Compared mixing at 0.5, 1 and 1.5 rpm for 5 min the bed not studied.
(presumably, every 24 h)d . Mixing at 0.5 rpm best.
Compared mixing every 6, 12 and 24 h (presumably, at
0.5 rpm for 5 min)d . Mixing every 24 h best.
Table 1 (Continued)
Organism, substrate and Bioreactor and operation
product
Aspergillus tamarii on coffee (i) Bottle reactors (4.5-cm diameter × 6.5-cm
pulp, producing spores and height, packed with 13 g of coffee pulp) used to
pectin methylesterase (PME). measure bonding of particles and PME
production. (ii) Glass column reactors (4.5-cm
diameter × 20-cm height, packed with 20 g of
coffee pulp) used to measure CO2 and spore
production. In all cases, cultures were static
during the first 12 h and then mixed for 1 min.
Then, mixing was done at intervals (every 3, 6,
9 or 12 h) with a vortex. Comparison with a
completely static control.
Aspergillus niger on apple 12-L rotating drum. Continuous agitation
pomace producing citric acid. compared with agitation of 1 h at 2 rpm, twice
daily at 12-h intervals.
Aspergillus sojae on wheat bran 250-mL Erlenmeyer flasks. Flasks agitated by
producing polygalacturonase. beating the flask on the palm of the hand for
∼1 min (1, 2, 3, 4 or 5 times a day), and
compared to a static control.
Starmerella bombicola on 500-mL Erlenmeyer flasks containing 45 g of
winterization oil cake from the substrates (oil cake and molasses) and 14 g of
refining of sunflower oil, wheat straw (as inert support). Flasks mixed at
supplemented with sugar beet days 3, 5 & 7 (flask contents placed in a 1-L
molasses, producing glass beaker, mixed with a sterile metal
sophorolipids spatula, and loaded back into the flask).
Compared with a static control.
a
From the information given, it is not possible to tell from where exactly in the bed the samples were removed.
b
The timings of the different phases were not specified.
c
The meaning of “discontinuous agitation” is not stated directly, however, the results section suggests that it means “the absence of agitation”.
d
From the information given, it is impossible to be sure about what was done.
Key measurements and findings related to performance Differences from the current Ref.
study
Sampling not explained clearly. Results expressed as single Effect of intermittent agitation [25]
values. In static culture, the “fraction of bonded particles” on product uniformity within
A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
reached 69% at 33 h. With intermittent mixing in bottle the bed not studied.
reactors, it was always lower than 33%. With intermittent
mixing in bottle reactors, PME production increased at
least 1.5-fold with mixing in comparison to static control,
but frequency of mixing did not affect PME production.
With intermittent mixing in columns, there were no
significant differences in the CO2 production rate or O2
uptake rate compared to static operation.
Insufficient information given about samplinga . Citric acid Effect of intermittent agitation [26]
production at 120 h was 33% higher with intermittent on product uniformity within
agitation than with continuous agitation. the bed not studied.
Sampling not explained clearly (presumably after the flask Effect of intermittent agitation [27]
contents were mixed, but this is unclear). Maximum on product uniformity within
enzyme activity obtained with 3 mixings per day (17% the bed not studied.
more than the static control), although mixing 1, 2, 4 or 5
times per day gave activities lower than the control.
One reactor sacrificed, after mixing, at each of days 3, 5, 7 Effect of intermittent agitation [28]
and 10. Intermittent mixing increased the maximum on product uniformity within
sophorolipid yield by 31% compared to the static control. the bed not studied.
At the end of the intermittent fermentation, overall O2
consumption 15% higher and 22% more fat consumed,
compared to the static control. Intermittent mixing
reduced the size of the solid aggregates.
11
12 A.T.J. Finkler et al. / Biochemical Engineering Journal 121 (2017) 1–12
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