Preparative Biochemistry & Biotechnology

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Influences of carbohydrate, nitrogen, and

phosphorus sources on the citric acid production
by fungal endophyte Aspergillus fumigatus P3I6

Jude Christian E. Francisco, Windell L. Rivera & Pierangeli G. Vital

To cite this article: Jude Christian E. Francisco, Windell L. Rivera & Pierangeli G. Vital (2019):
Influences of carbohydrate, nitrogen, and phosphorus sources on the citric acid production by
fungal endophyte Aspergillus�fumigatus P3I6, Preparative Biochemistry & Biotechnology, DOI:
10.1080/10826068.2019.1689510

To link to this article: https://doi.org/10.1080/10826068.2019.1689510

Published online: 17 Dec 2019.

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PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2019.1689510

Influences of carbohydrate, nitrogen, and phosphorus sources on the citric acid

production by fungal endophyte Aspergillus fumigatus P3I6
Jude Christian E. Franciscoa,b, Windell L. Riverab,c, and Pierangeli G. Vitala
a
Biological Research and Services Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman, Quezon City,
Philippines; bInstitute of Biology, College of Science, University of the Philippines Diliman, Quezon City, Philippines; cPathogen-Host-
Environment Interactions Research Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman, Quezon City,
Philippines

ABSTRACT KEYWORDS
Fungi are ecologically ubiquitous organisms on earth and regarded as one of the prolific sources Aspergillus fumigatus;
of natural products. Fungal endophytes may provide essential prerequisite molecules to plant bio- Aspergillus niger; citric acid;
chemical pathways which allow the efficient synthesis of primary and secondary metabolites. This fungal endophytes;
response surface
study characterized the influences of various combinations of process parameters namely, carbo- methodology
hydrate, nitrogen, and phosphorus sources on citric acid (CA) production by the isolated fungal
endophyte Aspergillus fumigatus P3I6 from Citrus microcarpa. Aspergillus fumigatus P3I6 had higher
CA concentration of 9.2 (± 0.9) g L
1 and 9.0 (± 5.0  10
15) g L
1 when supplemented with
sucrose and white refined sugar, respectively, than A. niger NRRL 599. Response Surface
Methodology (RSM) had shown that A. fumigatus P3I6 produced the highest CA (23.8 g L
1) in
Combination 4 (18.0% sucrose, 0.3 g L
1 ammonium sulfate, and 5.0 g L
1 dipotassium phosphate
(K2HPO4)). Analysis of variance showed that when K2HPO4 concentrations were increased, CA con-
tent in fermentation media was significantly elevated. Hence, K2HPO4 was the most critical variable
in the quadratic model (p < 0.05); however, sucrose concentration still has its role in production.
Aside from using A. niger in most fermentation processes, this discovered fungal strain can be
potentially used in biotechnological applications.

Introduction Aspergillus carbonarius, etc.), yeasts (e.g. Candida tropicalis,

Organic acids are extensively used in metallurgical treat-
ments, in the food industry as flavor enhancers, acidifiers,
stabilizers, preservatives, and disinfectants,[1] as well as in
pharmaceutical products and cosmetics.[2] Since synthetic-
ally-produced organic acids are very costly and natural sup-
plies are only limited, which may inflict significant costs to
the process, alternative sources are being used to generate
these compounds by fermentation processes using organic
substrates.[3–7] To address the demand for efficient and sus-
tainable alternatives, researchers are now resorting to bio-
logical tools such as fungi to produce high amounts of
industrially important organic acids. For example,
Aspergillus species, due to their ability to produce large
amounts of organic acids,[8] are typically used in the pro-
duction of citric, gluconic, malic, and itaconic acids. These
acids can be produced in large-scale processes showing high
potential of fungi in organic acid production systems.[1,9]
Among the organic acids, citric acid (CA) is the most
important acid produced through microbial fermentation by
means of a large number of microorganisms such as fila-
mentous fungi (e.g. Aspergillus niger, Aspergillus aculeatus,
Hansenula anamola, Yarrowia lipolytica, etc.), and bacteria
(e.g. Arthrobacter paraffinens, Bacillus licheniformis, etc.).
CA is a bulk product with an estimated global production of
over 900 thousand tons (8 million kilograms).[10] The yearly
production of CA is approximately 1.6 million tons (1.5 bil-
lion kilograms),[11] which is considered greatly in-demand
in the global markets for various purposes in industries.[12]
Worldwide demand for CA is increasing faster than its pro-
duction (as compared to the other acids); thus, it requires
more economical process models.[3,13] It is said that it is
continually increasing at a high annual rate of 5.0% and
with a steadily increasing consumption for numerous appli-
cations.[14,15] Therefore, in this study, a known biotechno-
logical strain and CA producer A. niger NRRL 599 and the
most robust CA producing-fungal endophyte Aspergillus
fumigatus P3I6 screened from Citrus microcarpa (calamon-
din), were compared in terms of their CA production.
Moreover, it was attempted to use the isolated A. fumigatus
P3I6 to statistically optimize the chemical variables such as
sugar, nitrogen, and phosphate sources, to produce CA in a
small laboratory-scale production setting.

CONTACT Pierangeli G. Vital pierangeli.vital@upd.edu.ph; piervital@hotmail.com Biological Research and Services Laboratory, Natural Sciences Research
Institute, University of the Philippines Diliman, Quezon City, 1101, Philippines
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.
ß 2019 Taylor & Francis Group, LLC
2 J. C. E. FRANCISCO ET AL.
Materials and methods
Chemicals and culture media
Potato dextrose agar (PDA), ethanol, bromothymol blue,
D-glucose, sucrose, ammonium sulfate ((NH4)2SO4), magne-
sium sulfate (MgSO47H2O), dipotassium phosphate
(K2HPO4), sulfuric acid, calcium chloride, hydrochloric acid
(HCl), ferrous sulfate, hydrogen peroxide, phenolphthalein,
and sodium hydroxide were purchased from Himedia
(Mumbai, India), Techno PharmaChem (Delhi, India), Ajax
Fine Chem (Australia), and QualiKems Fine Chem (Gujarat,
India). Brown raw sugar, white refined sugar, and antiseptic
hydrogen peroxide were store-bought.
Culture strains
An isolated fungal endophyte, A. fumigatus strain P3I6
(100.0% homologous with A. fumigatus strain Z1;
accession number: HQ331439.1) from Citrus microcarpa
(English common name calamondin) and A. niger NRRL
599 (ARS Culture Collection Peoria, Illinois) were used in
the fermentation processes. The fungal species were
cultured in PDA (200.0 g L
1 potatoes infusion, 20.0 g L
1
dextrose, and 15.0 g L
1 agar) (HiMedia Laboratories
Pvt. Ltd., Mumbai, India) with or without 100 g L
1 of
streptomycin and 150 g L
1 of ampicillin and incubated at
26  C for 7 days. After incubation, 7-day-old agar plugs
with diameter of 0.6 centimeters were cut with a flame
sterilized cork-borer and used as inoculum of fermenta-
tion media.
Isolation and screening of fungal endophyte strain
Plant samples from C. microcarpa were rinsed gently in
running water and were cut into 10  10 millimeter seg-
ments after surface sterilization in 70% ethanol and 50%
sodium hypochlorite. After sterilization, the samples were
washed with sterile distilled water and blot-dried on sterile
filter paper. The surface sterilized explants were inoculated
onto PDA and incubated at 26  C for 15 days.[16] The fungus
growing out from the explants were sub-cultured into new
PDA plates until pure cultures were obtained.
All the emerged fungal strains were qualitatively screened
for the production of organic acids using a plate assay
based on pH change. The fungal endophytes were inoculated
in the center of the Czapek-Dox agar (CDA: 30.0 g L
1
sucrose, 0.01 g L
1 FeSO4, 0.5 g L
1 KCl, 1.0 g L
1 KH2PO4,
0.5 g L
1 MgSO4, 2.0 g L
1 NaNO3, and 15.0 g L
1 agar)
supplemented with 0.5% bromothymol blue. The plates were
incubated at 30  C for 4–7 days. After incubation, the
diameter of the yellow zone formed was measured and
used as an indicator of the strain ability for organic acid
production.[17] A. niger strain NRRL 599 was used as posi-
tive control. The measurements were carried out in tripli-
cates in two biologically independent experiments.
Internal-transcribed spacer (ITS) sequencing of rDNA of
fungal endophyte strain
The DNA of the CA producer strain was extracted using the
Zymo Research Fungal/Bacterial Miniprep (The Epigenetics
Company, Irvine, CA, USA). The fungal DNA was analyzed
by sequencing the internal-transcribed spacer (ITS) region
of the rDNA, which used the universal primers ITS1 (50 -
TCC-GTA-GGT-GAA-CCT-GCG-G-30 ) and ITS4 (50 -TCC-
TCC-GCT-TAT-TGA-TAT-GC-30 ).[18,19] Amplification reac-
tions of 25 mL polymerase chain reaction (PCR) cocktail
mixtures were carried out with the following volumes:
12.5 mL PCR Master Mix, 1.0 mL each of the primers (ITS1
and ITS4), 7.5 mL PCR nuclease-free water, and 3.0 mL gen-
omic DNA. Amplifications in thermocycler was executed
with initial denaturation (95  C) for 5 min; followed by 35
cycles of 30 s denaturation (95  C), a 1-min annealing
(55  C), and an extension (72  C) for 1 min. The last cycle
was followed by an extension at 72  C for 6 min.
The PCR products for ITS rDNA gene were sent to
MACROGEN, Korea, for sequencing. The comparison and
description of the sequences of strains were identified using
the Basic Local Alignment Search Tool (BLAST) software of
the National Center for Biotechnology Information (NCBI).
The forward and reverse sequences were aligned and
assembled using BioEdit Software 7.0.5.3 to obtain the con-
sensus sequence.
Submerged fermentation medium
Phase 1 fermentation (preliminary screening)
In the preliminary screening, the production medium
consisted of the following components: carbon source
(D-glucose, brown raw sugar, and white refined sugar,
140 g L
1), (NH4)2SO4 (2.2 g L
1), MgSO47H2O (0.2 g L
1),
and K2HPO4 (1.0 g L
1). The pH of 350 mL medium was
adjusted to 3.0 using 0.1 N HCl. The fermentation medium
was inoculated with 3.0  106 spores/mL of either A. fumiga-
tus P3I6 or A. niger 599 and incubated at 30  C and
200 rpm agitation in a shaking incubator (Hei-MIX Shakers
and Mixers, Heidolph, Germany) for 7–8 days maximum.
After incubation, each fermentation medium was filtered by
passing through vacuum pump (Millipore WP6210060 High
Output). The obtained filtrate was then used for the deter-
mination of total CA.
Phase 2 fermentation (statistical optimization)
The preparations of production media were varied based on
the combinations obtained from the Central Composite
Design (CCD): sucrose (14%–22%), (NH4)2SO4
(0.1–0.4 g L
1), and K2HPO4 (0.5–5.0 g L
1), except
MgSO47H2O (0.2 g L
1) which remained constant in all fer-
mentation media. A 350-mL medium (pH 3.0) was inocu-
lated with 3.0  106 spores/mL of A. fumigatus P3I6 and was
incubated in a shaking incubator (Hei-MIX Shakers and
Mixers, Heidolph, Germany) with constant temperature of
30  C and 200 rpm agitation. The duration of incubation
was 7–8 days maximum, after which each fermentation

medium was filtered by passing through vacuum pump
(Millipore WP6210060 High Output). The obtained filtrate
was then used for the determination of total CA.
Response surface modeling
Individual and interactive influences of chemical factors on
the CA production were subjected to regression analyses by
Response Surface Methodology (RSM), a mathematical and
statistical method to solve multivariate problems by finding
out the optimum operating conditions for a given system.[20]
CCD,[21] a standard RSM, is able to find a suitable estima-
tion of the true functional relationship between the response
and the set of factors. The method was used to optimize
media constituents of fermentation. Chemical factors were
varied, such as substrate concentration, (NH4)2SO4, and
K2HPO4, as these significantly affect the CA production.
These variables were designated as x1, x2, and x3,
respectively.
A 23 factorial experiment was designed with one axial
point and seven replicates at the center points leading to a
total of 21 combinations, performed in duplicates. This
design was used for the optimization of laboratory-scale pro-
duction of CA. The second-order polynomial coefficients (ß)
were computed using the software package DESIGN
EXPERT software 7.0.0 (Stat-Ease Inc., MN) to estimate the
responses of the dependent variable. The experiments were
all performed in duplicates.
A CCD comprises of lk factorial points (coded ±1 nota-
tion), 2k axial points f(±A, 0, 0, … , 0), (0, ±A, 0, … , 0), (0,
0, ±A, … , 0), (0, 0, … , ±A)g, and nc center points (0, 0,
0, … , 0), where k is the number of controllable process vari-
ables, l is the number of levels for each process parameter, A
is equal to (nf)1/4, nf is the number of points used in factor-
ial position.[22] The independent variables used in this study
were coded based on the following equation:
Xi
X0
xi ¼ , i ¼ 1, 2, :::, k,
DX
where xi is the dimensionless value of a variable, xi is the
actual value of a variable, x0 is the value of xi at center
point, and Dx is steep change.[23]
The behavior of the system is explicated by the general
second-order polynomial:
Xk Xk Xk
Y ¼ A0 þ Ai xi þ A x2 þ Aij xi xij þ Ɛ
i i¼1 ii i i¼1
where x1, x2, … , xk are the input variables, which influence
on the response Y; Ai (i ¼ 1, 2, … , k) and Aij (i ¼ 1, 2, … , k;
j ¼ 1, 2, … , k) are unknown parameters, and Ɛ is a ran-
dom error.
The polynomial equation was validated by the analysis of
variance (ANOVA) statistical test for determining the sig-
nificance of each independent variable in the equation and
for estimating the goodness of fit. 3D surface, contour, and
perturbation plots were obtained from the effect of the inde-
pendent variables on CA content to define the possible
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 3
individual and cumulative effects of the variables together
with the mutual interactions between them.
CA determination
Special qualitative tests for the filtrates
Reaction with concentrated sulfuric acid. A mixture of
1.0 mL of filtrate of the fermented medium and 1.0 mL of
concentrated sulfuric acid was heated gently on a flame with
shaking. A positive result showed yellow coloration with no
charring (presence of black precipitates) as well as with the
release of carbon monoxide, carbon dioxide, and sulfur
dioxide gases.
Reaction with calcium chloride solution. The filtrate was
stored in 4  C refrigerator. About 1.0 mL of the cold filtrate
of the fermented medium was added to a few drops of cal-
cium chloride solution. A positive result showed the forma-
tion of white precipitate of calcium tartrate. This precipitate
dissolved in dilute HCl.
Reaction with Fenton’s reagent. About 1.0 mL of the fil-
trate of the fermented medium was added to one drop of
ferrous sulfate solution followed by two drops of hydrogen
peroxide solution. An excess of 10% of sodium hydroxide
solution was added until no intense violet color
was observed.
Neutralization reaction titration
Total organic acids (in terms of CA, g L
1) were quantified
by measuring the volumetric titratable acidity using 1.0%
phenolphthalein indicator. An analyte containing 30 mL fil-
trate of the fermented medium and 20 mL of distilled water
was titrated with 0.30 N (normality) NaOH solution. The
quantitation of organic acids was performed using CA as
the equivalent factor in grams. All experiments were carried
out in triplicates, and the data were calculated as mean-
(1)
s ± standard deviations. The CA content was calculated
according to the following formula[24]:
Normality of CA
Normality of NaOH  NaOH volume (3)
¼
Volume of analyte
Concentration of CA
 g 
(2) CA normality  Equivalent weight 64:0 mol  100
¼
Volume of distilllation
(4)
Results and discussion
Aside from A. niger which is the most widely applied micro-
organism for CA production, other Aspergillus species can
be used as workhorses in fermentation processes. It is
hypothesized that the isolated endophytic A. fumigatus P3I6
may have developed physiological co-evolution with the
4 J. C. E. FRANCISCO ET AL.

Table 1. Initial fermentation (CA content, mean ± SD) of A. niger NRRL 599 Table 2. CA content (g L
1) with different chemical compositions.
and A. fumigatus P3I6 in 14.0% of the carbon source (D-glucose, sucrose, white Response
refined sugar, and brown raw sugar).
Substrate AnCA (g L–1) AfCA (g L–1) Run x1 x2 x3 a
CA p
CA DCA
D-glucose 4.9 ± 0.8 7.3 ± 0.9 1 14.0 (–A) 0.3 (0) 2.8 (0) 16.9 ± 0.6 17.0 0.2
Sucrose 6.4 ± 0.7 9.2 ± 0.9 2 15.6 (–1) 0.2 (–1) 1.4 (–1) 12.1 ± 0.2 10.9 –1.2
White refined sugar 7.1 ± 0.1 9.0 ± 0.0 3 18.0 (0) 0.1 (–A) 2.8 (0) 17.1 ± 0.7 17.7 0.6
Brown raw sugar 8.8 ± 0.9 6.6 ± 0.6 4 18.0 (0) 0.3 (0) 5.0 (A) 23.8 ± 0.8 23.9 0.1
5 18.0 (0) 0.3 (0) 2.8 (0) 16.8 ± 1.3 17.7 0.8
Fermentation media were initially adjusted to pH 3.0 and temperature, agita- 6 18.0 (0) 0.3 (0) 2.8 (0) 17.9 ± 1.3 17.7 –0.2
tion, and incubation period withheld constant. A. niger NRRL 599 CA content 7 18.0 (0) 0.3 (0) 2.8 (0) 17.5 ± 1.3 17.7 0.1

(AnCA) and A. fumigatus P3I6 CA content (AfCA). 8 18.0 (0) 0.3 (0) 2.8 (0) 18.9 ± 1.3 17.7 –1.3
Significant (p < 0.05). 9 20.4 (1) 0.2 (–1) 4.1 (1) 23.0 ± 0.3 22.4 –0.6
10 18.0 (0) 0.3 (0) 2.8 (0) 17.6 ± 1.3 17.7 0.0
11 18.0 (0) 0.3 (0) 0.5 (–A) 5.0 ± 0.3 5.7 0.7
calamondin plant, providing its possible high organic acid 12 20.4 (1) 0.2 (–1) 1.4 (–1) 12.0 ± 0.0 11.7 –0.3
production. In the first phase of fermentation, A. niger 13 15.6 (–1) 0.3 (1) 1.4 (–1) 10.7 ± 0.3 10.9 0.2
NRRL 599 and fungal endophyte A. fumigatus P3I6 were 14 18.0 (0) 0.3 (0) 2.8 (0) 16.5 ± 1.3 17.7 1.2
15 15.6 (1) 0.3 (1) 4.1 (1) 22.0 ± 0.1 21.7 –0.3
compared based on their ability to convert substrates into 16 22.0 (A) 0.3 (0) 2.8 (0) 18.7 ± 0.4 18.3 –0.3
CA. A. fumigatus P3I6 was identified through phenotypic 17 15.6 (–1) 0.2 (–1) 4.1 (1) 21.9 ± 0.3 21.7 –0.2
and genotypic characterization through the use of ITS of 18 18.0 (0) 0.4 (A) 2.8 (0) 17.5 ± 0.6 17.7 0.2
19 20.4 (1) 0.3 (1) 4.1 (1) 21.9 ± 0.1 22.4 0.5
rDNA sequencing. The NCBI GenBank accession number of 20 18.0 (0) 0.3 (0) 2.8 (0) 17.6 ± 1.3 17.7 0.1
the strain is HQ331439.1. 21 20.4 (1) 0.3 (1) 1.4 (–1) 12.0 ± 0.9 11.7 –0.4
There were four different substrates used, namely, D-glu- Fermentation media were adjusted to pH 3.0 and the temperature, agitation,
cose, sucrose, white refined sugar, and brown raw sugar. A. and incubation period withheld constant.
x1 (% sucrose), x2 ((NH4)2SO4, g/L), and x3 (K2HPO4, g/L) presented in uncoded
niger NRRL 599 attained a relatively higher CA concentra- and coded variable combinations.
tion of 8.8 (± 0.9) g L
1 in consuming brown raw sugar

The runs or combinations were randomly tested to reduce statistical errors.
than the other three sugars. On the other hand, A. fumigatus The table presents the combinations in chronological order for reference
and clarity.
P3I6 had relatively and statistically significant increase in a
CA (actual CA content) ± standard deviation (SD) (obtained from at least 2
CA concentrations, 9.2 (± 0.9) g L
1 and 9.0 (± p
independent runs with at least 3 trials per run).
5.0  10
15) g L
1 when supplemented with sucrose and CA (predicted or theoretical CA content).
DCA (difference in pCA and aCA).
white refined sugar, respectively, as compared to D-glucose
and brown raw sugar (Table 1).
4.1 g L
1 K2HPO4) with CA content of 23.0 g L
1 and then
Based from the univariate analyses, there were significant
by Combination 15 (15.6% sucrose, 0.3 g L
1 (NH4)2SO4
differences in the CA production by A. niger NRRL 599 and
and 4.1 g/L K2HPO4) with CA content of 22.0 g L
1.
A. fumigatus P3I6 in the sugar-supplemented medium
Generally, chemical concentrations, physical conditions,
(Table 1), suggesting that the choice of fermentative sugars and type of microbial inoculum influence the production of
and fungal strains critically contributed to the response. A. CA. For example, in the study of Betiku and Adesina,[25]
fumigatus P3I6 significantly exceeded A. niger NRRL 599 in sweet potato starch hydrolysate (SPSH) was used and pre-
CA yield in terms of sucrose and white refined sugar. dicted that A. niger had attained highest CA concentration
However, sucrose was chosen when A. fumigatus P3I6 was (83.0 g L
1) at optimal conditions of 153.8 g L
1 SPSH,
used in the RSM because higher CA content was recorded 3.6 g L
1 (NH4)2HPO4, 2.6 g L
1 KH2PO4, 8 days incuba-
as compared to white refined sugar (Table 1). Hence, this tion, and pH 6. Barrington and Kim[26] reported that dry
study had selected the A. fumigatus as the strain for bio- peat moss (DPM), an inert support material, can enhance
production and sucrose as the substrate for the second phase CA production by A. niger NRRL 567. Three variables were
of fermentation. found to have significant effects to attain a maximum CA
CCD and RSM were used in the second phase of fermen- content of 354.8 g kg
1 DPM and these are with combina-
tation using A. fumigatus P3I6, to determine the possible tions of 19 g phytate/kg DPM, 49 g olive oil/kg DPM, and
and reliable combinations which would give statistically sig- 37 g methanol/kg DPM at 120 h.
nificant outcomes. Twenty-one[21] runs or combinations To determine the effects of the tested operable region of
were conducted in duplicates (two blocks). Table 2 summa- the variables on the dependent or response variable, the
rizes CA content from the 21 combinations formulated with measured actual response values were subjected to multiple
varying concentrations of sucrose (x1), (NH4)2SO4 (x2), and regression analysis. The individual and interactive influences
K2HPO4 (x3) and subjected to constant agitation, tempera- of independent variables such as sucrose, (NH4)2SO4, and
ture, and incubation periods. Results showed that the calcu- K2HPO4 on CA content were quantified by fitting the meas-
lated CA (g L
1) values ranged from 5.0 to 23.8 g L
1. The ured responses into a second-order polynomial model
lowest CA content (5.0 g L
1) was recorded in Combination (Eq. (2)).
11 with 18.0% sucrose, 0.3 g L
1 (NH4)2SO4, and 0.5 g L
1 Normal probability plots were generated to evaluate the
K2HPO4. Meanwhile, the highest CA content (23.8 g L
1) normality of the residuals (Figure 1). Studentized residual is
was observed in Combination 4 (18.0% sucrose, 0.3 g L
1 the residual divided by an estimate of its standard deviation.
(NH4)2SO4, and 5.0 g L
1 K2HPO4) followed by The residuals for default quadratic regression (Figure 1A)
Combination 9 (20.4% sucrose, 0.2 g L
1 (NH4)2SO4, and reduced quadratic regression (Figure 1B) followed
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 5

Figure 1. Normal probability plots of residuals for citric acid content (gL
1) in (A) default quadratic regression and (B) reduced quadratic regression.

normal distribution because the data points were closer to

Table 3. ANOVA for the reduced quadratic model1 for the effects of process
the fitted line with no reasonable outliers. variables on CA content (g L
1) in acidified fermentation media.
Sequential model sum of squares and model summary Source a
SS b
DF c
MS F-value p Value
statistics were both obtained to check the adequacy of the Model 414.7 3 138.2 315.8 <0.0001
model generated from the data. One of the features of these % Sucrose (x1) 2.0 1 2.0 4.6 0.05
two summaries is presenting the coefficient of determination K2HPO4 (g L–1) (x3) 396.8 1 396.8 906.7 <0.0001
x32 15.8 1 15.8 36.2 <0.0001
(R2). R2 is a statistical measure of how good the correlation Residual 7.4 17 0.4
is. Generally, the higher the R2, the better the model fits the Lack of fit 3.7 11 0.3 0.5 0.8
data. Model summary statistics showed that the calculated Pure error 3.8 6 0.6
1
values for R2 and adjusted R2 of the generated quadratic Goodness of fit to the model of the responses measured was characterized
with % coefficient of variation (%CV)¼3.9%, coefficient of determination
regression model were high (Table 3). However, R2 cannot (R2¼0.98, predicted R2¼0.97, adjusted R2¼0.98, and adequate precision
solely determine whether the coefficient estimates and pre- ¼ 62.8.
a
dictions are biased, which is why assessment of the residuals b
Sum of squares;
Degrees of freedom; cMean square.
is imperative. Additionally, R2 does not solely indicate 
Significant (p < 0.05).
whether a regression model is adequate. n
Not significant (lack of fit > pure error).
6 J. C. E. FRANCISCO ET AL.
Figure 2. Response surface model establishment of the effects of (A) ammonium sulfate and sucrose; and (B) dipotassium phosphate and sucrose, on citric acid
content (gL
1).
To further support the model validity, ANOVA analyses
were executed. Analyses show that the linear effect of
sucrose, and the linear and quadratic effects of K2HPO4
(Table 3) of the generated models were indeed significant.
Moreover, the adequate precision of the model of 62.8 for
the reduced quadratic regression that measures the signal-
to-noise ratio also indicated adequate signal since a ratio
greater than 4.0 is considered desirable. The model can,
therefore, be used to navigate the design space.
Table 3 shows the reduced quadratic regression ANOVA
analyses which indicate that the linear effect of sucrose and
the individual linear and quadratic effects of K2HPO4 sig-
nificantly influenced CA content in fermentation setups.
Only these significant influences were included in the
reduced predictive quadratic equation for CA content
(uncoded units of the independent variables, Eq. (5)). The
model F-value of 315.8 implies that the reduced quadratic
model was significant. There is only a 0.01% chance that a
“Model F-value” could occur due to noise. The p values
(Prob > F) of the model and its terms are less than 0.05
indicating their statistical significance.
Moreover, the “Lack of Fit” value of 0.5 means the Lack
of Fit value is insignificant comparative to the Pure Error.
There is 82.4% chance that a “Lack of Fit F-value” this large
could occur due to noise. The insignificance of Lack of Fit
is a good indication about the model significance (Table 3).
Hence, the null hypothesis (H0: bx1 ¼ bx2 ¼ bx3) was
rejected.
CA ðg L
1 Þ ¼
0:6 þ ½ð0:2Þ ð% sucroseÞ
þ ½ð7:2Þ ðP
source, g L
1 Þ (5)
þ ½ð
0:6Þ ðP
source, g L Þ 
1 2
(NH4)2SO4 had insignificant influences in the production
(Figures 2A and 3A), while K2HPO4 had significant influen-
ces in converting sucrose into CA (Figure 2B and 3B). The
validity of the response surface and contour plots was eval-
uated in the individual effects of each of the process-related

Figure 3. Contour plots establishment of the effects of (A) ammonium sulfate and sucrose; and (B) dipotassium phosphate and sucrose, on citric acid con-
tent (g L
1).
variables on the measured response using the perturbation
plot (Figure 4). Perturbation plot compared the effect of all
the factors at a particular point in the design space. A per-
turbation plot at a center point (18.0% sucrose, 0.3 g L
1
(NH4)2SO4, and 2.8 g L
1 K2HPO4) was obtained to show
the relative effect of the three independent variables as one
factor at a time on CA content. The plot indicated that the
steep increasing slope of the K2HPO4 has the most influen-
tial effect on CA content and then followed by sucrose.
Increasing K2HPO4 concentrations directly increased the
CA content in fermentation media, meaning, this is the
most critical variable among the tested variables (Figure 4).
Although, sucrose had less influence and (NH4)2SO4 was
not significant variable in the model (Table 3), they still
have respective roles in the production. For example,
sucrose is the sole carbon source of A. fumigatus P3I6 in the
media and (NH4)2SO4 is responsible for lowering the pH of
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 7
the media as well as contribute to the biomass, thereby,
favoring the production. Indeed, the model shows that
K2HPO4 is the significant factor (Table 3) in dictating the
bioconversion of sucrose to achieve a favorable increase in
CA content. However, a higher range of values for K2HPO4
should be implemented in the succeeding studies to attain
the maximum point of optimization.
It is critical to define the range of independent variables
(i.e. the physicochemical parameters) as these would dictate
the response variable. With these results, it can be observed
that the presence, type, and concentration of carbon source
or substrates strongly influenced CA accumulation.[27–29]
Several agro-industrial raw and waste materials can be used
for CA production, but there are some critical factors that
should be taken into consideration such as costs or the need
of pretreatment for choosing the type of substrate. For
example, molasses has trace elements that may act as
8 J. C. E. FRANCISCO ET AL.
Figure 4. Perturbation plot for citric acid content (gL
1) at center point of 18.0% sucrose, 0.3 gL
1 ammonium sulfate, and 2.8 gL
1 dipotassium phosphate. Line
segments show the (A) sucrose, (B) ammonium sulfate, and (C) dipotassium phosphate.
inhibitors to the fermentation processes and, hence, must be
precipitated by potassium ferrocyanide. Because of some
posing disadvantages of using agro-industrial products, this
study used instead the pretreated, commercially used sub-
strates, namely, D-glucose, laboratory-grade sucrose, white
refined sugar, and brown raw sugar (Table 1). Moreover, the
laboratory grade sucrose was used in the probabilistic mod-
eling because the substrate has better purity than white
refined sugar. Additionally, sucrose is regarded as the most
favorable carbon source followed by glucose, fructose, and
galactose,[30–32] because it can be cleaved by the invertase
enzyme into D-glucose and D-fructose. Thereby, through
invertase, sucrose-cleaved products would tend to produce
higher organic acids as compared to the other sugars.[33]
Therefore, the concentration of carbon source is crucial. The
highest CA productivities are usually achieved using 14–22%
sugar (Table 2). Using of high carbon concentrations can
enhance the glycolytic pathway as well as the suppression of
a-ketoglutarate dehydrogenase synthesis and thus inhibiting
the catabolism of CA via Krebs cycle.[34] This a-ketogluta-
rate dehydrogenase repression facilitates the accumulation of
CA in the medium.
Physiologically, ammonium salts are preferred (e.g. urea,
(NH4)2SO4, ammonium nitrate, peptone, etc.) because the
ammonium cations decrease the pH of the medium through
their dissociation in water[32,35] which is essential for the
CA fermentation. This study used nitrogen concentrations
of 0.1–0.4 g L
1 (Table 2), which is required for enhanced
CA production.[29,36] There is an increase in fungal growth
and sugar consumption when high nitrogen concentrations
(>0.4 g L
1) are used which subsequently decreases the
amount of CA products.[32] Low-level ranges of phosphorus
source (0.5–5.0 g L
1) (Table 2) was also used because it
would give a positive effect on the recovery of CA, as the
low concentrations enhance the catalytic activity of enzyme.
Whereas, the presence of excessive phosphate leads to a
decrease in the fixation of carbon dioxide which in turn
increases the formation of some other sugar acids such as
gluconic and oxalic acids, as well as the increase in
biomass.[32,35,36]
Also, it is indispensable to maintain the pH values of
media on the first day of fermentation before a certain
quantity of biomass production because the initial pH would
be the determining factor in successful fungal growth or ger-
mination. Because of that, the initial pH values of initial and
RSM (Phase 1 and Phase 2) fermentations were adjusted to
pH 3.0 to ensure spore germinations (Tables 1 and 2). The
pH value of 3.0 was chosen because changes in pH kinetics
also depend on the microorganism of choice. In case of
Aspergillus, Penicillium, and Rhizopus, the desired pH is
3.0–5.0. However, for other fungal groups, such as
Trichoderma, Sporotrichum, and Pleurotus, the pH is more
stable between 4.0 and 5.0. The nature of the substrate and
production technique also influences pH kinetics. Thus, ini-
tial pH should be well defined and optimized depending on
the microbial inoculant, substrate, and production
technique.[29]
Aspergillus niger has been the strain of choice over the
other microorganisms (i.e. yeasts and bacteria) because it is
easy to handle, can ferment a wide range of raw materials,
and increases the production of organic acids.[37] However,
other Aspergillus species can also be taken into consideration
such as a fungal endophyte A. fumigatus P3I6 isolated from
citrus plant (C. microcarpa). Generally, endophytes are non-
pathogenic microorganisms and they are potentially used in
various biotechnological applications. Several studies have
shown that A. fumigatus can manufacture certain industri-
ally important products such as CA,[38] cellulase,[39] tan-
nase,[40] and antimicrobial secondary metabolites[41] using
either submerged or solid-state fermentation. El-Gamal
et al.[38] used molasses as substrate for CA production by
soil inhabiting-A. fumigatus NA-1. Using RSM, it was found

out that the maximum CA production (1.2 g L
1) was at
45  C, pH 4.5, and 20% molasses concentration. Comparing
soil inhabiting-A. fumigatus NA-1 to the endophytic A.
fumigatus P3I6, the endophyte isolate was more efficient as
CA producer. However, industrial fungal strains, such as A.
niger ATTC 9142, A. niger IMI-41874, and Yarrow lipolytica
A101, still exhibit much higher CA production.[42–44]
Nonetheless, as this endophytic A. fumigatus P3I6 strain sur-
passed the A. niger NRRL 599, a known CA producer, in
CA production (Table 1), it may be used in future industrial
researches. Discovering new fungal species to act as indus-
trial workhorses would show a futuristic prospective as bio-
catalysts for industrial organic acid production.[45]
The results of this study can be used as a foundation of
fungal endophyte biotechnological research. Because endo-
phytic fungi of various plants chemically live in a special
environment in which they are typically surrounded by plant
molecules,[46–48] they may offer rich reservoir of chemical
compounds.[46] Furthermore, A. fumigatus P3I6 as an endo-
phyte can be used and genetically improved for CA produc-
tion in large-scale production in future researches.
Conclusion
This study explored the importance of fungal endophytes as
a biological tool for the production of CA primarily due to
the biochemical diversity of these organisms. A mathemat-
ical and statistical approach using the CCD was used to
optimize media constituents of fermentation of CA by A.
fumigatus P3I6. A 23 factorial experiment was designed with
one axial point and seven replicates at the center points
leading to 21 runs or combinations, performed in duplicates.
This design was used for the optimization of laboratory-
scale production of CA. The second-order polynomial coeffi-
cients (ß) were computed to estimate the responses of the
dependent variable. Results of CCD showed that the
obtained CA ranged from 5.0 to 23.8 g L
1. Hence, this
study provided a broad purpose of fungal endophyte of the
Class Eurotiomycetes in industrial biotechnology.
Funding
This study was supported by the Natural Sciences Research Institute
(NSRI) of University of the Philippines Diliman (UPD), Quezon City
Philippines (project code no.: BIO-18-1-06).
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