Biochemical Engineering Journal 65 (2012) 9095

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Short communication
Pectinase production from lemon peel pomace as support and carbon source
in solid-state fermentation column-tray bioreactor
Hctor A. Ruiz
a,
, Rosa M. Rodrguez-Jasso
a
, Ral Rodrguez
b
, Juan C. Contreras-Esquivel
b
,
Cristbal N. Aguilar
b,
a
IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b
Food Research Department. School of Chemistry, Universidad Autnoma de Coahuila, Unidad Saltillo, Blvd. V. Carranza e Ing. Jos Crdenas Valds, Saltillo, Coahuila C.P. 25001,
Mexico
a r t i c l e i n f o
Article history:
Received 8 March 2012
Received in revised form19 March 2012
Accepted 22 March 2012
Available online 30 March 2012
Keywords:
Solid-state fermentation
Bioreactor design
Pomace
A. niger
Pectinase
a b s t r a c t
Pectinase is an important enzyme that nds application in many food processing industries and solid-
state fermentation (SSF) is an attractive technology for enzyme production. In this work, a SSF process
is described for the production of pectinase by Aspergillus niger Aa-20 and lemon peel pomace (LPP) as
support and carbon source in a solid-state bioreactor. The process consists of three steps. (1) Selection
of microorganism for SSF. Eight different fungal strains from the genus Aspergillus and Penicillium were
screened for invasion ability on LPP; (2) Selection of particle size. Invasion ability of selected fungal strain
was analyzed on four particle sizes of LPP; (3) SSF process was operated in a column-tray bioreactor at
30

C and 70% moisture content, 194 mL/min of air ow rate and substrate particle size (20.7 mm) of LPP
for 96h. Results showed, that high levels of pectinase activities were obtained. The maximum pectinase
activity obtained was 2181 U/L. Maximum biomass and maximum specic growth rate of A. niger Aa-20
were X
max
= 8 mg glucosamine/g of LPP and
max
= 0.127 1/h. The LPP and the use of A. niger Aa-20 in SSF
suggest as a very promising process for pectinase production.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Pectinase or pectinolytic are enzymes, which degrade pectin
substances and are of great importance to the food industry. It
has been reported that microbial pectinases account for 25% of
the global food enzymes sales. These enzymes have been used
in several conventional industrial processes, such as textile, plant
ber processing, tea, coffee, oil extraction, treatment of industrial
wastewater [1]. An alternative for the production of these enzymes
is solid-state fermentation (SSF). SSF is a complex heterogeneous
three-phase (gasliquidsolid) andgenerally denedas the growth
of microorganisms, oftenfungi, onthe surface of a porous andmoist
solid substrate particle in which enough moisture is present to
maintain microbial growth and metabolism. This process is car-
ried out in absence or near-absence of visible liquid water between
the particles. This condition favors the development of lamentous

Corresponding author at: IBB-Institute for Biotechnology and Bioengineering,

Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057
Braga, Portugal. Tel.: +351 253 604 400; fax: +351 253 678 986.

Corresponding author at: Food Research Department, School of Chemistry, Uni-

versidad Autnoma de Coahuila, Blvd. V. Carranza and Gonzlez Lobo, ZIP 25280
Saltillo, Coahuila, Mexico. Tel.: +52 844 4161238; fax: +52 844 4159534.
E-mail addresses: hector ruiz@deb.uminho.pt (H.A. Ruiz),
cristobal.aguilar@uadec.edu.mx (C.N. Aguilar).
fungi, given their unique capacity to colonize the interparticular
spaces of solid matrices [2]. The spaces between the particles con-
tain a continuous gas phase. SSF generally employ a natural raw
material as carbon and energy source, moreover SSF can employ
an inert material as solid matrix, which requires supplementing a
nutrient solution, containingnecessarynutrients as well as acarbon
source [3]. SSF plays an important role, and has a great perspective
for the use and bioconversion of different agro-industrial residues
such as lemon peel pomace (LPP), this material is rich in pectin that
acts as the inducer and support, thus it can be used as a substrate
for the production of pectinolytic enzymes by microorganisms [2].
Utilization of agro-industrial residues for enzymes production
usingSSFminimizes thepollutionandallows obtaininghighadded-
value products using an economical technology. LPP is the main
solid by-product resulting from processing industry lemon and
constitute about 19.8% of the dry mass of lemon [4]. As per FAO
statistics, in Mexico, the lime production in 2009 was about 2 mil-
lion tons. It gives an estimation of about 396,000 tons of lemon
peel produced per year. The term pomace is referring the prod-
uct remain fromagro-industrial residues, principally to citrus peel,
apple bagasse, lemon skin, obtained after a thermal pretreatment
followed by several washes with water to lower the concentration
of soluble sugars and acids before dry [4]. SSF substrates require
characteristics (e.g. carbohydrates, nitrogen source, mineral salts)
for a good invasion capacity of lamentous fungus over the culture
1369-703X/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bej.2012.03.007
H.A. Ruiz et al. / Biochemical Engineering Journal 65 (2012) 9095 91
matrix. Other important characteristic of substrate is the parti-
cle size, may affect the ow pattern and porosity in the substrate.
Natural solid substrates generally need some kind of physical pre-
treatment such as chopping or grinding to reduce particle size to
make their chemical constituents more accessible and their phys-
ical structure more susceptible to mycelial penetration. The small
particles would have reduced porosity, leading to lowering of gas
diffusion, while the big particles would absorb less moisture, swell
less and thus by drying rapidly support only a sub optimal growth
of fungi. The available surface area will decrease with an increase
in particle size of the substrate [2,5,6]. For this reason a particle size
distribution that has these characteristics of nutrients, specic area
and porosity will be a potential substrate for SFF process as support
and carbon source.
In order to develop bioreactors for large-scale SSF, a quantita-
tive analysis of kinetics andstoichiometry of the reactionis needed.
This analysis is hampered in most natural SSF systems by exper-
imental difculties, because the substrate/support is structurally
and nutrionally heterogeneous and measurement of biomass dry
matter is impossible. In every fermentation process, the biore-
actor provides the environment for growth and activity for the
microorganisms, which cause the biological reaction. SSF employs
a great variety of matrices, which vary in composition, mechanical
resistance, porosity and water holding capacity. All these fac-
tors also affect the reactor design and the control strategy for
the parameters. In addition, the morphology of the fungus and
its resistance to mechanical agitation and the necessity or not
to have a sterile process should be considered in the election of
the bioreactor conguration. The most important advances in SSF
bioreactors for laboratory, bench and industrial scale have carried
out in the last decade being tray, packed bed and rotary drum
have being the most useful models [7]. However the majority of
the studies have occurred slowly, due to the operational, transport
phenomenon (heat and oxygen transfer) and scale-up complica-
tions. For that reason, it is necessary to develop and improve
bioreactors designed for SFF in order to cover the microorgan-
ismgrowth metabolic necessities for obtaining high concentration
of enzymes [8]. The main aim of this work was development
a process for the production of pectinase enzyme by a selected
Aspergillus niger and particle size of LPP through SSF technology.
The design and performance of a column-tray bioreactor also was
considered.
2. Materials and methods
2.1. Preparation of lemon peel pomace (LPP)
Mature limes (Citrus aurantifolia) were obtained in the local
market (Saltillo, Coahuila, Mexico). LPP preparation was realized
following the procedure reported by Rodrguez-Jasso [4]. Sub-
sequently the LPP was milled using a laboratory knife mill and
separated into fractions >2.0mm (mesh 10); 2.00.7mm (mesh
25); 0.70.3mm (mesh 50) and 0.30.17mm (mesh 180) using a
vibratory sieve shaker. A homogeneous blend of this particle size
was used for the selection of the microorganism(see below).
2.2. Chemical characterization of LPP
The different particle size distribution of LPP was analyzed for
their chemical composition. Physicochemical analysis included the
evaluation of total solids, moisture, crude ber, protein and fat
were carried out using the methods described by AOAC meth-
ods [9]. Total sugars were determined using colorimetric method
(phenolsulfuric).
Table 1
Filamentous fungi isolated fromnortheast Mexican desert.
Source Species Symbology
Quercus spp Penicilliumpinophilum EH
2
Quercus spp Penicilliumpinophilum EH
3
Larrea tridentate Aspergillus niger GH
1
Larrea tridentate Penicilliumpurpurogenum GH
2
Larrea tridentate Aspergillus fumigatus GS
Pinus cembroides Aspergillus niger PSH
Pinus cembroides Aspergillus ustus PSS
2.3. Screening for selection of microorganismand particle size of
LPP
2.3.1. Microorganismand culture medium
Eight lamentous microorganisms of Aspergillus and Penicillium
genus were used in radial growth kinetic. Seven were isolated from
Mexico semi-desert plants (Table 1) and obtained fromDIA/UAdeC
(Food Research Department/University of Coahuila) collection and
one fungus strain A. niger Aa-20 was obtained from IRD, France-
UAMI, Mxico (Institut de Recherche pour le Dveloppement
Universidad Autnoma Metropolitana-Iztapalapa) collection
[10,11]. For inoculumpreparation the culture was grown in potato
dextrose agar (PDA) onaskat 30

Cfor 5days. The growthmedium

usedwas Czapek-Doxmodied(g/L): NaNO
3
(7.66); KH
2
PO
4
(3.04);
MgSO
4
(1.52); KCl (1.52); KH
2
PO
4
(2.47); (NH
4
)
2
SO
4
(6.60); CaCl
2
(0.48). The initial pH of the medium was adjusted to 5.0. The
selected microorganism was used in solid-state fermentation for
pectinase production.
2.3.2. Radial growth kinetic for microorganismselection
Fungal strains screeningwas performedfor determinationof the
microorganismgrowthrate inPetri dishes measuringradial growth
ona homogenous blendparticle size of LPP. The moisture (70%) was
adjusted with Czapek-Dox modied (g/L): NaNO
3
(7.66); KH
2
PO
4
(3.04); MgSO
4
(1.52); KCl (1.52) and 30% of a homogenous blend
of particle size distribution were added to Petri dishes. The LPP
was used as sole energy and carbon source. Spores suspension con-
taining around 210
7
spores/mL was inoculated at the center over
dishes with LPP, monitored the support invasion capacity every 8h
during 4days. Ingeneral, the radial growthcanbe describedandt-
tedusingtheexponential model. Inthis model, reportedbyMitchell
et al. [12], X is the radial growth (cm), is the maximum specic
growth rate constant (1/h) and t is time (h).
2.3.3. Radial growth kinetic for LPP particle size selection
Fungal strain selected with highest (see above) was used
for the selection of LPP particle size. Four particle size distribu-
tion of LPP were used >2.0mm(mesh 10); 2.00.7mm(mesh 25);
0.70.3mm(mesh 50) and 0.30.17mm (mesh 180). The microor-
ganismgrowthrate was performedinPetri dishes measuring radial
growth on each particle size distribution. For the determination
of invasion capacity was used the same method for radial growth
kinetic in the microorganism selection evaluating maximum spe-
cic growthrate . The selectedparticle size was usedinsolid-state
fermentation for pectinase production.
2.4. Solid-state fermentation for pectinase production
2.4.1. Column-tray bioreactor design
The schematic of the column-tray bioreactor, which consisted
of a vertical cylindrical, was constructed of acrylic with diameter
(25cm) and height L (38cm). As shown in Fig. 1A, the bioreactor
containing eight perforated base trays (Fig. 1B) that supported the
solid substrate (LPP) and through which forced aeration is applied
using an aerator pump to increase the accessibility to O
2
. The
92 H.A. Ruiz et al. / Biochemical Engineering Journal 65 (2012) 9095
Fig. 1. Schematic representation of column-tray bioreactor. (A) The bioreactor
includes: (1) sterilized lter, (2) air inlet, (3) air distributor, (4) lime pomace, (5)
perforated trays, (6) water-bath jacket, (7) water outlet, (8) sample carrier, (9) gas
exit, (10) culture mediumlevel, (11) water inlet, (12) thermometer port, (13) water
container. (B) Superior viewof the perforated trays conguration.
reactor includes a water ow jacket for control temperature con-
nected to an external water pump. The air-ow was sterilized by
passing through a 0.45m cellulose acetate membrane and the
humidier systemwas regulated by air diffuser in the bottombub-
bling contained sterilized distilled water. In the below part of the
bioreactor, the temperature was monitored and in the upper part
of the bioreactor the lid was removed for taking the samples. The
advantage of plastic trays over wooden trays would make steril-
ization and cleaning easier and therefore reduce the possibility of
contamination or spoilage.
2.4.2. Solid-state fermentation in the column-tray bioreactor
Solid-state fermentationwas performedina column-tray biore-
actor (Fig. 1AB) using A. niger Aa-20 and as support/substrate
LPP with a particle size distribution of 2.00.7mm(mesh 25). The
microorganism and particle size distribution were select based
on its invasion capacity measured as maximum specic growth
rate . The solid substrate LPP and Czapek-Dox medium were
sterilized in an autoclave 120

C for 20min. Each bioreactor trays

contained 3g of LPP and the initial moisture content of the solid
substrate was adjusted to 70% with Czapek-Dox medium con-
taining 210
7
spores/g LPP. The fermentation was carried out at
301

C and samples were withdrawn at 12h intervals until a

total of 96h. Relative humidity regulation was maintained with an
air-owrate at 194mL/min.
2.4.3. Biomass evaluation
Solid cultured samples were rst suspending in water (20mL),
subsequently the fraction solid (biomass) and liquid (enzymatic
extracted) were separated via vacuum ltration. The solid was
washed with distilled water and dried at 60

C (usually 24h).
Mycelial biomass was estimated for these dry samples by an indi-
rect methodology based on glucosamine content of A. niger [13,14].
Biomass concentration is reported as mg glucosamine/g LPP. The
residual substrate concentration was determined by total sugars,
using colorimetric method (phenolsulfuric).
2.4.3.1. Kinetic biomass growth parameter estimation. Although
various mathematical models can describe the kinetic biomass
growth, the most frequently used model describing the kinetic of
fungal growth in SSF is the logistic model, is also sometimes called
the VerlhurstPearl equation, originally developed for population
growth[12]. Biomass productioninthe column-traybioreactor was
estimated as X (mg glucosamine/g LLP),
m
is the maximal spe-
cic growth rate (1/h) and X
m
is maximal microbial biomass level
achieved. X
0
is the biomass (t =0). The estimate parameters X
m
and
reported by Mitchell et al. [12] were determined using a non-
linear regression data analysis of MATLAB Version 7.6.0, R2008a
software (MathWorks, Inc., Natick, Massachusetts, USA).
2.4.4. Pectinase assay
The crude enzymatic extract was assayed for pectinase activ-
ity. Pectinase activity was determined by measuring the reduction
groups liberated from polygalacturonic acid substrate. The corre-
lation between growth and pectinase production was established
using the LuedekingPiret model [15]. Furthermore, in order
to estimate the MichaelisMenten kinetic parameters, the ini-
tial hydrolysis rates were measured by varying concentration of
polygalacturonic acid (0.4, 0.8, 1.6 and 3.2g/L, respectively). The
reaction mixture contained 245L of polygalacturonic acid (dis-
solved in 50mM acetate buffer, pH 4.5) and 245L of the enzyme
extract. The reaction mixture was incubated at 37

C. After 30min
of incubation, the reducing sugars released were quantied. The
standard curve was established using galacturonic acid as reducing
sugar. One unit of pectinase activity was dened as the amount of
enzymerequiredtoreleased1mol of galacturonic acidper minute
under the assay conditions. All the measurements were made in
triplicate.
2.4.4.1. Determination of kinetic parameters for pectinase. The
MichaelisMenten constant (K
m
) and maximum velocity (V
m
)
values of pectinase were determined by measuring the activity
reactions rates (under the conditions given earlier). The kinetic
parameters K
m
and V
max
were determined using MATLAB software.
3. Results and discussion
3.1. Physicochemical characterization of LPP
The physicochemical analysis performed on different LPP par-
ticle size showed (Table 2) that the content of total sugars was
highest at 2.00.7mm(mesh 25) with 59.15% on a dry matter basis.
Higher levels of crude ber were found about of (21.1425.57%) in
the different particle size. Total protein and fat for a particle size of
0.30.17mm(mesh 180) were higher, 6.15% and 7.8% respectively.
The protein content was lower than 6.15% in the rest of particle
H.A. Ruiz et al. / Biochemical Engineering Journal 65 (2012) 9095 93
Table 2
Physicochemical characterization of LPP (% dry weight).
Particle size distribution of LPP (% in dry matter basis)
>2.0mm 2.00.7mm 0.70.3mm 0.30.17mm
Mesh 10 25 50 180
Total solids 93.0 0.14 94.7 0.77 95.0 0.49 94.50 0.56
Moisture 7.00 56 5.3 0.28 5.00 0.56 5.50 0.21
Total sugars 37.59 1.42 59.15 2.22 37.83 1.25 44 1.27
Total protein 3.89 0.28 4.07 0.51 5.63 0.40 6.15 0.52
Crude ber 25.78 1.1 23.29 2.19 21.14 1.59 21.30 1.48
Fat 3.46 0.45 3.86 0.31 5.76 0.38 7.8 0.42
size. The nitrogen source can play an important role as nutrient.
Moreover, affecting the pH changes in the substrate during the
fermentation. Moisture content did not showdifferences between
eachparticle size distribution. This physicochemical compositionis
in good agreement with other values found in the literature for this
material [16]. Grigelmo-Miguel and Martn-Bellose [17] reported
similar values of lipids using oranges as raw material and Nassar
et al. [18] showed similar values of fat in the chemical composi-
tion of oranges peel. Only few reports have been published on the
physicochemical composition of LPP. This characterization showed
that the LPP is a good prospect that could be used as carbon source
for SFF.
3.2. Microorganismselection for SSF
Growth of the fungal strains (Table 1) on the particle size blend
of LPP (Fig. 2A) was performed for the selection of microorganism
with the higher adaption. A. niger Aa-20 and GH
1
had a com-
plete capacity invasion in 48h with a maximum specic growth
rate (
max
) of 0.068 and 0.0631/h, respectively. Radial growths
were 0.098 and 0.095cm/h correspondingly, while the GS and PSS
strains invaded 18.5% of total Petri dishes at 56h. A. niger Aa-20
was selected for the SSF experiment in the column-tray bioreactor.
Robledo et al. [19] reported higher growth rate for GH
1
and PSH
on pomegranate husk (0.04 and 0.041cm/h, respectively). Trevi no-
Cueto et al. [20] reported a rate of invasion of 0.015 and 0.019cm/h
using A. niger Aa-20 on Larrea tridentate Cov.
3.3. Particle size selection for SSF
Theselectionof anadequatesupport for performingSFFis essen-
tial, since the success of the process depends on it. Inter-particular
spaces or porosity of substrate that depends onthe particle size dis-
tribution is one of the parameters that govern the mass and heat
transfer during the bioconversion of substrate. Small particles, or
particles withlargeat surfaces, tendtopacktogether closely, mak-
ing it difcult to aerate the substrate mass. If the microorganism
can penetrate into the particle this increases the directly accessible
substrate and decreases the distances over which diffusion needs
to occur. In this case the optimal particle size will be inuenced
by the depth of penetration. The optimal particle size often repre-
sents a compromise between the accessibility of nutrients and the
availability of oxygen. After to selected the microorganismA. niger
Aa-20, were performed the kinetics of invasion capacity for the
selection the better particle size distribution (Fig. 2B). The highest
specic growth rate (
max
) was 0.0991/h for a particle size dis-
tribution of 2.00.7mm (mesh 25) this behavior probably can be
assumed to the nutritional composition, and maybe there is more
specic area, porosityandaerationconditions. Moreover, inaerobic
fermentations, oxygen transfer to the fungus is an important phe-
nomenon to sustain microbial growth. Aerobic growth takes place
in a thin layer near the gasliquid interface, while in deeper regions
anaerobic growth and anaerobic conversions take place [21].
According to Tao et al. [22], reported that the maximumenzyme
productivity was obtained from 400m sized particles and was
lower with bigger (>0.9mm) and smaller particles (0.076mm) and
concluded that with signicantly smaller particles, the specic sur-
face area was greater but the porosity was less for lamentous
fungus attack that could not penetrate deep into the pores and
hence into the substrate particles. Larger particles provide bet-
ter aeration/respiration opportunities but provide lesser surface
area. The quantities of total sugar maybe also affect the growth
of microorganismas a relation of carbohydrates in the medium. At
particle size distribution of 0.30.17mm (mesh 180) the growth
rate was of 0.0551/h, probably the oxygen transportation are
Fig. 2. Growth rate on LPP: (A) at different types of microorganism: () GH1; ()
GH2; () GS; ( ) PSH; ( ) Aa-20; ( ) EH3; () EH2; () PSS. (B) A. niger Aa-20
growth over different particle size distribution: mesh () 10; ( ) 25; () 50; ()
80.
94 H.A. Ruiz et al. / Biochemical Engineering Journal 65 (2012) 9095
decient and in the smaller particles, the specic surface area was
greater but probablytheporositywas less. EachLPPfractionhas dif-
ferent properties that can affect the SSF process. Different authors
have studied the effect of particle size on growth and product for-
mation. RecentlyMembrilloet al. [6] showedthat geometrical ratio,
shape and size of sugar cane bagasse bers have a strong inuence
in packing density with an impact in the production of extracellu-
lar enzymes, growth rates and composition changes in substrate.
Bari et al. [5] used oil palm empty fruit bunches as substrate and
reported that the citric production increased with the decrease
of particle size from 3 to 0.25mm, obtained the maximum citric
production at 0.5mmof particle size.
3.4. Solid-state fermentation in the column-tray bioreactor
3.4.1. Biomass production in column-tray bioreactor
Biomass production prole for A. niger Aa-20 on LPP particle
size distribution of 2.00.7mm in function of the glucosamine
content that is the index parameter of the cell growth. The high-
est glucosamine content is obtained at 60h, correspondent to
7.55mg glucosamine/g LPP and it is remarkable that at the end
of the culture, biomass reached a value of 8.017mg glucosamine/g
LPP at 96h. The data adjustment to logistic equation with a high
correlation coefcient R
2
=0.997; R
2
adj
= 0.996, thus allowed to
determined specic growth rate of 0.1271/h and showing that
the model was adequate for this process. As part of this analysis,
it is shown that, for logistic growth kinetics, the time at which the
biomass reaches 94.17% of its maximum possible value is a good
estimate of the optimumharvesting time for maximizing produc-
tivity.
Membrillo et al. [6] reported similar amount of glucosamine at
the endof fermentationandthe specic growthrates values of 0.05,
0.049 and 0.0431/h for particles of 0.92, 1.68 and 2.9mm, respec-
tively. Daz-Godnez et al. [23] reported specic growth rate of
0.471/h, using sucrose as carbon source and polyurethane foamas
the solid support and A. niger C28B25 as microorganism.
On the other hand, the carbohydrate is an important parameter
for growth of microorganism. The quantity of total sugars before
fermentationwas 70.65mg of total sugar/100mg of LPP, decreasing
to 37.02mg of total sugar/100mg de pomace, this represent about
of 50% of the initial total sugars. Solis-Pereyra et al. [24] used high
initial glucose concentration of 100, 250, 350 and 450g/L for pecti-
nases production in SSF and reported that the maximum amount
of glucose was consumed and less than 3% of the initial glucose
remained unutilized after 24h of fermentation.
3.4.2. Enzyme production and kinetic parameters
Product formation was related to the growth by a modied
LuedekingPiret equation (Fig. 3). A reasonably good correlation
R
2
=0.949 was obtained. This result assumed that the rate of prod-
uct formation is related to the rate of growth. According to Aguilar
et al. [15], this kind of behavior showed that the formation of pecti-
nase is in the stationary phase of the culture process. According
to Paul and Thomas [25], the growth and metabolite produc-
tion from lamentous fungi are associated with complex and not
well-understood processes as the multicellular structure of the
mycelium, the morphological heterogeneity and the heterogeneity
in physiology and differentiation along the length of the hyphae
and during fermentation process. In a recent work, Martnez-
Trujillo et al. [26] reported the pectinase production using the
LuedekingPiret and founded that the exopectinases are produced
mainly during the growth phase where they release degradation
products fromcarbonsource, whichserve as inducers of endopecti-
nases that are produced at the end of this phase.
Table 3
Enzyme activity at different concentrations of substrate.
Substrate concentration (g/L) Enzyme activity (U/L)
0.4 1232.42
0.8 2181.23
1.6 863.1
3.2 991.65
SSF enzymatic extract was produced in the presence of the
inducer (pectin) fromLPP and evaluated at different substrate con-
centrations (Table 3). At 0.4g/L the highest production was 1232.
42U/L with 36h being stable until 60h when start to decrease
almost (238.3U/L), at 0.8g/L the highest production were at 40 and
60hwithvalues of 2146.54and2181.23U/L, respectively. However
at 1.6g/L pectinase activity were at earlier times observed (24h)
but with lower production (863.1U/L), this decrement was similar
at 3.2g/L with a higher value at 48h of 991.65U/L, which can be
assumed to a possible inhibition for product formation caused for
higher concentration of substrate.
These results are in agreement with the data reported for pecti-
nase activities. In a recent work Zeni et al. [27] reported an activity
of polygalacturonases above 3000U/L. Martos et al. [28] usedpectin
agar as substrate in submerged fermentation for polygalacturonase
enzyme by A. niger, the activity reported was 1032U/L. According
to several works, the pectinase productionhas beenreportedindif-
ferent types of bioreactors (i.e. stirred tank, airlift, column) [29,30].
Panda and Naidu [31] reported the pectinase production using a
batch stirred tank bioreactor and the maximum production was
1.313U of polygalacturonase. Huerta et al. [32] reported a pecti-
nase specic activity of 790U/mg of protein using a packed bed
bioreactor with A. niger CH
4
.
The bioreactor proposed in this work has advantages, the forced
aeration allows better control of environmental conditions in the
bed, due to the ability to manipulate the temperature and owrate
of the process air. Therefore the bioreactor should be operated so as
to minimize drying of the bed, because drying can eventually lead
to the moisture content in the bed reaching values which restrict
the growth of the microorganism. Smits et al. [33] modeled the
diffusion of water vapor in the void spaces of the bed. When it
was assumed that the air surrounding the tray was maintained at
a high humidity, then the combination of metabolic water produc-
tion with the relatively slow water vapor diffusion meant that the
predicted water content of the substrate remained above the initial
Fig. 3. Correlation between biomass growth and pectinase activity using the
LuedekingPiret model.
H.A. Ruiz et al. / Biochemical Engineering Journal 65 (2012) 9095 95
value. Under such conditions there will be no danger of the growth
rate being limited by lowwater activities of the solid substrate. For
that reason, the column-tray bioreactor represents a simple and
excellent technology for SSF.
In respect to, kinetic parameters, the pectinase activity
showed a typical MichaelisMenten prole. Kinetic parame-
ters of pectinase were determined as K
M
=0.155mol/L and
V
max
=38.84mol/(L/min), respectively. In this case the graphic
(data not showed) was linear with a correlation coefcient (R
2
) of
0. 996. Showing a higher afnity between enzyme and substrate.
4. Conclusions
In the present study, a promising solid-state fermentation pro-
cess for pectinase production has been performed in laboratory
scale. Lemon peel pomace is an attractive agro-industrial residues
and an alternative for the production of high levels of pectinolytic
enzymes, taking into account the adaptation of microorganism
and the signicant effect of particle size. The SSF process allowed
obtained high levels of fungal biomass and enzyme production in
SSF column-tray bioreactor.
Acknowledgments
The authors H.A. Ruiz and R.M. Rodrguez-Jasso thank to Mex-
ican Science and Technology Council (CONACYT, Mexico) for PhD
fellowship support (CONACYT grant number: 213592/308679 and
206607/230415, respectively). This work was supported in part
by the Food Research Department, Universidad Autnoma de
Coahuila, Mxico.
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