Anais da Academia Brasileira de Ciências (2019) 91(2): e20180333

(Annals of the Brazilian Academy of Sciences)

Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201920180333
www.scielo.br/aabc | www.fb.com/aabcjournal

Comparison of acid and enzymatic hydrolysis of pectin, as

inexpensive source to cell growth of Cupriavidus necator

GABRIEL OLIVO LOCATELLI1,2, LEANDRO FINKLER1 AND CHRISTINE L.L. FINKLER1

1
Universidade Federal de Pernambuco/UFPE, Centro Acadêmico de Vitória, Rua Alto do
Reservatório, s/n, Bela Vista, 55608-680 Vitória de Santo Antão, PE, Brazil
2
UNIBRA, Centro Universitário Brasileiro, Núcleo de Nutrição, Rua Padre Inglês, 257, Boa Vista, 50050-230 Recife, PE, Brazil

Manuscript received on April 12, 2018; accepted for publication on July 24, 2018

How to cite: LOCATELLI GO, FINKLER L AND FINKLER CLL. 2019. Comparison of acid and enzymatic
hydrolysis of pectin, as inexpensive source to cell growth of Cupriavidus necator. An Acad Bras Cienc 91:
e20180333. DOI 10.1590/0001-3765201920180333.

Abstract: The present work investigated what the appropriate methods of hydrolysis of pectin for reducing
compounds (RCs) production, employed as a substrate for cell growth of Cupriavidus necator. This
microorganism has great importance industrial, because besides potential single cell protein (SCP), is the
most studied microorganism for production of polyhydroxybutyrate (PHB), and both processes require high
cell concentration with inexpensive substrates For this, it was compared to acid and enzymatic hydrolysis
procedures, through rotational central composite experimental design, using pectin concentration (1.0%). It
was analyzed as a variable response for both experimental design, the RCs’ production. The best conditions
of each procedure were used in study kinetics of RCs’ production and as a substrate for cell growth of
C. necator. The results indicated that the enzymatic hydrolysis method was the most efficient, with a
93.0% yield of RCs, while the yield for acid hydrolysis was 60.0%. The optimum conditions for enzymatic
hydrolysis were an enzyme concentration of 10.01 UI/g (International Unit of enzyme per gram of pectin)
and an agitation speed of 230.3 rpm. C. necator showed satisfactory growth in the media containing pectin
hydrolysates, with specific growth rates (μMax) similar to those reported for other substrates.
Key words: Cupriavidus necator, galacturonic acid, pectin depolymerization, pectin hydrolysates,
polygalacturonase.

INTRODUCTION apple, sugar beet, and sunflower are considered

of special interest, due to the physicochemical
Pectins are among the most abundant natural
quality and the availability of their biomasses
polysaccharides, present as a component of the
in agroindustrial wastes (Muzzarelli et al. 2012,
primary cell wall and middle lamella of fruits and
Adetunji et al. 2017).
vegetables and normally found associated with
Pectins are high molecular weight
cellulose, hemicellulose, and lignin. Although
ubiquitous in almost all plants, pectins of the citrus, heteropolymers, with a high content of galacturonic
acid (GalA) – an oxidized form of D-galactose,
which constitutes the main monomeric unit (around
Correspondence to: Gabriel Olivo Locatelli
E-mail: [email protected] 65.0%) of the pectin molecule. The structure
ORCID: https://orcid.org/0000-0002-5109-7485 of pectins can change according to the material

Biological Sciences An Acad Bras Cienc (2019) 91(2)

GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

of origin, and its understanding is currently al. 2002). As an application alternative, previous
characterized by much speculation and has not work by our research group demonstrated the
yet been fully resolved. A general model proposes capability of C. necator to grow using GalA as the
linearly alternating chains of homogalacturonans, sole source of carbon as well as using the products
composed of repeating units of (1→4)-α-D-GalA, of acid hydrolysis of pectin (Locatelli et al. 2011),
interrupted by branched regions composed of
but optimizing the hydrolysis conditions should be
(1→2)-α-L-rhamnose units, to which are bound
further studied.
neutral sugars including galactose, arabinose,
C. necator was famous as a potential single
xylose, and fructose (May 2000, Caffall and
Mohnen 2009). cell protein (SCP) in the 1970s, studies evaluated
Pectin can be degraded by either acid or that bacterial cells would average 50.0% protein,
enzymatic hydrolysis. Acid methods are commonly with 93.0% of digestibility by animals and high
used in analytical procedures; however, divergent concentration of important amino acids similar to
results suggest that these techniques still require those found in casein. However, the competition
significant improvement. The maintenance of from soybased protein resulted in SCP not receiving
strongly acidic conditions for prolonged periods much attention, but in recent years has resurgence
can result in a rate of destruction of free GalA that the interest in SCP and PHA as a component of
exceeds the rate of polymer release (Garna et al. animal feed to increase the metabolizable energy
2006). content (Kunasundari et al. 2013).
Enzymatic procedures are widely used
Although hundreds of species of
in industry to improve the yield during the
microorganisms are capable of accumulating
extraction and clarification of juices. The (endo)
polyhydroxyalkanoates (PHAs), C. necator is
polygalacturonases (E.C.3.2.1.15) are probably the
most important pectinases for biocatalysis, since the most important microorganism and has been
they are able to hydrolyze both pectin and pectic extensively studied for industrial production of
acids. However, few kinetic studies have been PHAs because it can accumulate up to 80.0% of its
reported concerning the mode of action of these dry mass in the form of biopolymers (Akaraonye
enzymes (Kiss et al. 2008, Kohli and Gupta 2015). et al. 2010, Wang et al. 2014). However, the
Agroindustrial pectin-rich waste, such as fruit polyhydroxyalkanoates production is still 5 to 10
pulp, husks, and bagasse, are potentially suitable times more expensive than chemically synthesized
feedstocks for bioconversion into products of polymers, and the substrate may represent more
biotechnological interest. Often bioconversions of than 40.0% of the cost of production (Akaraonye et
these residues can be done by acid or enzymatic
al. 2010, Albuquerque and Malafaia 2018).
hydrolysis to provide a useful source of carbon
Thus, either for SCP production or for PHA
and energy for use in biotransformation processes
production is very important to identify a new and
(Pinto et al. 2006).
inexpensive substrates source that can be used to
Although few studies have been undertaken
concerning the saccharification of pectin, cell growth of C. necator. Like this, the objective
D-galacturonic acid is an important primary this work was to compare methods of acid and
material in the food, pharmaceutical, and cosmetic enzymatic hydrolysis for the saccharification of
industries and can be used to produce vitamin C, pectin and investigate its use as a substrate for the
acidification agents, and surfactants (Jörneding et growth of C. necator.

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

MATERIALS AND METHODS the mixture was refluxed for 5 hours. At the end of
each test, the hydrolysate was cooled in an ice bath
A commercial citrus pectin with a high degree
and immediately neutralized with NaOH 50.0%
of esterification (67.0%) was purchased (Vetec,
(w/v).
Brazil). Sulfuric acid (Vetec, Brazil) was employed
The results of the experimental design were
in the acid hydrolysis and a polygalacturonase
used to define the hydrolysis conditions that
(Sigma-Aldrich, St. Louis, US, E.C.3.2.1.15), with
favored RCs’ production. Experiments were then
an enzymatic activity of 1.32 UI per mg of enzyme,
performed in triplicate under these conditions, with
was used in the enzymatic hydrolysis, the value of
samples removed at the start, and then after every
enzymatic activity was confirmed experimentally
15 minutes during the first hour, and subsequently
(data not shown).
after every 30 minutes up to a final time of 5 hours.
In order to refine the conditions for pectin
The samples were immediately neutralized with
hydrolysis and maximize the yield of reducing
NaOH 50.0% (w/v), cooled in an ice bath, diluted
compounds (RCs), two independent variables
for analysis of RCs, and stored at -18 oC until the
(defined separately for each hydrolysis method)
chromatographic procedures were performed.
were evaluated using a full 22 experimental design
The final hydrolysate obtained was neutralized,
(rotational central composite design – RCCD),
sterilized in an autoclave, and used as the substrate
with three central points (level 0) and four axial
in the microorganism culture medium.
points (levels ± α, where α = 1.4142), totaling 11
experiments. This experimental design model, it ENZYMATIC HYDROLYSIS
allows a greater comprehension of the parameters
According to the information provided by Sigma,
tested, minimizing the experiments number. The
the polygalacturonase utilized for the enzymatic
experiments were performed randomly, and the
hydrolysis experiments presented optimum activity
data were analyzed using Statistica 8.0 software
at pH 4.0 and a temperature of 50 oC. To enable
(StatSoft, Dell Software, US), with a 95.0%
comparison with the acid hydrolysis method,
confidence level. The experimental error was
the same pectin concentration (1.0% w/v) was
obtained from the mean and standard deviation
of the central points. The software calculate an TABLE I
empirical model described by Equation, through Codified levels and actual values of the variables studied
the experimental data, allowing prediction of the in the acid hydrolysis experiments.
experimental value at any point within the study Concentration of H2SO4
Test Temperature (oC)
(% v/v)
area.
1 -1 (1.9) -1 (74.4)
ACID HYDROLYSIS 2 +1 (6.1) -1 (74.4)
3 -1 (1.9) +1 (95.6)
The acid hydrolysis of pectin was based on the 4 +1 (6.1) +1 (95.6)
method proposed by Wenzel (2001). The tests were 5 -1.41 (1.0) 0 (85.0)
performed using a rotary evaporator reflux system 6 +1.41 (7.0) 0 (85.0)
(Marconi). The initial pectin concentration was 7 0 (4.0) -1.41 (70.0)
1.0% (w/v), and temperature and acid concentration 8 0 (4.0) +1.41 (100.0)
were the independent variables (Table I). For this, 9 0 (4.0) 0 (85.0)
a 5.0 g portion of pectin was added to 500 mL of a 10 0 (4.0) 0 (85.0)

solution of sulfuric acid in a distillation flask, and 11 0 (4.0) 0 (85.0)

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

employed, and the independent variables were MICROORGANISM AND MAINTENANCE OF THE
the enzyme concentration and the agitation speed CULTURE

(Table II).
The strain of Cupriavidus necator was obtained from
The tests were performed using 125 mL
the Deutsche Sammlung von Mikroorganismen und
Erlenmeyer flasks containing 0.5 g of pectin and
Zellkulturen (DSMZ 545), and maintained in the
50 mL of sodium acetate buffer (50 mM) and
culture collection of the Department of Antibiotics
incubated under orbital agitation for 24 hours. At
of the Federal University of Pernambuco (UFPEDA
the end of the reaction period, the enzyme activity
0604). During the tests, the bacterium culture was
was interrupted by placing the sample in a boiling
frequently subcultured on tubes containing nutrient
water bath for 5 minutes.
agar slants and stored in a refrigerator (4–8 oC).
The optimum conditions for enzymatic
hydrolysis were identified from the results, and PREPARATION OF THE INOCULUM
experiments were then performed in triplicate,
For the production of the inoculum, one loopful
with removal of sample aliquots at the start of the
experiment, and then after every 15 minutes during of the bacterial culture was transferred from a
the first hour, and subsequently after every hour slant culture into an Erlenmeyer flask (250 mL)
during a total period of 24 hours. After removal, containing 100 mL nutrient broth (NB) medium.
the samples were immediately placed in a boiling The flask was incubated in a shaking incubator
water bath for 5 minutes, diluted for analysis of at 30 °C and 300 rpm for 10 hours. This time of
RCs, and stored at -18 oC until the chromatographic cultivation had previously been established for
procedures were performed. The final hydrolysate attainment of the exponential growth phase (data
was neutralized, sterilized in an autoclave, and it not shown).
was used as the substrate in the microorganism CULTURE CONDITIONS
culture medium.
The culture was performed under the same
TABLE II conditions described previously, employing an
Codified levels and actual values of the variables studied inoculum of 5.0% (v/v) of the cellular material
in the enzymatic hydrolysis experiments.
obtained in the previous step. The mineral medium
used was described by Ramsay et al. (1990),
Test Enzyme conc. (UI/g) Agitation speed (rpm)
modified by Aragão et al. (1996). The medium was
composed of a mixture of four solutions, as follows
1 -1 (6.9) -1 (211.6)
(with concentrations as g/L): Nitrolactic acid (0.19),
2 +1 (11.1) -1 (211.6)
ferrous ammonium citrate (0.06), MgSO4.7H2O
3 -1 (6.9) +1 (268.4)
4 +1 (11.1) +1 (268.4)
(0.5), CaCl2.2H2O (0.01), solution of oligoelements
5 -1.41 (6.0) 0 (240.0) (1.0 mL) (Solution 1); Na2HPO4.12H2O (8.95),
6 +1.41 (12.0) 0 (240.0) KH 2PO 4 (1.5) (Solution 2); (NH 4) 2SO 4 (5.0)
7 0 (9.0) -1.41 (200.0) (Solution 3); pectin hydrolysate (Solution 4).
8 0 (9.0) +1.41 (280.0) The solution of oligoelements consisted of (g/L):
9 0 (9.0) 0 (240.0) H 3BO 3 (3.0), CoCl 2.6H 2O (0.2), ZnSO 4.7H 2O
10 0 (9.0) 0 (240.0) (0.1), MnCl2.4H2O (0.03), Na2MoO4.2H2O (0.03),
11 0 (9.0) 0 (240.0)
NiCl2.6H2O (0.02), and CuSO4.5H2O (0.01).

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

The pH of the solutions was adjusted to 7.0 with RESULTS AND DISCUSSION

KOH (5.0 M). The solutions were then autoclaved ACID HYDROLYSIS OPTIMIZATION
separately and mixed aseptically to produce the
medium. Samples were withdrawn at intervals of RCs’ concentrations for different experiment
conditionals were evaluated based on results of
2 hours for analyses of pH, cell concentration, and
experimental design. The best experimental results
consumption of RCs.
obtained was 4.6 g/L of RCs, with 1.0% (v/v)
ANALYTICAL METHODS sulfuric acid at 85 oC, but the empirical model
predict increase RCs’ production with 1.0% (v/v)
Cell growth was measured using a Marconi sulfuric acid at 100 oC (Figure 1a). The Pareto chart
spectrophotometer, operated at a wavelength of (Figure 1c) and ANOVA (Table III) showed that all
600 nm. The optical density values were correlated of the effects were significant (p < 0.05), and were
to the dry mass using a calibration curve. The therefore used in the prediction of an empirical
determination of bacterial cell dry mass was model described by Equation 1. The parameter
(1) L – Concentration of H2SO4 (% v/v) exerted
performed by drying in an oven at 70 oC to a
the greatest influence on RC production, but this
constant weight, after filtration using a 0.22 μm
effect was negative, decreasing RC production
membrane. The pH was monitored using a Marconi with increasing of concentration of H2SO4. On the
potentiometer. Measurements of RCs were other hand, the parameters (2) L – Temperature
performed according to the 3,5-dinitrosalicylic (oC) and Q – Temperature (oC) exerted a positive
acid (DNS) method described by Miller (1959). influence on RC production. The distribution
Glucose was used as a standard to produce the of the residuals (values predicted by the model
calibration curve. versus observed values) showed that the deviations
were normally distributed, and that there was a
The RCs were identified by high-performance
satisfactory correlation between the theoretical and
liquid chromatography (Varian, Walnut Creek, CA,
experimental values (Figure 1b).
US), using a refractive index detector. A column
suitable for organic acids was employed (Aminex RC = 14.870 + 0.169*H2SO4 - 0.019*(H2SO4)2 -
0.292*T + 0.002*T2 - 0.005*H2SO4*T (1)
HPX – 87H, 300 x 780 mm, Bio-Rad, Hercules,
CA, US), maintained at 65 ºC (Klein and Leubolt, Therefore, the formation of RCs was favored
1993). The mobile phase was an aqueous solution by decreasing the sulfuric acid concentration and
of H2SO4 (8.0 mM), and the flow rate was 0.6 increasing the temperature. Leitão et al. (1995),
mL/min. Retention times were determined using who used hydrochloric acid and trifluoroacetic acid
standard solutions of GalA, fructose, galactose, to hydrolyze sunflower pectin, obtained similar
results. These authors observed that the GalA yield
xylose, rhamnose and arabinose (Sigma), at
increased at higher temperatures and lower acid
concentrations in the range 0.2–10 g/L. The RCs’
concentrations. Garna et al. (2006) studied the
concentrations were expressed as a separate hydrolysis of pectin using different concentrations
galacturonic acid group (GalA) and the sum of of sulfuric acid and achieved the best results at
neutral sugars (NeutralS), compounds for fructose acid concentrations around 1.0 M at 100 oC and
+ xylose + galactose + rhamnose + arabinose. lower hydrolysis rates using acid concentrations

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

Figure 1 - Experimental design to pectin acid hydrolysis optimization – response surface (a), distribution of residuals (b), and
Pareto chart (c) as a function of independent variables: temperature and H2SO4 concentration, and response variable: reducing
compounds (RCs) production.

of 0.2 and 2.0 M. Other researchers also indicated greater resistance of the glycosidic bonds under
the high stability of PGalA in acidic environments milder hydrolysis conditions (Biermann 1988,
(Lim, et al. 2012, Min et al. 2011). De Ruiter et al. 1992). The negative effect of
Garna et al. (2006) also observed the positive high acid concentrations can be explained by the
effect of temperature on the yield of free GalA. The decomposition of the RCs by other products. The
positive influence of temperature on hydrolysis combination of acid and high temperature may
can be explained by greater solubilization of the cause the formation of furfural derivatives, resulting
polysaccharides at higher temperatures, and the in an imprecise determination of sugars (Wikiera

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

TABLE III
Acid hydrolysis optimization - ANOVA statistics data of independent variables and their interactions; dependent variable
(RC concentration g/L).
Factor SS Df MS F P

(1) L - Concentration of H2SO4 (% v/v) 5.149892 1 5.149892 4076.829 0.000245

(1) Q - Concentration of H2SO4 (% v/v) 0.039797 1 0.039797 31.505 0.030306

(2) L - Temperature (oC) 0.710374 1 0.710374 562.356 0.001774

o
(2) Q - Temperature ( C) 0.281626 1 0.281626 222.945 0.004455
1 L by 2 L 0.043972 1 0.043972 34.810 0.027546
Pure Error 0.002526 2 0.001263
Total SS 6.976098 10

et al. 2015). Such secondary reactions not only hydrolysis of pectin after 24 hours of bioreaction,
reduce the yield of the desired monosaccharides is given by Equation 2.
but also produce toxic compounds that prohibit the
use of these hydrolysates in biological conversion RC = -24.892 + 1.705*Enz – 0.067*Enz2 +
processes. 0.219*Agit – 0.00044*Enz2 (2)
The results clearly showed that optimum
Thus, can conclude that both variables have a
hydrolysis was achieved at higher temperatures and
acid concentrations of up to 1.0% (v/v). Since it great influence on the enzymatic activity. Higher
was not practically feasible to raise the temperature pectin concentrations increase the viscosity of the
above 100 oC, the conditions chosen for the RC- medium, agitation being indispensable to promote
release kinetics experiments were a temperature contact of the enzyme with the substrate. But at low
of 100 oC and an acid concentration of 1.0% (v/v) concentrations such as that used in this work, high
H2SO4. agitation speeds may hinder the formation of the
ENZYMATIC HYDROLYSIS OPTIMIZATION enzyme-substrate complex. Songpim et al. (2010),
working with pectate lyase enzyme, studied the
The best experimental results obtained was 8.8 g/L
effect of agitation speeds from 150 at 250 rpm and
of RCs, with 11 UI/g of pectin and an agitation
speed of 211.6 rpm, but the empirical model predict maximum response was obtained with 200 rpm,
maximum concentration of RCs can be achieved demonstrating the effect of this variable on the
using an enzyme concentration of 10.01 UI/g of enzymatic activity.
pectin and an agitation speed of 230.3 rpm (Figure In general, the enzyme activity increases as
2a). All of the effects were significant (p < 0.05) as enzyme concentration increases. However, this
showed the Pareto chart (Figure 2c) and ANOVA
increase is limited until the level of substrate
(Table IV), the observation that the second order
saturation, as was demonstrated by Michizoe et al.
effects were negative indicated that there was an
(2001) when they studied the laccase concentration
optimum point for the two variables. Figure 2b
shows that there was a good agreement between effect from 0 at 15 μM over degradation of
experimental data and numerical predictions. o-chlorophenol. The authors observed that after 11
The codified model, optimized for the enzymatic μM, the degradation rate was practically stable.

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

Figure 2 - Experimental design to pectin enzymatic hydrolysis optimization - response surface (a), distribution of residuals (b), and
Pareto chart (c) as a function of independent variables: agitation speed and enzyme concentration, and response variable: reducing
compounds (RCs) production.

COMPARISON BETWEEN KINETICS OF ACID AND The release profiles of the carbohydrate groups were
ENZYMATIC HYDROLYSIS
in agreement with the results obtained for RCs,
The kinetics during at acid hydrolysis (Figure 3a) with the concentration of GalA (maximum 3.2 g/L
revealed a higher rate of hydrolysis during the reached in a 4-hour hydrolysis period) exceeding
first 15 minutes, with a gradual increase up to 4 that of NeutralS (maximum concentration 1.8 g/L
hours, when a maximum concentration of 6.0 g/L reached in a 1-hour hydrolysis period).
was achieved (which was higher than the 5.3 g/L Pectins with a high degree of esterification are
predicted by the model). Then there was a slight commonly extracted using hot water (60 at 100
o
decline until the end of the 5-hour hydrolysis period. C) at pH ranges from 1.5 to 3.0 for several hours

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

TABLE IV
Enzymatic hydrolysis optimization - ANOVA statistics data of independent variables and their interactions; dependent
variable (RC concentration g/L).
Factor SS Df MS F P

(1) L - Enzyme (UI/g) 0.524779 1 0.524779 724.5920 0.001377

(1) Q - Enzyme (UI/g) 0.513524 1 0.513524 709.0513 0.001407

(2) L - Agitation (rpm) 0.314697 1 0.314697 434.5194 0.002293

(2) Q – Agitation (rpm) 0.705515 1 0.705515 974.1444 0.001025

1 L by 2 L 0.034157 1 0.034157 47.1628 0.020552
Pure Error 0.001448 2 0.000724
Total SS 2.061697 10

(Koubala et al. 2008). These extraction conditions formation of these degradation products was low
of pectin could explain the elevated initial rate of for up to 4 hours of hydrolysis under the conditions
free GalA. The resistance to acid hydrolysis of the employed, so that the hydrolysate could therefore
glycosidic linkages is variable and runs as follows: be used in the culture medium.
GalA---GalA > GalA---Rha > Rha---GalA > sugar In order to confirm the results obtained
neutral–sugar neutral, GalA monomers being the in enzymatic hydrolysis optimization, it was
last to be released (Novoselskaya et al. 2000). This performed an experiment in triplicate using the
could explain the gradual release of RCs up to the optimized conditions achieved previously. The RC
end of the hydrolysis process. The lower resistance release kinetics was monitored during 24 hours
of the glycosidic linkages between neutral sugars of enzymatic hydrolysis (Figure 3b). The rate of
are perceived in the behavior curve of the NeutralS hydrolysis was faster during the first 30 minutes
that have a higher rate of release in the first 30 min and remained high for 8 hours. This was succeeded
of hydrolysis. by a gradual hydrolysis for the remaining period of
These results are similar to those obtained 24 hours, and at the end the RC concentration was
in earlier studies by Garna et al. (2006), who 9.34 ± 0.16 g/L (which was greater than the value
hydrolysed a highly esterified pectin using different of 8.85 g/L predicted by the model).
concentrations of sulfuric acid at 100 oC. For all The release profiles of the GalA and NeutralS
treatments, a gradual release of GalA was observed groups were broadly similar to that of the RCs.
during the first hours of the process, a decline in There was a greater initial release of the GalA
the concentration of free GalA being observed after during the first hour of hydrolysis, followed by
different times for each treatment. According to the gradual release for the remaining period of 24 hours
authors, the continuation of hydrolysis conditions and reaching a maximum concentration of 8.4 g/L.
for long periods led to rates of destruction of free The NeutralS group showed a gradual release up to
GalA that exceeded the release rates. 19 hours, after which the concentration remained
In addition to the GalA monomers, the pentoses constant up to 24 hours of hydrolysis (maximum
present in pectin can be degraded to formation concentration 1.4 g/L).
of furfural derivatives, which makes the precise Bélafi-Bakó et al. (2007) also observed a
determination of sugars impossible (Wikiera et al. decrease in the RC and GalA release rate during
2015, Medina et al. 1942). The results indicated that the course of the hydrolysis process. Those authors

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

Figure 3 – Release kinetics of reducing compounds during acid hydrolysis of pectin (a) – with 1.0% v/v H2SO4; 100 oC; 1.0%
(w/v) pectin; and during enzymatic hydrolysis of pectin (b) - with 10.01 UI/g of enzyme; 230.3 rpm; 1.0% (w/v) of pectin.

utilized different initial concentrations of free GalA concentration, pH, and substrate consumption are
and demonstrated that the product of hydrolysis shown in Figures 4 and 5.
inhibited the enzymatic activity. The neutral When the chemical hydrolysate was used
sugars, found at lower concentrations in pectin, are (Figure 4a), growth of the microorganism began
probably depolymerized due to the lower resistance immediately after cell inoculation and continued
of their glycosidic bonds (Novoselskaya et al. for around 10 hours, with μMax of 0.26 h-1. For the
2000), with an acidity of pH 4.0 and a temperature culture using the enzymatic hydrolysate (Figure
of 50 oC being sufficient to cause their release. 5a), there was an adaptive phase lasting for around
Given an initial pectin concentration of 1.0% 2 hours, followed by exponential growth up to
(w/v), the average yields of the enzymatic and acid 12 hours, with μMax of 0.29 h-1. The results can be
hydrolyses were 93.0% and 60.0%, respectively. compared with those obtained by other authors
The enzymatic method was therefore more efficient (Table V).
for the production of RCs from the hydrolysis of The pH remained at around 7.0 throughout
pectin. the culture period, using both formulations. The
consumption of RCs was related to the growth
CELL GROWTH
phase, with residual values of 2.8 and 3.7 g/L for
The hydrolysates obtained by both methods media formulated with the hydrolyzed, acid and
were diluted for use in the culture medium enzymatic, respectively (Figure 4b and 5b). We see
formulations. Initial concentrations of RCs were a residual amount of RCs that can’t be metabolized
different because it was considered the initial by C. necator, which was also observed by other
substrate concentration in the hydrolysis process authors using other carbon sources (Baei et al.
(pectin 1.0% w/v). Therefore, it achieved 3.82 and 2009, 2011, Locatelli et al. 2011, Lagunes and
5.30 g/L RCs in the initial media formulated with Winterburn 2016).
the hydrolyzed acid and enzymatic, respectively. This RC residual in both hydrolysates could
The growth of C. necator and parameters kinetics be related to the presence of oligomers with
of process were followed as a function of time. Cell reducing terminal residues that the microorganism

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GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

Figure 4 – Cell growth and pH (a); and consumption of Figure 5 - Cell growth and pH (a); and consumption of
substrates (b), during culture of C. necator in mineral media substrates (b), during culture of C. necator in mineral media
containing chemical pectin hydrolysate. containing enzymatic pectin hydrolysate.

is unable to metabolize. The furanic aldehydes with a yield 33.0% higher than that achieved using
formed by degradation of GalA and sugar affect acid hydrolysis. The release profiles of the GalA
the microorganism’s metabolism, being toxic and NeutralS groups were broadly similar to that of
for fungus (Szengyel and Zacchi 2000), yeasts the RCs, to both hydrolysis processes. Moreover,
(Taherzadeh et al. 1999) and bacteria (Zaldivar it proves that drastic conditions as the high acid’
et al. 1999) and undesirables in culture medium concentration can be negative over RC’ production.
formulations, which could explain the low final The mineral medium formulation containing
cellular concentration using the medium formulated an enzymatic hydrolysate provided a higher final
with the hydrolyzed acid. cell concentration during growth of C. necator,
with a specific growth rate that was superior to
CONCLUSIONS
that obtained using a chemical hydrolysate. In
The production of RCs from the hydrolysis of pectin this way, enzymatic hydrolysis can be used in the
was more efficient using an enzymatic method, saccharification of agroindustrial waste pectin,

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 TABLE V
Cell growth rates of Cupriavidus necator using Chemical and Enzymatic Hydrolysate, compared with other carbon sources.

Initial Carbon
Carbon source
References Microbial Strain concentration Yx/s μMax
Type
(g L-1)

Chemical hydrolysate 3.82 (in reducing compounds) 0.58 0.26 h-1

This paper C. necator-DSM 545
Enzymatic hydrolysate 5.30 (in reducing compounds) 0.62 0.29 h-1

Yousuf and Winterburn 2016 C. necator H16 Date seeds extract 10.80 (of fructose) 0.68 0.13 h-1

An Acad Bras Cienc (2019) 91(2)

GABRIEL OLIVO LOCATELLI et al.

Lagunes and Winterburn 2016 C. necator H16-DSM 428 Orange juicing waste 14.94 (of fructose) 0.40 0.179 h-1

Arabinose
Glucose
Aramvash et al. 2015 C. necator-ATCC 17699 20.00 (of each) --- ---
Fructose
Sucrose

C. necator-IPT 026

Residual Glycerin from Biosiesel

Figueiredo et al. 2014 C. necator-IPT 027 30.00 (of each) --- ---
Glucose

C. necator-IPT 028

Galacturonic acid 15.00 --- ---

Locatelli et al. 2011 C. necator-DSM 545
Pectin hydrolysate 3.82 (in reducing compounds) 0.55 0.26 h-1
Glucose 0.53 0.17 h-1
Baei et al. 2011 C. necator-DSM 545 Fructose 40.00 (of each) 0.50 0.125 h-1
Sugarcane Molasses (Inverted sugar) 0.55 0.42 h-1

Dalcanton et al. 2010 C. necator-DSM 545 Rice starch hydrolysate 30.00 (in reducing sugars) --- 0.238 h-1

Waste glycerol (GRP) 0.45 0.15 h-1

e20180333
Cavalheiro et al. 2009 C. necator-DSM 545 ---
Commercial Glycerol (PG) 0.37 0.12 h-1
Inverted sugar 0.26 h-1
INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

12 | 14
R. eutropha-DSM 545 Glucose 0.23 h-1
Marangoni et al. 2001 20.00 (of each) ---
(Nowdays C. necator) Fructose 0.21 h-1
Galactose 0.13 h-1
Source: The authors.
GABRIEL OLIVO LOCATELLI et al. INEXPENSIVE SOURCE TO CELL GROWTH OF Cupriavidus necator

and the hydrolysis product can be applied in the by Cupriavidus necator. Int J Environ Sci Technol 12:
2307-2316.
process of C. necator growth, with potential for
BAEI MS, NAJAFPOUR GD, YOUNESI H, TABANDEH
SCP production or for PHA production. F AND EISAZADEH H. 2009. Poly(3-hydroxybutyrate)
synthesis by Cupriavidus necator DSMZ 545 utilizing
ACKNOWLEDGMENTS various carbon sources. World Appl Sci J 7: 157-161.
BAEI MS, NAJAFPOUR GD, YOUNESI H, TABANDEH
The authors are very grateful for financial support F, ISSAZADEH H AND KHODABANDEH M.
from the Fundação de Amparo à Ciência e 2011. Growth kinetic parameters and biosynthesis of
polyhydroxybutyrate in Cupriavidus necator DSMZ 545
Tecnologia do Estado de Pernambuco (FACEPE), on selected substrates. Chem Ind Chem Eng Q 17: 1-8.
for the master’s degree scholarship, and to Professor BÉLAFI-BAKÓ K, ESZTERLE M, KISS K, NEMESTÓTHY
Nelson Medeiros de Lima Filho, for assistance with N AND GUBICZA L. 2007. HYDROLYSIS of pectin
by Aspergillus niger polygalacturonase in a membrane
the chromatographic analysis.
bioreactor. J Food Eng 78: 438-442.
BIERMANN CJ. 1988. Hydrolysis and other cleavages of
AUTHOR CONTRIBUTIONS
glycosidic linkages in polysaccharides. Adv Carbohydr
Chem Biochem 46: 251-272.
All authors conceived and planned the experiments. CAFFALL KH AND MOHNEN D. 2009. The structure,
G.O. Locatelli carried out the experiments. L function, and biosynthesis of plant cell wall pectic
Finkler and C.L.L Finkler contributed to samples polysaccharides. Carbohydr Res 344: 1879-1900.
CAVALHEIRO JMBT, DE ALMEIDA MCMD, GRANDFILS
preparation. All authors contributed to the C AND DAFONSECA MMR. 2009. Poly(3-
interpretation of the results. G.O. Locatelli took hydroxybutyrate) production by Cupriavidus necator
the lead in writing the manuscript. All authors using waste glycerol. Process Biochem 44: 509-515.
DALCANTON F, IENCZAK JL, FIORESE ML AND
provided critical feedback and contributed to the
ARAGÃO GMF. 2010. Produção de poli(3-hidroxibutirato)
final version of the manuscript. por Cupriavidus necator em meio hidrolisado de amido de
arroz com suplementação de óleo de soja em diferentes
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