Environmental Science and Pollution Research

https://doi.org/10.1007/s11356-020-07928-9

RESEARCH ARTICLE

Pomegranate peel waste: a new substrate for citric acid production

by Aspergillus niger in solid-state fermentation under non-aseptic
conditions
Triantafyllos Roukas 1 & Parthena Kotzekidou 2

Received: 22 October 2019 / Accepted: 28 January 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Citric acid production from dried and non-dried pomegranate peel wastes by the fungus Aspergillus niger B60 in solid-state
fermentation (SSF) under non-aseptic conditions was investigated. The maximum amount of citric acid (278.5 g/kg dry peel) was
achieved using dried (at 45 °C for 48 h) pulverized pomegranate peels with moisture content 75% and initial pH 8.0, after 8 days
of fermentation at 25 °C. Under the same fermentation conditions, a higher amount of citric acid (306.8 g/kg dry peel) was
observed during SSF of non-dried peels as a substrate. The addition of methanol as an inducer at a concentration of 3% (w/w) into
the dried and non-dried pomegranate peel wastes increased the amount of citric acid to 300.7 and 351.5 g/kg dry peel, respec-
tively. The non-dried pomegranate peel waste in SSF under non-aseptic conditions is a cheap and useful substrate for the
commercial production of citric acid with low energy cost. The utilization of inexpensive agro-industrial wastes through SSF
can contribute to achieve industrially feasible and environmentally sustainable bio-production of citric acid.

Keywords Agro-industrial wastes . Non-sterilized substrate . Bioprocessing . Methanol . Fermentation parameters . Waste
valorization

Introduction a sustainable production of chemicals can be achieved

(Papadaki et al. 2018).
The food waste generated during food processing, consisting Pomegranate (Punica granatum L.) is consumed as an ed-
39% of total food waste in EU, can cause environmental prob- ible fruit or juice; it is widely grown in many tropical and
lems by greenhouse gas emission, pollution of water and soil, subtropical countries. It is extensively cultivated in
and lead to impacts on human health and ecosystems (Yousuf Argentina, China, Egypt, France, India, Iran, Japan, Russia,
et al. 2018; Ghinea et al. 2019). Thus, the valorization process Spain, and USA (Ullah et al. 2012). The production of pome-
by conversion of solid waste to bulk chemicals can lead in 7.5 granate is reached around 2 million tons due to the constant
times higher value compared with its present use as animal global demands (Faour-Klingbeil and Todd 2018). The pro-
feed (Singh and Choudhury 2019). Using renewable biomass, portion of pomegranate juice compared with the fruit weight is
low as the juice yield is lower than half of the fruit weight
(Kaderides et al. 2015). For this reason, large amounts of peel
wastes consisting mainly from rind and fleshy mesocarp are
Responsible editor: Ta Yeong Wu
generated during extraction of juice from pomegranate fruits
in industrial scale. Traditionally, pomegranate by-product
* Triantafyllos Roukas
roukas@agro.auth.gr wastes have been valorized as animal feed; but, recently, they
are used for the production of pectin (Pereira et al. 2016;
1
Güzel and Akpinar 2019), phenolic compounds and caroten-
Laboratory of Food Engineering and Processing, Department of
Food Science and Technology, Aristotle University of Thessaloniki,
oids (Goula et al. 2017), flavonoids, vitamin C, fertilizers,
Box 250, 54124 Thessaloniki, Greece dietary fibers, and tannins (Pathak et al. 2017), reducing
2
Laboratory of Food Microbiology and Hygiene, Department of Food
sugars (Pocan et al. 2018) and biochar (Siddiqui et al. 2019).
Science and Technology, Aristotle University of Thessaloniki, Box Pomegranate peel powder consists mainly of reducing sugars
250, 54124 Thessaloniki, Greece
 Environ Sci Pollut Res

30.4% of dry matter, crude fiber 21%, fat 9.4%, and protein moisture content, pH, temperature, and methanol supplemen-
8.7% (Ullah et al. 2012). tation) was studied in order to improve the production of citric
Citric acid, a generally recognized as safe (GRAS) organic acid.
acid, is used as flavor and acidifying agent in the food, bever-
age, and pharmaceutical industry (Dhillon et al. 2013a;
Roukas 2000). Due to numerous applications, the production Materials and methods
of citric acid is increasing at an annual growth rate of 5%, and
the citric acid production by fermentation continues to be of Microorganism and culture conditions
interest for extensive study (Dhillon et al. 2011; Angumeenal
and Venkappayya 2013). The production of citric acid using a Aspergillus niger B60 kindly provided by Prof. C.P. Kubicek
food processing by-product as a fermentation substrate re- (Technische Universitat Wien, Vienna, Austria) was used for
duces the production cost and contributes to the waste material the inoculation of the substrates. The fungus was grown on
management. In industrial scale, the most important substrates potato dextrose agar (PDA) Petri dishes for 4 days at 28 °C.
used for citric acid production are synthetic media and molas- The spores formed were suspended in 10 ml of distilled water
ses. Recently, the high cost of raw materials and energy has to obtain a concentration of 1.0 × 108 spores/ml.
transformed the once lucrative citric acid production sector
into an unprofitable market (Dhillon et al. 2013b). Thus, the Treatment of pomegranate peel wastes
reduction of the production cost using inexpensive non- and fermentation conditions
sterilized food processing wastes as substrate for citric acid
production is of considerable interest. Pomegranates (cultivar Ermioni) were obtained from a
Currently, citric acid is mainly produced by Aspergillus local farm. An integrated process for the production of
niger using submerged fermentation. But, the solid-state fer- citric acid from pomegranate peel wastes is presented in
mentation (SSF) of food processing by-products seems to Fig. 1. After extraction of juice with a pomegranate press,
have some advantages. The growth of the microorganism in the peel wastes were used for citric acid production by
a solid substrate is similar to its natural environment, and the A. niger. Three different substrates were prepared.
production of citric acid is more efficiently than in submerged Substrate I: the peels were cut in pieces (0.5–1.0 cm
fermentation (Barrington and Kim 2008). Thus, there has been length), dried at 45 °C for 48 h in a laboratory scale hot
an increasing interest on the use of SSF as an alternative to air oven, and ground in a blender (Type Colorato, high
submerged fermentation. Because, several advantages are of- p e r f o r m a n c e c o m m e r c i a l b l e n d e r s , C L B - 1 5 0 0 P,
fered over submerged fermentation, such as (a) low risk of Thessaloniki) at high speed (particle size 0.08 mm).
bacterial contamination under low substrate humidity and re- Substrate II: the peels were cut in pieces (0.5–1.0 cm
duced liquid phase; (b) the volumes of waste material and the length), dried at 105 °C for 4 h in an oven, and ground
liquid effluents are reduced; (c) operation under non-sterile as above. Ten grams of the peel powder of the substrates I
conditions can be applied; and (d) simple fermentation facili- and II was added in 500-ml conical flasks. The moisture
ties are used, and less energy consumption is required content (75%) and the initial pH (8.0) were adjusted with
(Barrington and Kim 2008). the addition of the appropriate amount of distilled water
Citric acid production by the fungus A. niger is one of the and NaOH 1N, respectively, under non-aseptic conditions.
most utilized examples of biological valorization of agricul- The non-sterilized substrates were inoculated with the
tural or food processing wastes in order to achieve waste man- spore suspension in order to contain 1.0 × 107 spores/g
agement and environmental sustainability. A considerable in- wet substrate. The conical flasks were incubated at
terest has been shown in using different agro-industrial wastes 25 °C in an incubator without forced aeration under static
for citric acid production such as pineapple waste (Imandi conditions for 2, 4, 6, 8, 10, and 12 days. In the substrate
et al. 2008), banana peel (Karthikeyan and Sivakumar I at the optimum fermentation time (i.e., when maximum
2010), apple pomace, brewer’s spent grain, citrus waste, citric acid production was obtained), the following factors
sphagnum peat moss (Dhillon et al. 2013a), and cheese whey were studied: (a) the effect of the moisture content of the
(Arslan et al. 2016). substrate recorded at 70, 75, 80, and 85%; (b) the effect of
The application of SSF for the production of citric acid the initial pH of the medium (at pH values 4.0, 5.0, 6.0,
under non-aseptic conditions by A. niger using peels of pome- 7.0, and 8.0), whereas the moisture content of the sub-
granate as a novel substrate could lead to the reduction of the strate was kept at the optimum level; (c) the effect of
production and energy cost. The present work aims to exam- incubation temperature by fermentations carried out at
ine the valorization of pomegranate peel waste for citric acid 20, 25, 30, 35, and 40 °C while moisture content and
production by A. niger in SSF under non-aseptic conditions. pH of the substrate were kept at the optimum values. At
In addition, the effect of various fermentation parameters (i.e., the optimum defined parameters, the effect of methanol at
Environ Sci Pollut Res

Pomegranate fruit the above treatments of the substrate were carried out
under non-aseptic conditions.
Pressing Juice
Determination of dry biomass, citric acid, residual
sugars, pH, moisture content, and phenolic
Peel waste compounds

The kinetic parameters were measured in the fermentation

Chopping
medium. The extraction of citric acid and the residual sugars
from the fermented mash was carried out after removing the
Grinding Drying mycelium as reported by Roukas (1999). Briefly, the substrate
Substrate III 45 oC for 48 h (Substrate I ) was mixed with 200 ml of distilled water and shaken on a
105 oC for 4 h (Substrate II) rotary incubator at 250 rpm for 1 h at 30 °C. The extract
was centrifuged at 5000 rpm for 10 min, and the sediment
Adjustment was treated again as described above. The supernatants of
Moisture Grinding
the two treatments were mixed, and the extract was used for
pH
the determination of citric acid and residual sugars. The citric
acid was determined as described by Saffran and Denstedt
Adjustment (1948) using acetic anhydride and pyridine reagents. The
Moisture
pH
absorbance was measured at 420 nm using a
spectrophotometer. The residual sugars expressed as glucose
were measured as reported by Dubois et al. (1956) using phe-
Inoculation by A. niger B60 - Fermentation nol and concentrated sulfuric acid. The method is used for the
determination of sugars and their methyl derivatives, oligosac-
Fermentation mash Removal of mycelium charides, and polysaccharides. The absorbance was measured
at 490 nm. The mycelium was washed with distilled water and
Extraction of citric acid
dried at 105 °C overnight. During the production of citric acid
from non-dried pomegranate peels, the biomass of the fungus
Supernatant
attached very strongly on the substrate, and it was very diffi-
Centrifugation Sediment
cult to detach the mycelium from the medium. Thus, the dry
Recovery of citric acid biomass of the fungus was not determined during fermenta-
tion. In this case, 200 ml of distilled water was added into each
Fig. 1 Integrated process for the production of citric acid from
pomegranate peel wastes
flask containing the fermentation mash (i.e., the biomass and
the substrate), and the flasks were shaken at 250 rpm for 1 h at
30 °C. The extract was used for the determination of citric acid
different concentrations (i.e., 1, 2, 3, 4, and 5%, w/w) was and residual sugars as described above. Sugar utilization (%)
studied. Substrate III: the peels were cut in pieces (0.5– was expressed as g sugar consumed per 100 g initial sugars.
1.0 cm diameter) and pulverized without drying in a The pΗ of the fermentation medium was measured with a
blender as described in the case of the substrate I. Ten pH meter using a glass electrode. During fermentation, the
grams of the pulverized particles was added in 500-ml moisture content of the substrate was measured using 2 g of
conical flasks. The moisture content and the pH of the the wet substrate, after drying at 105 °C overnight. The total
medium were adjusted to 75% and 8.0, respectively. The phenolic compounds were extracted from the pomegranate
non-sterilized substrate was inoculated with the spore sus- peels as described by Kaderides et al. (2015). Briefly, the dried
pension containing 1.0 × 107 spores/g wet substrate and peels with particle size 0.08 mm were mixed with distilled
incubated at 25 °C for 2, 4, 6, 8, 10, and 12 days. In order water in a ratio of 1:33, and the phenolic content was extracted
to study the effect of methanol supplementation of sub- using a sonicator (model VCX130, Sonics and Materials Inc.,
strate III on citric acid production, the moisture content U.S.A) for 10 min at room temperature. The phenolic content
and the pH of the medium were adjusted to 75% and 8.0, was determined according to the methodology reported by
respectively, and different concentrations of methanol (1, Chun et al. (2005) using the Folin-Ciocalteu phenol reagent
2, 3, 4, and 5% w/w) were added into the fermentation and 5% Na2CO3 solution. The absorbance was measured at
medium. The substrates were inoculated with the spore 750 nm. The result was expressed as g gallic acid/l solution.
suspension containing 1.0 × 107 spores/g wet substrate, All data are the average values ± SD of three independent
and the flasks were incubated at 25 °C for 8 days. All experiments.
 Environ Sci Pollut Res

Dry biomass (g/kg wet substrate)

Results and discussion 200 a 30

Citric acid (g/kg dry

25
160
Effect of fermentation time on citric acid, dry biomass,
20
pH, and residual sugar concentration 120

peel)
15
80
The changes in citric acid, dry biomass, pH, and residual sugar 10
concentration during dried pomegranate peel waste fermenta- 40 5
tion by A. niger B60 are shown in Figs. 2 and 3. The peels 0 0
were dried in the case of substrate I at 45 °C for 48 h, whereas 0 2 4 6 8 10 12
in substrate II at 105 °C for 4 h as described in the “Treatment Time (days)
of pomegranate peel wastes and fermentation conditions” sec-
tion. In both substrates, the citric acid concentration increased

Residual sugars (g/kg wet substrate)

100
up to the 8th day of the fermentation and then decreased (Figs. 8 b
2a and 3a). The decline in citric acid concentration was due to 80
the exhaustion of the initial sugar concentration (Figs. 2b and
6
3b). In the substrates I and II, the highest concentration of pH 60
citric acid was 278.5 and 207.5 g/kg dry peel, respectively,
40
after 8 days of incubation and then decreased (Figs. 2a and 4
3a). This was due to the better growth of the fungus in the 20
substrate I than in substrate II (Figs. 2a and 3a). This may be
explained by the fact that in the substrate I which dried at 2 0
0 2 4 6 8 10 12
45 °C for 48 h, the loss of the nutrient constituents was lower
than in substrate II which dried at 105 °C for 4 h, as the high Time (days)
temperature during drying (105 °C) involves the degradation Fig. 3 Citric acid production from pomegranate peel wastes dried at
of important nutritional ingredients. In addition, an increase in 105 °C for 4 h (substrate II). a -○-, citric acid; -□-, dry biomass. b -△,
residual sugars; -X-, pH. Fermentation conditions: moisture content 75%,
drying temperature caused important degradation of reducing
pH 8.0, temperature 25 °C. Bars indicate SD

280 40 sugars mainly because of Maillard reactions (Calín-Sánchez

a
(g/kg wet substrate)

240 35 et al. 2013). Moreover, the total phenolic compounds deter-

Dry biomass
(g/kg dry peel)

200 30 mined in the substrates I and II were 1.32 and 1.45 g, respec-
Citric acid

160 25 tively. Increased concentration of phenolic compounds sup-

20
120 presses the growth of the fungus A. niger (Leontopoulos
15
80
10
et al. 2015). Thus, the fungus A. niger B60 grown in the
40 5 substrate II, which contained higher concentration of pheno-
0 0 lics than substrate I, undergoes a higher toxic effect resulting
0 2 4 6 8 10 12 in a lower citric acid concentration.
Time (days) In both substrates, the dry biomass concentration was in-
creased up to the 8th day of the incubation and then remained
Residual sugars (g/kg wet substrate)

100 constant (Figs. 2a and 3a). The highest dry biomass concen-
8 b
tration in substrates I and II (32.0 and 18.5 g/kg wet substrate,
80
respectively) was observed after 8 days of incubation. The
6 60 above results show that the biosynthesis of citric acid was
pH
carried out during the growth phase of the fungus (Figs. 2a
40
4
and 3a). The pH of the substrates I and II decreased during
20 fermentation from the initial value of 8.0 to 3.0 and 3.7, re-
spectively, and then increased slightly (Figs. 2b and 3b). The
2 0
slight increase of pH value after 8 days of incubation (Figs. 2b
0 2 4 6 8 10 12
and 3b) was due to citric acid oxidation by A. niger (Roukas
Time (days)
1999).
Fig. 2 Citric acid production from pomegranate peel wastes dried at During fermentation, the residual sugar concentration de-
45 °C for 48 h (substrate I). a -○-, citric acid; -□-, dry biomass. b -△,
residual sugars; -X-, pH. Fermentation conditions: moisture content 75%, creased (Figs. 2b and 3b). Using the substrates I and II, the
pH 8.0, temperature 25 °C. Bars indicate SD lowest concentration of residual sugars (24.6 and 32.8 g/kg
Environ Sci Pollut Res

300 45
wet substrate, respectively) was observed after 12 days of
40
incubation (Figs. 2b and 3b). At this time, the sugars con-

Dry biomass/Residual sugars

250 35

Citric acid (g/kg dry peel)

sumed were 74.6 and 66.2%, respectively.

(g/kg wet substrate)

30
200
25

20
Effect of temperature, initial pH, and moisture 150
15
content on citric acid and dry biomass production 10
from dried pomegranate peel wastes by A. niger B60 100
5

50 0
The effect of temperature, initial pH, and moisture content on 3 4 5 6 7 8 9

citric acid production and dry biomass from pomegranate peel Initial pH

wastes dried at 45 °C for 48 h (substrate I) by A. niger B60 is Fig. 5 Effect of initial pH of pomegranate peel wastes dried at 45 °C for
shown in Figs. 4, 5, and 6. The concentration of citric acid and 48 h (substrate I) on the fermentation parameters. The initial conditions of
the experiment were moisture 80% and temperature 25 °C. The
the dry biomass increased with the increase of fermentation
fermentation time was 8 days. -○-, citric acid; -□-, dry biomass; -△-,
temperature from 20 to 25 and 30 °C, respectively, and then residual sugars. Bars indicate SD
decreased (Fig. 4). The maximum citric acid concentration
(240.0 g/kg dry peel) and the highest dry biomass (25.4 g/kg
wet substrate) were obtained at 25 and 30 °C, respectively. As the maximum dry biomass (24.5 g/kg wet substrate) were
the fermentation temperature increased from 20 to 25 °C, the obtained at an initial pH value of 8.0 and 7.0, respectively
final pH of the substrate after 8 days of fermentation decreased (Fig. 5). In cultures grown at different initial pH (4.0–7.0),
from 3.2 to 2.9 while further increase of temperature resulted the sugar concentration decreased after 8 days of incubation.
in a slight increase of the pH value of the fermentation medi- At initial pH values of 7.0 and 8.0, the concentration of resid-
um (data not shown). On the other hand, at 20, 25, and 30 °C, ual sugars was almost the same (Fig. 5). In all cases, the final
the residual sugar concentration remained almost constant and pH value of the substrate fluctuated between 2.8 and 3.5 after
increased significantly as the fermentation temperature in- 8 days of fermentation (data not shown). Roukas (1999,
creased from 30 to 40 °C (Fig. 4). This was due to the drastic 2000), Hamdy (2013), and Kim (2014) studied the production
decrease in citric acid concentration and dry biomass when the of citric acid from carob pod, fig, orange peel, and lignocellu-
temperature was higher than 30 °C (Fig. 4). losic by-products by different strains of A. niger using SSF
The concentration of citric acid and the dry biomass in- and found that the maximum citric acid concentration was
creased significantly after 8 days of fermentation when the obtained at different initial pH values of the substrates (i.e.,
initial pH value of the substrate increased from 4.0 to 7.0 6.5, 7.0, 5.0, and 5.5, respectively). These differences were
(Fig. 5). The dry biomass remained almost constant by a fur- due to the different compositions of the substrate used for the
ther increase of the initial pH value of the substrate to 8.0. The production of citric acid. In the case of pomegranate peel
highest concentration of citric acid (248.5 g/kg dry peel) and waste, the fungal growth is not favored at low initial pH values
of the substrate due to the high concentration of phenolic
300 45
300 45
40
40
Citric acid (g/kg dry peel)

250 Dry biomass/Residual sugars

Dry biomass/Residual sugars
Citric acid (g/kg dry peel)

35
250 35
(g/kg wet substrate)
(g/kg wet substrate)

30
200 30
25 200
25
20
150 20
150
15
15

100 10
100 10
5
5
50 0 50 0
15 20 25 30 35 40 45 65 70 75 80 85 90
Temperature (oC) Moisture (%, w/w)

Fig. 4 Effect of temperature on kinetic parameters during fermentation of Fig. 6 Effect of moisture content on kinetic parameters during
pomegranate peel wastes dried at 45 °C for 48 h (substrate I). -○-, citric fermentation of pomegranate peel wastes dried at 45 °C for 48 h
acid; -□-, dry biomass; -△-, residual sugars. The initial conditions in the (substrate I). -○-, citric acid; -□-, dry biomass; -△-, residual sugars. The
experiment were moisture 80% and pH 7.0. Fermentation time 8 days. initial conditions in the experiment were pH 8.0 and temperature 25 °C.
Bars indicate SD Fermentation time 8 days. Bars indicate SD
 Environ Sci Pollut Res

compounds. At initial pH values between 7.0 and 8.0, the 300 a

biomass and citric acid production are positively affected as 250

Citric acid (g/kg dry

the above pH values are near the cytoplasmic pH value of the
200
fungus and favor the metabolism contributing to the maxi-

peel)
mum activity of the intracellular key enzymes (Hesse et al. 150
2002). 100
Moisture content and particle size distribution affect the
50
progress of solid-state fermentation. As shown in Fig. 6, the
citric acid concentration and the dry biomass increased with 0
0 2 4 6 8 10 12
the increase of the moisture content from 70 to 75% and then
decreased significantly with a further increase of the moisture Time (days)
level. The highest citric acid and dry biomass concentration

Residual sugars (g/kg wet

(278.5 g/kg dry peel and 32.0 g/kg wet substrate, respectively) 100
8 b
were obtained at a moisture content of 75%. As indicated, the
80
availability of nutrients to the fungus is reduced at low mois-

substrate)
ture levels causing reduction of citric acid production. The low 6 60
pH
product formation is a consequence of low diffusion of nutri-
ents and oxygen to the cells of the fungus during fermentation. 40
4
On the other hand, at increased moisture, the porosity of the 20
substrate is reduced thereby limiting the oxygen transfer. The
residual sugar concentration decreased as the moisture content 2 0
0 2 4 6 8 10 12
of the substrate increased from 70 to 75% and increased with
an increase in moisture content from 75 to 85% (Fig. 6). This Time (days)
was due to the fact that the increased citric acid concentration Fig. 7 Citric acid production from non-dried pomegranate peel wastes
is correlated with decreased dry biomass production during (substrate III). a -○-, citric acid. b -△-, residual sugars; -X-, pH.
Fermentation conditions: moisture content 75%, initial pH 8.0,
SSF. At the end of fermentation, the final pH of the substrate
temperature 25 °C. Bars indicate SD
fluctuated between 2.9 and 3.3 (data not shown). The evalua-
tion of substrate moisture profile during fermentation was
studied. The results showed that when the initial moisture of citric acid (306.8 g/kg dry peel) was achieved after 8 days
content of the substrate was 70, 75, 80, and 85%, the moisture of incubation (Fig. 7a). This means that the citric acid concen-
content during the fermentation was 70.0–69.7%, 75.0– tration produced from non-dried pomegranate peels (substrate
74.8%, 80.0–79.6%, and 85.0–84.5%, respectively (data not III) was higher compared with that obtained from pomegran-
shown). It is indicated that the moisture content of the sub- ate peels dried at 45 °C for 48 h or 105 °C for 4 h (substrates I
strate remained almost constant during fermentation. This was and II, respectively). This may be explained by the fact that
due to the mycelium formed on the surface of the substrate and the non-dried pomegranate peels contain higher concentration
the diffusion of citric acid from the mycelium into the of nutrient constituents than dried pomegranate peels, and the
substrate. concentration of phenolic compounds (0.37 g) was lower than
In previous publications, reporting on citric acid production in the dried substrates I and II (1.32 and 1.45 g, respectively).
by A. niger grown on carob pod and fig (Roukas 1999, 2000), Thus, the fermentation conditions were more favorable for the
orange peel (Hamdy 2013), agricultural by-products (Kim fungus grown in the non-dried substrate resulting in a higher
2014), and a chemically defined medium (Kola et al. 2018), citric acid production. The highest concentration of citric acid
the optimum temperature was 30 °C, the initial pH fluctuated achieved by A. niger ATCC 9142 and ATCC 10577 using SSF
between 5.0 and 7.0, and the moisture level was 65–75%. The was 176.0 g/kg dry pod and 64.0 g/kg dry figs (Roukas 1999,
above differences were due to the different composition of the 2000). Imandi et al. (2008), Karthikeyan and Sivakumar
substrates used for the production of citric acid. (2010), and Dhillon et al. (2013b) found that a maximum citric
acid concentration of 202.3 g/kg dry pineapple, 175.0 g/kg dry
Citric acid production from non-dried pomegranate banana peel, and 183.0 g/kg dry apple pomace was obtained
peel wastes by A. niger B60 when Yarrowia lipolytica NCIM 3589, A. niger MTCC 282,
and A. niger NRRL 567 were grown in SSF, respectively.
The citric acid production from non-dried pomegranate peel The pH of the fermentation substrate and the residual
wastes (substrate III) by A. niger B60 is shown in Fig. 7. The sugars decreased during incubation (Fig. 7b). The lowest sug-
citric acid concentration increased up to the 8th day of the ar concentration (32.0 g/kg wet substrate) was obtained at the
fermentation and then decreased. The highest concentration maximum citric acid concentration (Fig. 7a, b). Before
Environ Sci Pollut Res

fermentation, the total sugars expressed as glucose were to 3% (w/w) and decreased after this value. The highest
16.8 g/100 g of the raw peel. After fermentation, the residual citric acid concentration in dried and non-dried pomegran-
sugars were 5.5 g/100 g of the raw peel (data not shown). The ate peels, i.e., 300.7 and 351.5 g/kg dry peel, respectively,
above results show that 67% of the initial sugars consumed was achieved at methanol concentration of 3% w/w (Fig.
during fermentation. The total phenolic compounds expressed 8a). This means that when the fungus A. niger B60 was
as gallic acid were 3.7 g/ 100 g of the raw peel before fermen- grown in non-dried pomegranate peels, the citric acid con-
tation and reduced to 2.5 g/100 g of the raw peel after the centration was 16.8% higher than that obtained in dried
incubation (data not shown). The results showed that the phe- pomegranate peels. The above results show that the addi-
nolic compounds decrease 32.4% during the fermentation. tion of methanol as an inducer stimulates significantly the
This was due to the phenolic degradation by A. niger B60. production of citric acid. Pazouki et al. (2000) reported
Chebaibi et al. (2019) studied the bioconversion of the olive that methanol affects the permeability of the cell, allowing
cake by different fungi and found significant decreases of the excretion of the citric acid from the cell resulting in an
phenolic compounds up to 43% of the initial phenolic content. increase in citric acid production. Roukas (1999, 2000)
reported that a methanol concentration of 6% (w/w) in-
creased significantly the production of citric acid by
Effect of methanol supplementation on citric acid A. niger from fig and carob pod using SSF. Dhillon
production from dried and non-dried pomegranate et al. (2013a) and Kumar et al. (2003) found that the
peel wastes by A. niger B60 addition of methanol into the medium at concentrations
of 3% and 4% (w/w) resulted in the production of 312.3
The effect of methanol supplementation on citric acid pro- and 113.0 g citric acid/kg dry apple pomace and pineapple
duction from dried at 45 °C for 48 h (substrate I) and non- waste, respectively, by A. niger using SSF.
dried (substrate III) pomegranate peel wastes is shown in During fermentation of non-dried pomegranate peel wastes
Fig. 8. In both substrates, the citric acid concentration (substrate III), the pH value of the substrate at the maximum
increased with the increase in methanol concentration up citric acid production was 2.8–2.9 (data not shown). In the
case of dried pomegranate peel (substrate I), the dry biomass
concentration decreased with the increase of methanol con-
Citric acid (g/kg dry peel)

400 centration (Fig. 8b). The maximum dry biomass concentration

a (32.0 g/kg wet substrate) was obtained in substrate I without
350
the addition of methanol. On the other hand, the highest con-
300
centration of citric acid (300.7 g/kg dry peel) was achieved in
250 substrate I when the dry biomass of the fungus was 22.3 g/kg
wet substrate (Fig. 8a, b). This means that there is not a linear
200
0 1 2 3 4 5 correlation between dry biomass and citric acid concentration.
Methanol (%, w/w) In all cases, using dried pomegranate peels (substrate I), the
sugar utilization remained almost constant while in non-dried
peels (substrate III), the sugar utilization fluctuated between
b
65.5 and 77.5% (Fig. 8b).

Conclusions

The non-dried pomegranate peel waste was the better sub-

strate for citric acid production by A. niger B60 using SSF
under non-aseptic conditions. The highest citric acid level was
produced at 75% moisture and initial pH 8.0 following 8 days
of fermentation at 25 °C. Methanol supplementation of the
non-dried pomegranate peel waste at a concentration of 3%
Fig. 8 Effect of methanol concentration on dry biomass and citric acid
production from dried at 45 °C for 48 h and non-dried pomegranate peel (w/w) stimulated the citric acid biosynthesis resulting in a
wastes (substrate I and substrate III, respectively). a -○-, citric acid (dried notable increase in citric acid production. The production of
substrate, moisture content 75%, initial pH 8.0, temperature 25 °C); -●-, citric acid using a food processing waste as a fermentation
citric acid (non-dried substrate, moisture content 75%, initial pH 8.0,
temperature 25 °C). b -□-, dry biomass (dried substrate); -△- and -▲-
substrate reduces the production cost and contributes to the
sugar utilization (%) (dried and non-dried substrate, respectively). conversion of this waste into a high value-added product used
Fermentation time 8 days. Bars indicate SD in industry.
 Environ Sci Pollut Res

Compliance with ethical standards Güzel M, Akpinar Ö (2019) Valorisation of fruit by-products: production
characterization of pectins from fruit peels. Food Bioprod Process
115:126–133. https://doi.org/10.1016/j.fbp.2019.03.009
Conflict of interest The authors declare that they have no conflict of
Hamdy HS (2013) Citric acid production by Aspergillus niger grown on
interest.
orange peel medium fortified with cane molasses. Ann Microbiol
63:267–278. https://doi.org/10.1007/s13213-012-0470-3
Ethical approval This article does not contain any studies with human Hesse SJ, Ruijter GJG, Dijkema C, Visser J (2002) Intracellular pH ho-
participants or animals performed by any of the authors. meostasis in the filamentous fungus Aspergillus niger. Eur J
Biochem 269:3485–3494. https://doi.org/10.1046/j.1432-1033.
2002.03042.x
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