Biochemical Engineering Department

THE HARNESSED OF WASTE: Production of galacturonic acid from plantain peel.

a
Julián Ayala, aLuisa Caicedo, aMarcelo Parra, aMauricio Bechara.
Department of Biochemical Engineering, Universidad Icesi, Cali, Colombia.

ARTICLE INFO ABSTRACT

Keywords:

Pectin
Currently, in Valle del Cauca 300 tons of plantain from the Dominico-Harton group are
Pectinase produced monthly, this agro-industrial production involves the generation of 105 tons of
plantain peels per month which are not harnessed. For this reason, this article will evaluate
Galacturonic Acid the best design of a bioreactor to produce galacturonic acid from these residues and provide
them with added value. To obtain galacturonic acid, it is necessary to degrade the pectin
Kinetic parameters present in the plantain peel, this was achieved using the enzyme pectinase 831L and the
following kinetic parameters were obtained: Vmax=21.097 uM/min, Km=4.31435 g/L and
Bach bioreactor finally Ks=7.6698 g/L. Subsequently, two bioreactors were modeled in Matlab, the first one
is a batch type bioreactor, in which a volume of 1,9758 m^3 was obtained, and the second
CSTR bioreactor one is a CSTR type bioreactor where the volume was 3,2833m^3. From these data, the
conversion and production of each one of them were calculated, and finally the BATCH
bioreactor was chosen for achieving better results.
Matlab

1. Introduction
In the world, the diversity of industries has made an
unstoppable source of waste with great potential for the
economy. Although, in Colombia the industries have these
characteristics, the potential of agrobusiness is not so far
behind generating a large amount of organic waste.
Specifically, in Valle del Cauca, the great variety of thermal
soils makes this department a perfect area for the
development of agro-industries for the use of raw materials
such as sugar cane, plantain, and chontaduro among others,
that currently generates usable wastes such as sugarcane
bagasse or plantain and chontaduro peels.
105 tons per month of plantain peel waste are useful for said
process.
Therefore, a development of the industrial process is
required taking into account the capacity and characteristics
of two reactors of common use in the industries (BATCH
and CSTR), for which an analysis was made using the
MATLAB software for the sake of designing the process and
choosing the best bioreactor.
2. Materials and methodology
2.1 Extraction of Pectin from plantain

In this article, the ability of the plantain peel to obtain
galacturonic acid will be evaluated using the pectin polymer
present in said waste, since it presents 20.68% of pectin [1],
the Asociación de Productores Agropecuarios de Argelia
(Asproagro) is supplying 300 tons of plantain for the
companies Yupi and Fritolay [8], from which approximately
1
The pectin is extracted from the Dominico-Hartón plantain
peel. This process begins by selecting the green peels of the
plantains, after that the peels were washed so the impurities
could be remove, in the next step the clean peels were taken
to a bioreactor where a enzymatic deactivation will take
place, this occurs at a temperature of 90°C for ten minutes.
Next an acid hydrolysis was performed in a solution of citric
acid for 40 minutes to reach a pH of level 3 at a temperature
of 80°C. As a result of this a filtration was used to separate
the bagasse and citric acid from the concentrated liquid, the
later one was then brought to the centrifuge where it was
exposed to a speed of 400 rpm, then the liquid went to a
cooling process with a temperature of 4°C. Right after that a
precipitation with ethyl alcohol of 98GL was performed,
after that by filtration of the liquid phase pectin is separated
from the solid. Finally, it was taken to a drying oven at 60 °
C, to obtain dry pectin, which must be mixed with water to
solubilize before entering the reactor. The diagram where the
extraction is illustrated is found as an annex in which this
procedure is explained in more detail.
2.2 Obtaining the enzyme complex
The degradation of pectin was obtained through the enzyme
pectinase 831L complex, this enzyme showed a substrate-
dependent inhibition at concentrations greater than 4 mg / ml
pectin, it works in an optimal way at a pH range of 2.5 to 5.5
and an optimum temperature level of 40 to 55°C. The
enzyme has endogalacturonase pectinase and
polygalacturonase activity. The enzyme is provided by the
company Biocatalysts [3].
2.3 D-galacturonic acid yield
2.5 Mathematical model
2.5.1 kinetic parameters
In order to model batch Batch and CSTR bioreactors, it is
necessary to determine the kinetic parameters, to correctly
model the kinetic behavior, two models were used;
Michaelis-Menten (eq.1) and noncompetitive inhibition
by substrate (eq. 2); (d)equation 1, you get the value
of Vmax using the Lineweaver-
Burk linearization in Excel; the values of Km and Ks are
obtained from equation 2 by modeling them in MATLAB.
V=(Vmax[S])/(Km+[S])
(eq 1): Michaelis Menten equation.
V=(V_max [S])/(K_M+[S]+[S]^2/K_S )
(eq 2): Substrate uncompetitive inhibition equation.
2.5.2. Batch reactor
For the modeling of the Batch bioreactor, MATLAB was
used and the following assumptions were made: It is a
closed and non-stationary system; also used the non-
competitive substrate inhibition kinetics to model the Batch
bioreactor, to find the reaction time (tb), the following
equation was used:

The process to purify the galacturonic acid consists of using

electrodialysis with bipolar membranes. Thanks to the
positive charge that this acid possesses all the components
(eq 3): Batch reaction time equation.
that are not necessary can be separated from the desired
product.
2.5.3. CSTR reactor
2.4 Experimental determination
For the CSTR modeling, MATLAB was used and the
following assumptions were made: It has an open system,
After the extraction of pectin, the enzymatic activity was
also the input flow is equal to the output flow, the equation
evaluated with 7 tests, using different substrate
of the overall balance of the substrate was reduced, and the
concentrations, which were taken from the document Pectin-
inhibition kinetics of the non-competitive substrate was
to-Galacturonic Acid: Waste-to-Bioproducts [5]; and using
included in the reaction rate. For this process, the following
the same concentration of enzyme. For the conduct of these
equations were used:
tests, the indications of the ICESI University Bioreactor
Design Laboratory Guide: Procedimientos generales para
V ̇=F⁄([So])
medir la actividad enzimática de la Pectinliasa [4].
(eq 4): Volumetric Flow.
2.5 calibration curve
V=V ̇*(So-Sf)/rs
Where
In order to find the conversion of galacturonic acid, a
rs=(Vmax*Sf)/(Km+Sf+(Sf^2)/Ks)
galacturonic acid calibration curve was developed, the
procedure of which was performed following the
(eq 5): CSTR volume equation.
instructions of the bioreactor design laboratory of the
university ICESI: Medición de Ácido Galacturónico
Liberado por Hidrolisis enzimática [6].

2
Fig 1: General diagram of pectin transformation
S [mg/mL] Vo [uM/min]
3. Results and discussion

To carry out the different processes for the development of 0,35 0,858487003
the project, different key aspects were considered to obtain
galacturonic acid. These aspects are the kinetic parameters
in the Michaelis-Menten kinetics, the concentration and 0,56 2,403763607
performance of galacturonic acid with respect to pectin, and
the modeling of bioreactors.
1,77 6,95374472
3.1 Determination of kinetic parameters
3,3 8,241475224
The kinetics of the biochemical reaction was taken into
account as kinetics that starts from the fundamental
Michaelis-Menten kinetics, however, this presents certain 4,5 10,38769273
variants since a competitive inhibition was demonstrated
experimentally at the substrate level at close concentrations
and above 4.5 mg / mL of pectin, which makes pectin lyase 5,5 9,443357028
a hard enzyme to be used for modeling in industrial reactors.
It should not be overlooked that the distributed enzyme
Pectinase 803-L was chosen for this process and that these 7,1 7,554685622
enzymes have optimal conditions at pH between 3.5 and 6.0 Table 1. Enzymatic activity of Pectinase 831-L obtained
and temperatures ranging between 10 and 50 ° C. from the document Pectin-to-Galacturonic Acid: Waste-to-
Bioproducts.
The enzymatic activity that occurs during the process is
evidenced in table number 1 through which it was possible
to calculate the kinetic parameters by linearization methods,
in this case, Lineweaver-Burk (L-B). This same table allows
us to obtain an approach of the behavior of the enzyme
concerning the amount of substrate, considering the
Michaelis-Menten graph as shown in graph 1.

3
 Although a conversion above 90% in any process on an
industrial scale is ideal, it is necessary to take into account
that factors such as the high viscosity of the pectin solution,
that can negatively influence the design of a large-scale
bioreactor since this will affect mainly stirring and mixing
within the bioreactor; It is for this reason that it is
recommended to work with a conversion close to 80% on a
large scale.

3.3 Bioreactor evaluation

To develop a comparison between the design of a Batch and

a CSTR bioreactor to obtain galacturonic acid from pectin
from plantain peel, it was necessary to simulate both designs
using MATLAB software. For these simulations, the same
calculus base was used, that is, the same kinetic parameters
were used, the same amount of enzyme and the initial
concentration of substrate in the medium was varied. In the
Batch bioreactor modeling, the operating time and the
Graph 1: Michaelis Menten plot using the velocity
volume of the reactor were obtained; on the other hand, for
obtained from the document Pectin-to-Galacturonic Acid:
the CSTR reactor, the volume of the reactor was obtained.
Waste-to-Bioproducts.
3.3.1. Batch reactor
As can be seen in figure 1 and as previously mentioned, the
first section of the graph presents kinetics like that of MM,
As mentioned above, in batch modeling the batch operation
however after the concentration of substrate reaches 4.5
time and reactor volume were obtained for an initial
mg/mL the graph decreases because of the competitive
substrate concentration of 10 g/mL. It should be noted that
inhibition.
for this to be carried out, a series of assumptions were made,
such that the degradation of pectin is constant due to the
Using the linearization method, it was possible to obtain
good mixing of the reactor, and that the dead time to sterilize
certain apparent parameters for the enzymatic reaction, and
the reactor after each batch was 4 hours.
with the help of the MATLAB software, it was possible to
Based on the design equation (eq 6) and the variation of
conclude a constant affinity for the enzyme Km = 3.54 mg /
parameters such as the reaction time of each cycle and the
mL, a maximum speed of 21,097 IU and since an inhibition
initial pectin concentration, the minimum required volume
zone is presented, an inhibition constant Ks = 7.67 mg / mL
of the reactor was determined to achieve a conversion of
is obtained.
92%, in the following graphs show the results produced by
MATLAB [7].
3.2 Galacturonic acid yield

According to the results obtained in the experimental

laboratory 3, the recovery yield of galacturonic acid was
92% w/w, this means that more than 90% of the total weight
of pectin was converted to galacturonic acid.
Subsequently, to find the yield at the end of the process, the
galacturonic acid of the enzymatic reaction for the enzyme
Pectinase 831-L was quantified from the total weight of dry
shells that entered the process, which was 4.2 ton/month,
later it was found that 20.68% w / w of these shells
correspond to pectin, this means that there is a total of
0.86856 ton/month of pectin and of that pectin as previously
mentioned 92% w / w is galacturonic acid; thus reaching a
total of 0,799 tons/month or what is equal to 799,000
kg/month of galacturonic acid.
4

Graph 2: Variant times of each Bach depending on the
concentration of initial substrate
Finally, to determine the appropriate volume for this reactor
type, (eq 6) was used, which made it possible to obtain a
graph 3 of concentration vs. time with which it was possible
to determine the desired volumes for the process.
Graph 3: Volume vs initial concentration of substrate for
BATCH.
In accordance with the graph above, it was determined that
the required volume is 1,9758 m ^ 3 and that the time of each
cycle is 12.61 hours plus 4 hours of dead time after each
batch.
5
3.3.2. CSTR reactor
To determine the required volume of the CSTR, the molar
flow was calculated based on the amount of monthly pectin
to be processed. Likewise, the initial substrate concentration
was varied as shown in figure 5 maintaining the 92%
conversion. Finally, having the characteristics of the CSTR
bioreactor in steady state and the design equation x, we have
that the volume of the reactor is 3.2833 m ^ 3 as evidenced
by the following graph [7].
Graph 4: Volume vs initial concentration of substrate for
CSTR.
3.4. Criteria for reactor selection
As previously mentioned, for this project it is required to
process 0.86856 ton/month of pectin, therefore the design of
the previous reactors is based on this principle, in addition
of assuming that the enzymatic kinetics used for both
reactors respond when modeling inhibition by substrate,
modeling also takes into account the same initial
concentration for both substrates and the same conversion.
Therefore, it is essential to consider the magnitude of the
space required in both reactors as a crucial factor for the
correct choice of this, that is, which reactor has a smaller
volume. For this reason, even though the CSTR reactor is
carried out in a continuous process and achieves a steady
state, it is not suitable for this process, since compared to the
BATCH, the CSTR has a higher volume as previously
reported. In addition to this, for BATCH it is easier to control
the reactor conditions such as pH, temperature, pressure,
among others, which are crucial for the enzyme. Therefore,
for the process to be carried out a BATCH reactor is chosen
to process the pectin.

4. Conclusions
The plantain peels of Algeria-Valle del Cauca, from Frito
Lay and Yupi, will decrease their negative impact on the
environment, due to the great potential they have to become
a raw material for the production of pectin and galacturonic
acid, because they are of great interest in the industrial
sector, such as the production of gelling agents, emulsifiers,
ascorbic acid (Vitamin C), among others; this is done
through an efficient extraction process in which 0.87 tons /
month of pectin were extracted, which the 12.72%, with high
extraction and purification standards, is galacturonic acid.
Observing the previous results, it can be concluded that the
reactor that best adapts to the transformation of pectin is the
Batch, in addition, the enzymatic kinetics used for both
reactors respond to the modeling of substrate inhibition, also
the modelling takes into account the same initial
concentration for both substrates and the same conversion.
Therefore, the magnitude of the space required in both
reactors should be considered as a crucial factor in the
correct choice of the reactor (The one with the lower
volume). Even though the CSTR reactor is carried out in a
continuous process and reaches a stable state, it is not
suitable for this process, because of its high volume. Besides
this, batch makes it easier to control reactor conditions, such
as pH, temperature, pressure, among others, which are
crucial for the enzyme. As a result of all this, a BATCH
reactor is chosen to process the pectin.
6
5.Referencias
[1]A. Arellanes, M. J. (6 de octubre de 2011). Obtained
from
https://produccioncientificaluz.org/index.php/agron
omia/article/view/26899
[2]ARIAS, D. H. (2019). EXTRACCIÓN Y
CARACTERIZACIÓN DE PECTINA DE
CÁSCARA DE PLÁTANO CULTIVADO EN
COLOMBIA Y DE LA ESPECIE Musa paradisíaca
PARA SU APLICACIÓN EN LA PREPARACIÓN
DE NANOPARTÍCULAS. Obtained from
https://repository.unad.edu.co/bitstream/handle/105
96/27152/dhvalenciaa.pdf?sequence=1&isAllowed
=y
[3]Biocatalysts. (19 de abril de 2016). Obtained from
https://www.icesi.edu.co/moodle/mod/folder/view.
php?id=118990
[4]Bioquímica, D. d. (22 de febrero de 2020). Obtained
from
https://www.icesi.edu.co/moodle/mod/folder/view.
php?id=118990
[5]Carlos Alvarez, A. C. (28 de april de 2020). Obtained
from
https://www.icesi.edu.co/moodle/mod/folder/view.
php?id=231094
[6]Departamento de Ingeniería Bioquímica . (21 de febrero
de 2020). Obtained from
https://www.icesi.edu.co/moodle/mod/folder/view.
php?id=118990
[7]Fogler, H. S. (2006). Elements of Chemical Reaction
Engineering, 4th ed. PrenticeHall PTR.
[8]Gutiérrez, C. (8 de Agosto de 2019). Gobernación del
Valle. Obtained from
https://www.valledelcauca.gov.co/publicaciones/63
807/en-el-norte-del-valle-producen-300-toneladas--
de-platano-con-apoyo-de-la-gobernacion/
Annexes:

Other equations:
(eq 6): volumen= (mPectina_ciclo. /So)

Detailed calculations:
In order to obtain the value of Vmax, which will be used as a constant, in the modeling of the kinetics of inhibition
by substrate to obtain the values of Km and Ks, the following calculations were performed in excel:

LB linearization:

M-M / LB linealización
0,45
0,4 y = 0,2045x + 0,0474
0,35 R² = 0,992
0,3
0,25
1/Vo

0,2
0,15
0,1
0,05
0
0 0,5 1 1,5 2
1/[S]

Vmax= 1/0.0474 = 21,0970464

Apparent km: Vmax*0.2045 = 4,314

7
MATLAB code for substrate inhibition:

% modelamiento de la cinetica de M_M con inhibición por sustrato,

%dejando constante la Vmax
% El dato de vMax, el cueal es constante, es obtenido mediante la
% linealización de LB en excel
function [Vmax, Km, Ks]=parametros_cin(Datos)
Data.S=[0;0.35;0.56;1.77;3.3;4.5;5.5;7.1]; %(g/l)
Data.Vo=[0;0.8584870025;2.403763607;6.95374472;8.241475224;10.38769273;9.443357028;7.554685622]; % mg
acid G L/h
plot(Data.S,Data.Vo,'ro')
xlabel('[S] (mg/mL)')
ylabel('Vo (uM/min)')
title('M-M Model')

Km_0=input('Escriba el valor para Km');

Ks_0=input('Escriba el valor para Ks');

theta_0=[Km_0; Ks_0];

theta=fminsearch(@SSMIN,theta_0,[],Data);

Km=theta(1)
Ks=theta(2)

sys.Km=theta(1);
sys.Ks=theta(2);

S=linspace(0,8,1000);
Voi=Vel(sys,S);
hold on
plot(S,Voi)
return

function sse=SSMIN(theta,Data);
sys.Km=theta(1);
sys.Ks=theta(2);
Vos=Vel(sys,Data.S);
res=Data.Vo-Vos;
sse=0.5*dot(res,res);
return

function Voi=Vel(sys,S);
Voi =( 21.0970464*S)./(sys.Km+S+(S.^2)/sys.Ks);

return

8
MATLAB code for Batch :

%Calculo de volumen del reactor Batch

mPectina=10422.72% Masa de pectina en kg (year)

%parametros cineticos-Pectinase 831L

Vm=0.3516; % Velocidad maxima de la cinetica uM/s
Vmax=(Vm*3600*(10^(-6))*194.139)/0.1272 %Velocidad maxima g de pectina/Lh
Km=3.435; % Constante de afinidad por sustrato g/L
Ks=7.6698; % constante de inhibición por sustrato g/L
X=0.92;% conversion

So=linspace(1,10,100); %Concentracion de sustrato inicial g/L

Sf=So.*(1-X); %Concentracion de sustrato final g/L

tb=(Km./Vmax.*log(So/Sf))+((So-Sf)./Vmax)+(((So.^2)-(Sf.^2))./(2.*Vmax.*Ks));% tiempo batch en horas

tm_batch=4; %tiempo muerto del reactor batch, se tiene en cuenta tiempo de preparacion, esterilizacion, limpieza
y adecuación del reactor batch en horas
t_ciclo=tm_batch+tb; % Tiempo total que dura un ciclo batch
ciclos_year=(24./t_ciclo).*365; % Cantidad de ciclos batch en un año
mPectina_ciclo=mPectina.*1000./ciclos_year;% masa de pectina por ciclo batch (g)
volumen=(mPectina_ciclo./So)./1000;%m3
V_BATCH=min(volumen)

figure(3)
plot(So,volumen,'r')
xlabel('Initial substrate concentration(g/L)')
ylabel('Volume (m3)')
title({'BATCH';'Volume vs. Initial substrate concentration'})

figure(4)
plot(So,tb,'r')
xlabel('Initial substrate concentration(g/L)')
ylabel('Batch time (h)')
title({'BATCH';'Batch time vs. Initial substrate concentration'})

9
MATLAB code for CSTR:

%Calculo del volumen del CSTR

tm=25; % dias tiempo muerto
mPectina= 10422,72 % Masa de pectina en kg (kg/año)
mPectina_hora=mPectina./((365-tm)*24); %Flujo masico de pectina por hora (kg pectina/hora)
mPectina_seg=mPectina_hora./3600; % Flujo masico de pectina por segundo ( kg pectina /s)

%parametros cineticos-Pectinase 831L

Vm=0.3516; % Velocidad maxima de la cinetica uM/s
Vmax=(Vm*(10^(-6))*194.139)/0.1272; %Velocidad maxima g de pectina/Lh
Km=3.435; % Constante de afinidad por sustrato g/L
Ks=7.6698; % constante de inhibicion por sustrato g/L
X=0.92;% conversion
So=linspace(1,10,100);% g/L;

%Calculo del volumen del reactor CSTR

flujo_pectin= (mPectina_seg)*1000; % g pectina/s
Sf=So.*(1-X); % g pectina/L
rate_o=(Vmax*Sf)./(Sf+Km+(Sf.*Sf./Ks)); % g/Ls
Volumen=((flujo_pectin.*X)./rate_o)./1000; %m3
vo=(flujo_pectin./So).*3600./1000; % flujo volumetrico de entrada al reactor m3/h
tao=(Volumen./vo)./3600; % tiempo de residencia (s)
V_CSTR=min(Volumen)%volumen minimo que se obtiene de la grafica

figure(7)
plot(So,Volumen,'r')
title( 'CSTR')
xlabel('Initial Substrate Concentration (g/L)')
ylabel('Volume (m3)')

10