Application of calcium carbonate nanoparticles and microwave irradiation in submerged fermentation production and recovery of fumaric acid: A novel approach

Article abstract of DOI:10.1039/c6ra00316h

The aim of the present study was to explore the possible application of calcium carbonate nanoparticles (CCNPs) and microwave irradiation (MWI) in fumaric acid (FA) production and recovery, respectively. The fungal strain Rhizopus oryzae 1526 was employed as the biocatalyst for FA production. A glucose-basic salt medium was used as the fermentation medium. Scanning electron microscopy (SEM) analysis of CCNPs displayed the spherical shapes, while zetasizer measurements showed the CCNPs to be around 190 ± 20 nm in size. FTIR analysis of CCNPs confirmed the chemical composition. BET analysis confirmed higher specific surface areas of CCNPs (11.95 ± 0.03 m² g^-1) compared to calcium carbonate microparticles (CCMPs) (3.51 ± 0.02 m² g^-1). FA neutralization timing for CCNPs was much lower than CCMPs (190 and 350 seconds, respectively). CCNPs enhanced the volumetric productivity of FA from 0.47 g L^-1 h^-1 to 0.74 g L^-1 h^-1. At 20, 40 and 60 g L^-1 concentrations and at 25 °C, viscosities of the CCNPs were found to be lower than respective CCMPs. Moreover, the CCNPs did not exhibit any toxicity towards the fungus. FA production obtained with CC micro and CCNPs were 67.34 ± 2 g L^-1 and 66.92 ± 2.7 g L^-1, respectively. Under MWI heating, 10 ± 1 min was found to be sufficient for recovery of FA and this was much lower than conventional heating timing of 28 ± 1 min.

Full text of DOI:10.1039/c6ra00316h

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Application of calcium carbonate nanoparticles and
microwave irradiation in submerged fermentation
production and recovery of fumaric acid: a novel
approach
Cite this: RSC Adv., 2016, 6, 25829
a
a
Ratul Kumar Das, Satinder Kaur Brar and Mausam Vermab
*
The aim of the present study was to explore the possible application of calcium carbonate nanoparticles
(CCNPs) and microwave irradiation (MWI) in fumaric acid (FA) production and recovery, respectively. The
fungal strain Rhizopus oryzae 1526 was employed as the biocatalyst for FA production. A glucose-basic
salt medium was used as the fermentation medium. Scanning electron microscopy (SEM) analysis of
CCNPs displayed the spherical shapes, while zetasizer measurements showed the CCNPs to be around
190 ꢀ 20 nm in size. FTIR analysis of CCNPs confirmed the chemical composition. BET analysis
confirmed higher specific surface areas of CCNPs (11.95 ꢀ 0.03 m2
g
ꢁ1) compared to calcium carbonate
g
ꢁ1). FA neutralization timing for CCNPs was much lower than
microparticles (CCMPs) (3.51 ꢀ 0.02 m2
CCMPs (190 and 350 seconds, respectively). CCNPs enhanced the volumetric productivity of FA from
0.47 g Lꢁ1 hꢁ1 to 0.74 g Lꢁ1 ꢁ1. At 20, 40 and 60 g Lꢁ1 concentrations and at 25 ꢂC, viscosities of the
h
Received 5th January 2016
Accepted 2nd March 2016
CCNPs were found to be lower than respective CCMPs. Moreover, the CCNPs did not exhibit any
toxicity towards the fungus. FA production obtained with CC micro and CCNPs were 67.34 ꢀ 2 g Lꢁ1
and 66.92 ꢀ 2.7 g Lꢁ1, respectively. Under MWI heating, 10 ꢀ 1 min was found to be sufficient for
recovery of FA and this was much lower than conventional heating timing of 28 ꢀ 1 min.
DOI: 10.1039/c6ra00316h
www.rsc.org/advances
research on different aspects of upstream and downstream
processing of fermentative FA production has uncovered the key
1. Introduction
factors that contribute to the higher yield of this multifaceted
organic acid.9,10 Apart from the safety issues of chemically
derived FA, newly explored bio-medical applications of FA and
its ester derivatives has necessitated the biological production
of FA.11,12 In the last two decades, different research groups have
explored the cost-effective FA production through SmF by
introducing new concepts, such as carbon-economy and white
biotechnology. Low cost carbon sources have been utilized for
FA production. Different strains of the lamentous fungus
Rhizopus oryzae have been employed as the most efficient bio-
catalysts in SmF based FA production.9,10 Strategies, such as
immobilization of fungal strains on different solid supports,
genetic engineering and metabolic shi in tricarboxylic cycle
have been experimented for enhanced FA production under
SmF conditions.13–16 The overall progress made on the upstream
processing of fermentative FA production is remarkable.
However, fermentative approach of FA production is yet to
address the bottlenecks in its downstream processing domain.
This issue has been highlighted by different experts from time
to time.9,10 Efforts have been made for easy recovery of FA from
fermentation broth. In a study by Zhang et al., activated carbon
was used as the adsorbent for FA recovery from the fermenta-
tion broth and later desorbed into acetone. The FA recovery
Fumaric acid (FA) [IUPAC ID: (E)-butenedioic acid] is an organic
acid with diverse applications in the eld of food, resin and
dairy industries.1–3 Because of the non-hygroscopic and non-
toxic properties and higher buffering capacity than other food
acids, FA is preferably used as an acidulant and nutritive
additive in the food industry.2 The chemical structure makes FA
suitable for polymerization and esterication reactions and this
has led to the extensive application of FA in resin industry.
About 56% of the total annual production of FA is used in the
resin industry for the production of paper resins, alkyd resins
and unsaturated polyester resins.4,5 In the dairy industry, FA has
been recommended for use in ruminant diet for reducing
enteric methane (CH4) emission.6,7 Moreover, FA has been
recently experimented as highly efficient promoters of the
Beckmann rearrangement (from benzophenone oxime Ia to
benzamide IIa).8 In the production domain, FA production
through submerged fermentation (SmF) has emerged as the
most explored biological alternative to chemical route. Active
a
´
´
´
INRS-ETE, Universite du Quebec, 490 Rue de la Couronne, Quebec, QC G1K 9A9,
Canada. E-mail: satinder.brar@ete.inrs.ca; Fax: +1 418 654 2600; Tel: +1 418 654
3116
bCO2 Solutions Inc., 2300, rue Jean-Perrin, Quebec, Quebec G2C 1T9, Canada
´
´
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yield was found to be around 93%.17 In another study, a xed microwave irradiation (MWI) can be a good option for time-
bed column of amberlite ion exchange resin (IRA900) was effective recovery of FA from the broth. MWI is an electromag-
applied with a stirred-tank bioreactor for intermittent in situ FA netic spectrum with a frequency in the range of 300 MHz to 300
recovery from the fermentation broth. Compared to the results GHz. For a variety of inorganic synthesis and biomedical
of the control experiment (without adsorption), this approach applications, MWI is a well proven and widely accepted pro-
enhanced the recovery yield and productivity of FA by 25% and cessing technology.24,25 As compared to general heating treat-
59%, respectively.18 Techniques such as nanoltration and ment, a rapid and homogenous heating can easily be achieved
bipolar electrodialysis have also been applied for the recovery of by employing MWI even in materials exhibiting low heat
FA from model solutions and real fermentation broth. The conductivity as the transfer of energy does not rely on heat
combination
of
nanoltration
(nanoporous
ceramic diffusion.26,27 On the other hand, dissolution enhancement is
membrane) with bipolar electrodialysis (membrane PC 200bip an innovative concept which additionally accelerates the solu-
and the anion-exchange membrane PC 200D) efficiently recov- bility and dissolution.27,28 However, a good dielectric property is
ered and concentrated FA from the fermentation broth.19
required for excellent efficiency of heat transfer.29 Water (in the
To the specic interest of the present study, application of fermented broth) represents the solvent with a dielectric
calcium carbonate (to be called as CC hereaer) as a neutral- constant value of 80.37 (at 25 ꢂC) and the major byproduct
izing agent in SmF based FA production is unavoidable. There ‘ethanol’ formed during FA fermentation has a dielectric
has been serious concern regarding the disadvantages of using constant value of 24.3 (at 25 ꢂC).30 Both solvents can thus
CC over other neutralizing agents, such as Na2CO3, NaHCO3, certainly enhance the MWI efficacy of heating the fermented
(NH4)2CO3, and Ca(OH)2. However, pertaining to the higher broth in a shortened time. Moreover, acidic condition favors
yield of FA, CC has been established as the default neutralizing MWI heating. Considering the bottlenecks in the production
agent in FA production.9 Lower solubility and higher viscosity of and recovery of FA, CCNPs and MWI were thus investigated in
CC slurries causes problems in the heat, mass and oxygen the present study.
transfer throughout the broth during SmF. Non-stoichiometric
addition of CC for neutralization of FA and maintenance of
broth pH near to 6–7 is a regular practice during SmF based FA
2. Experimental
2.1 Materials
production. The fermentation product ‘calcium fumarate’
(CaC4H2O4) has low water solubility (15.6 g Lꢁ1) and this makes
The fungal strain, Rhizopus oryzae NRRL 1526 (to be called R.
oryzae hereaer) was procured from Agricultural Research
fermentation broth even more viscous.9 CaC4H2O4 has
commercial application as calcium fortier in beverages.
Studies have been carried out on the relationship between the
water solubility of CaC4H2O4 and pH with different acidulants.20
By stoichiometric ratio, 20 g of CC can neutralize 23.2 g of FA
and this is irrespective of any SmF conditions. As FA concen-
tration (g Lꢁ1) in the SmF broth is not predictable, this stoi-
chiometric ratio cannot be followed and this necessitates the
addition of extra CC during SmF. Sticking to this principle, it
might be possible to change the physical state of the CC but not
the required concentration (g Lꢁ1). Application of CC nano-
particles (CCNPs) instead of CC microparticles (CCMPs) can be
an interesting and noble approach to experiment with. Nano
dimension imparts new physical properties lacking in the bulk
form (including micro size). Higher surface to volume ratio is
one of the common advantages of nanoform over their micro-
sized counterparts. For CCNPs, apart from higher surface
area, lower viscosity and increased rate of FA neutralization
might be the technical advantages over CCMPs. There has been
no prior study in this sense but studies have been carried out on
the advantages of using small sized CC in acid neutralizing
kinetic studies.21–23
Services (ARS) culture collection, IL, USA. The inorganic salts
(CaCl2, Na2CO3, KH2PO4, MgSO4$7H2O, ZnSO4$7H2O, FeSO4-
$7H2O and CaCO3), urea and dextrose (dextrorotatory glucose)
used in this study were of analytical grade (with min 99.5%
purity) and purchased from Fisher Scientic (Ottawa, Ontario,
Canada).
2.2 Culture and maintenance of Rhizopus oryzae 1526
For cultural practices (revival, sub-culturing and regular main-
tenance) of R. oryzae, the procedure mentioned by Das et al.
(2014) was followed.15 Briey, PDA medium and incubation
ꢂ
conditions of 37 ꢀ 1 C for 4 days were used for revival. Spor-
angiospores were collected by spread plate method aer incu-
bating 37 ꢀ 1 ꢂC for 72 h. Mycelium free spore suspension was
ꢂ
kept at 4 C and the glycerol stock (20%) was preserved at ꢁ80
ꢂC. For inoculation, a stock of 1 ꢃ 108 spores per mL was
maintained.
2.3 Media and inoculum preparation
Glucose-basic salts medium consisting of glucose (50 g Lꢁ1),
Apart from the CC associated bottlenecks of FA production, urea (2 g Lꢁ1), KH2PO4 (0.6 g Lꢁ1), MgSO4$7H2O (0.5 g Lꢁ1),
the fermentation products CaC4H2O4 needs attention for easy ZnSO4$7H2O (0.11 g Lꢁ1) and FeSO4$7H2O (0.0088 g Lꢁ1) was
recovery of FA. Conventionally, CaC4H2O4 in broth is simulta- used for spore germination. The medium nal pH (4.6) was not
ꢂ
neously heated (around at 80 C) and acidied (mineral acids adjusted, unless specically indicated. To avoid Maillard reac-
such as HCl or H2SO4) that release FA from CaC4H2O4 and also tion, the two media components: (a) glucose; and (b) urea +
increases the solubility of FA in the broth. However, this salts, were heat sterilized (20 min, 15 psi, 121 ꢀ 1 ꢂC) separately
method is time consuming. The dissolution of FA into water and mixed aseptically at room temperature. For SmF medium
depends on uniform heating and rate of heating. In this regard, preparation, the same procedure was followed but with a higher
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carbon (80 g Lꢁ1 glucose) to nitrogen (0.2 g Lꢁ1 urea) ratio (C : N a refractive index detector (PDA-100 DIONEX, UV, 210 nm).
¼ 400 : 1) was maintained.
2.5 mM methanesulfonic acid (CH3SO3H) at a ow rate of 1 mL
Around 50 mL of sterilized growth medium was inoculated minꢁ1 was used as the mobile phase and column temperature
ꢂ
(2%, v/v) with sporangiospores of R. oryzae in a 250 mL Erlen- was 30 C.
meyer ask and then incubated at 30 C, 200 rpm for 24 h.
ꢂ
2.7 Statistical analysis
2.4 SmF conditions
Data are represented as mean ꢀ SD of three independent
Around 7.9 mL (nal inoculum concentration 5%, v/v) of cell
pellet inoculum was transferred into 500 mL Erlenmeyer asks
containing 142.1 mL of SmF medium. The inoculated asks
were incubated at 25 ꢂC, in a rotary shaker at 200 rpm for
a maximum of 84 h. Heat sterilized CCMPs (5–10 mm) and
CCNPs (190 ꢀ 20 nm) were used as neutralizing agents at 60 g
experiments. Correlations were considered signicant at p <
0.05.
3. Results and discussion
3.1 Characterization of CCNPs
Lꢁ1
.
SEM analysis displayed roughly spherical CCNPs with solid
dense structure. A SEM image of the CCNPs is shown in Fig. 1.
As no stabilizing agent was used during the preparation of
CCNPs, aggregation of CCNPs was observed aer drying of the
sample on SEM grid. The size distribution curve of CCNPs
originating from the zetasizer nano analysis indicated uniform
size distribution of particles with a mean size of 190 ꢀ 20 nm as
shown in Fig. 2. FTIR analysis of CCNPs sample showed the
three distinctive infrared transmission bands corresponding to
their chemical composition. The results are displayed in Fig. 3.
The infrared bands at 1452 cmꢁ1, 871 cmꢁ1 and 714 cmꢁ1
wavenumbers corresponded to the asymmetric stretching
vibration (v3), asymmetric stretching vibration (v2) and
symmetric stretching vibration (v4) of CO3, respectively; and
also conrmed the calcite crystalline state of CCNPs.34–36 The
BET analysis conrmed the larger specic surface area of
CCNPs as compared CCMPs. The specic surface areas
measured for CCMPs and CCNPs were 3.51 ꢀ 0.02 m2 gꢁ1 and
11.95 ꢀ 0.03 m2 gꢁ1 respectively. Previously, it has been shown
that changes in the specic surface area of CCNPs depend on
preparation method and concentration of the inorganic salts
(CaCl2 and Na2CO3 in the present study).37
2.5 Preparation of CCNPs
The CCNPs were prepared following the method of Ueno et al.
with some modications.31 Briey, 6.50 mL of 5 M CaCl2 and
25 mL of 1 M Na2CO3 were mixed and stirred vigorously for
20 min, followed by addition of 50 mL of d. H2O. The large
CCNPs were rst discarded by precipitation without centrifu-
gation. The suspension was centrifuged (34 000 ꢃ g, 10 min)
and the supernatant and precipitate (CCNPs) was separated.
The prepared CCNPs were then dispersed in d. H2O and freeze-
dried into powder form for further application in SmF.
2.6 Analytical methods
Physicochemical characterization of the prepared CCNPs was
carried out by different instrumental analysis. The size and
shape of the CCNPs was investigated by a scanning electron
microscope (Zeiss EVO® 50 Smart SEM system). For average size
(diameter) measurement, zetasizer nanoZS (Malvern instru-
ments Ltd., UK) was employed. Fourier transform infrared
(FTIR) analysis of the CCNPs was carried with a Nicolet™
iS™50 spectrophotometer (Thermo Scientic) with He–Ne
laser, DTGS detector and KBR beamsplitter with a built-in
Miracle-ATR (Diamond). The ATR-FTIR spectra of the CCNPs
samples were collected and analyzed using OMNIC Specta™
soware. The Brunauer–Emmett–Teller (BET) surface area
analysis of CCMPs and CCNPs samples was carried out with
a Quantachrome, AUTOSORB-1 series model instrument. The
changes in the pHs of FA solution aer addition of CC (CCNPs
or CCMPs) were recorded as a function of time (seconds, s) and
compared for CCNPs and CCMPs. Viscosities of the CC slurries,
CaC4H2O4 solutions and broth samples were measured by
a rotational viscometer (Fungilab, Premium Series, NY, USA). L1
spindle was used with a sample cup volume of 30 mL. Viscosi-
ties were expressed in the unit of cP. Acidied broth samples
were irradiated for different time durations (2–20 minutes) at
the maximum power output (900 W) of the microwave oven. For
comparison, conventional heating method was followed for FA
recovery.32 HPLC analysis was carried out for quantication of
FA in the fermented broth samples. For HPLC sample prepa-
ration, the method of Zhou et al. was followed.33 The technical
details of the HPLC were as follows: System: DIONEX DX500,
3.2 Measurement of pH of FA solutions
Solubility of FA in water changes with temperature and this in
turn, controls the nal pH of the solution. In the present study,
different amounts (g Lꢁ1) of FA were dissolved in water at 25 ꢂC
Fig. 1 SEM micrograph of CaCO3 nanoparticles prepared in the
with an Acclaim OA, 5 mm, (4.6 ꢃ 150 mm) column with present study.
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6.1 g Lꢁ1 at 25 ꢂC, the pH was not affected by further addition of
FA. At half (3.05 g Lꢁ1) of the maximum soluble concentration,
less production of H+ ions from the dissociation of FA caused
higher pH (2.4 ꢀ 0.3). This basic information was extrapolated
for the pH vs. FA production under SmF conditions.
3.3 Formation of CaC4H2O4 and reaction time
The reaction between CC and FA can be represented as follows
in eqn (1):
CaCO3 + C4H4O4 / CaC4H2O4 + H2O + CO2
(1)
This reaction conrms that 20 g (200 mM) of CaCO3 (CCNPs
or CCMPs) neutralizes approximately 23.2 g (200 mM) of FA. In
the present study, CC was used as CCMPs and CCNPs against FA
at above mentioned stoichiometric ratio. The time (s) required
for change in the pH (2.5) of FA aqueous solution was recorded
and the results are shown in Fig. 4. The neutralization reaction
between FA and CCMPs took around 350 ꢀ 19 s and the pH
reached to 6.2. Thereaer, changes in pH were negligible and
indicated completion of reaction. When experimented with
CCNPs, the changes in pH were rapid and a stable pH of 6.23
was achieved aer 190 ꢀ 12 s. Thus, in comparison to CCMPs,
the neutralization reaction time for CCNPs was reduced by 160
s. Particle size of neutralizing agent can have impacts on the
reaction time. Previously, it has been shown that when applied
as antacids; acid-consuming capacity and acid neutralizing
velocity are inuenced by the available specic surface area of
the particles of CC. As the specic surface areas of CCNPs (11.95
ꢀ 0.03 m2 gꢁ1) were larger to CCMPs (3.51 ꢀ 0.02 m2 gꢁ1), the
FA neutralization reaction was faster.21–23
Fig. 2 Size distribution of CaCO3 nanoparticles prepared in the
present study.
3.4 Viscosity measurement
Measurements of viscosities (cP) of different concentrations of
ꢂ
the CCMPs and CCNPs slurries were carried at 25 C and the
Fig. 3 ATR-FTIR spectrum for CaCO3 nanoparticles prepared in the
present study.
and the corresponding pH was measured. The results are pre-
sented in Table 1. The solubility of FA in water was around 6.1 g
Lꢁ1 at 25 C and it corresponded to the pH of 2.1 ꢀ 0.2. Lower
ꢂ
concentration (3.05 g Lꢁ1) increased the pH to 2.4 ꢀ 0.3.
However, above 6.1 g Lꢁ1 FA, the pH remained almost
unchanged. These results were in accordance with the literature
on FA properties.38 Aer reaching the maximum solubility of
Table 1 pH of different fumaric acid aqueous solutions measured at
25 ꢂ
C
Fumaric acid concentration (g Lꢁ1
)
pH
3.05
6.1
10
20
30
2.4 ꢀ 0.3
2.1 ꢀ 0.2
2.2 ꢀ 0.1
2.1 ꢀ 0.1
2.1 ꢀ 0.2
Fig.
4 Effect of CaCO3 particle size on the neutralization time
(seconds) of fumaric acid aqueous solution.
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ꢂ
results are displayed in Table 2. The concentrations (20, 40 and CaC4H2O4 in water at 25 C was 1.56 grams per 100 grams of
60 g Lꢁ1) of CC was selected on the basis of the stoichiometric water.42 Thus, the viscosities measured for CaC4H2O4 in water at
ratio to FA. For CCMPs, the viscosity was 1.39 ꢀ 0.12 cP at 20 g 25 ꢂC actually represented the rheological behaviour of
Lꢁ1 and increased with further increase in CC concentration. At CaC4H2O4 with a solubility of 15.6 g Lꢁ1 in water solvent. The
60 g Lꢁ1, viscosity reached 1.9 ꢀ 0.11 cP. Compared to CCMPs, formation of CaC4H2O4 in water at 25 C was found to be irre-
ꢂ
the viscosities of CCNPs slurries at same corresponding spective of the size (MPs and NPs) of CC. As pH lowered,
concentrations were lower. At 20 g Lꢁ1 of CCNPs concentration, viscosity increase/decrease and formation of CaC4H2O4 in water
the viscosity was 1.15 ꢀ 0.1 cP and at the next two applied at 25 ꢂC during FA production should follow the same stoi-
concentrations (40 and 60 g Lꢁ1), the viscosities recorded were chiometric ratio, the information on the viscosities of CaC4H2O4
1.48 ꢀ 0.15 cP and 1.72 ꢀ 0.1 cP, respectively. The lowering of solutions was vital before real SmF application of CCNPs.
viscosity of CCNPs slurries can be discussed on the ground of
general acceptance of solid particle properties vs. viscosity. It has
been shown that small particles generate larger inter-particle
free space that helps in lowering viscosity. For larger particles,
3.5 SmF with CCMPs and CCNPs and effects on FA
production
due to the greater inertia of interaction, more energy is required It is a consensual view that broth viscosity can inuence the
to momentarily accelerate or retard. This energy accounts for the productivity of FA during SmF.43 Previous studies have conrmed
extra viscosity.39,40 Moreover, recently it has been proven that that CC slurries (concentration dependent) increase broth
CCNPs can enhance surface hydrophobicity and decreases the viscosity which leads to oxygen transfer problem throughout the
yield stress and viscosity.41 Based on these theories, it can be broth.9,10 In an aerobic process such as FA production, high
ascertained that lowering of viscosity of CCNPs slurries, as was viscosity of CC slurry can lower FA production. Meanwhile, pellet
found in the present study, was pertinent.
morphology of FA producing strains is considered as the most
Apart from CCMPs and CCNPs slurries, viscosities were also favourable form that allows easy mass, heat and oxygen transfer
measured for the reaction products (i.e. calcium fumarate, during SmF. Thus, SmF with pellet rules out morphological
CaC4H2O4) formed aer the reactions between different applied interference in FA production. To practically correlate all these
concentrations of CCMPs and CCNPs and FA in a stoichiometric vital factors to FA production prole under real SmF conditions,
ratio. As shown in Table 2, the reaction product CaC4H2O4 viscosities of different samples were measured before and during
formed from the reaction between 20 g Lꢁ1 of CCMPs + 23.2 g SmF. The obtained results are shown in Table 3. The glucose
Lꢁ1 of FA showed a viscosity of 2.2 ꢀ 0.28 cP at 25 ꢂC. At double basic-salt medium with around 80 g Lꢁ1 of glucose showed
stoichiometric concentrations of CCMPs and FA, the viscosity viscosity of 1.25 ꢀ 0.12 cP at 25 ꢂC. This value was in accordance
increased to 2.67 ꢀ 0.21 cP. For the reaction product of 20 g Lꢁ1 with previous ndings.44 Aer addition of 5% pre-cultured cell
of CCNPs + 23.2 g Lꢁ1 of FA, the viscosity recorded was 2.18 ꢀ pellets (inoculum), the viscosity of the medium changed
0.19 cP and increased to 2.58 ꢀ 0.26 cP at 40 g Lꢁ1 of CCNPs + marginally. However, mixing of CCMPs (60 g Lꢁ1) considerably
46.4 g Lꢁ1 of FA stoichiometric ratio. Overall, there was no effect increased the viscosity to 3.27 ꢀ 0.18 cP. In case of CCNPs,
of the size of CC on the viscosity of the reaction product. mixing of same amount to the medium increased the viscosity
Previous study conrmed that the maximum solubility of upto 2.98 ꢀ 0.1 cP. In the rst 24 h of SmF, the viscosity changes
were monitored aer every 12 h. For CCMPs, the initial (0 h)
viscosity lowered from 3.27 ꢀ 0.18 to 3.1 ꢀ 0.08 cP aer 12 h of
SmF. It further decreased to 2.28 ꢀ 0.2 cP aer 24 h of SmF. From
Table
2 Viscosities of different samples of calcium carbonate
measured at 25 ꢂCa
Sample analyzed
Water
24–48 h, the viscosity increased again and reached 2.41 ꢀ 0.18 cP.
There was a marginal increase (0.14 cP) from 48–72 h. CCNPs
also showed similar changes of viscosity during SmF. However,
in the rst 12 h of SmF with CCNPs, the change in the viscosity
was around 0.6 cP and this was much higher than 0.17 cP, as was
obtained with CCMPs. The decrease in the viscosity during zero
to 12 h of SmF for both CCMPs and CCNPs was caused by the
consumption of CC by FA. This trend continued upto 24 h of
SmF. Aer reacting with FA, CCMPs and CCNPs formed
CaC4H2O4 and this product has higher solubility (15.6 g Lꢁ1) as
compared to CCMPs or CCNPs (6.1 g Lꢁ1) at 25 ꢂC. Further
accumulation of CaC4H2O4 caused viscosity increase from 24–72
of SmF for both CCMPs and CCNPs.
Viscosity (cP)
0.85 ꢀ 0.08
1.39 ꢀ 0.12
1.7 ꢀ 0.18
1.9 ꢀ 0.11
1.15 ꢀ 0.1
1.48 ꢀ 0.15
1.72 ꢀ 0.1
2.2 ꢀ 0.28
CCMPs (20 g Lꢁ1
CCMPs (40 g Lꢁ1
CCMPs (60 g Lꢁ1
)
)
)
)
)
CCNPs (20 g Lꢁ1
CCNPs (40 g Lꢁ1
CCNPs (60 g Lꢁ1
)
Calcium fumarate (20 g Lꢁ1 of CCMPs +
23.2 g Lꢁ1 of FA)
Calcium fumarate (40 g Lꢁ1 of CCMPs +
46.4 g Lꢁ1 of FA)
2.67 ꢀ 0.21
2.18 ꢀ 0.19
2.58 ꢀ 0.26
Calcium fumarate (20 g Lꢁ1 of CCNPs +
23.2 g Lꢁ1 of FA)
The production prole of FA obtained with glucose basic-salt
medium and CCMPs and CCNPs, is shown in Fig. 5. In the rst
12 h of SmF, the concentrations of FA were 6.44 ꢀ 1.23 and 5.6 ꢀ
1.8 g Lꢁ1 for CCMPs and CCNPs, respectively. From 12–24 h of
Calcium fumarate (40 g Lꢁ1 of CCNPs +
46.4 g Lꢁ1 of FA)
a
CCMPs, calcium carbonate microparticles; CCNPs, calcium carbonate
nanoparticles; FA, fumaric acid.
SmF, as compared to FA concentration (11.34 ꢀ 2.4 g Lꢁ1
)
obtained with CCMPs, production of FA was higher (17.89 ꢀ 3.3
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Table 3 Viscosities of different samples at various time intervals (h) of submerged fermentationa
Viscosity (cP)
Sample analyzed
0 h
12 h
24 h
48 h
72 h
Glucose basic salt medium
Glucose basic salt medium + 5% (v/v) cell
pellets
1.25 ꢀ 0.12
1.28 ꢀ 0.05
NA
NA
NA
NA
NA
NA
NA
NA
Glucose basic salt medium + 5% (v/v) cell
3.27 ꢀ 0.18
2.98 ꢀ 0.1
3.1 ꢀ 0.08
2.28 ꢀ 0.2
2.41 ꢀ 0.18
1.95 ꢀ 0.14
2.55 ꢀ 0.13
2.11 ꢀ 0.15
pellets + CCMPs (60 g Lꢁ1
)
Glucose basic salt medium + 5% (v/v) cell
2.38 ꢀ 0.19
1.65 ꢀ 0.23
pellets + CCNPs (60 g Lꢁ1
)
a
NA, not applicable; CCMPs, calcium carbonate microparticles; CCNPs, calcium carbonate nanoparticles.
g Lꢁ1) with CCNPs. This corresponded to the increase of volu- CCNPs were comparable, it strongly suggested that the meta-
metric productivity from 0.47 g Lꢁ1 hꢁ1 to 0.74 g Lꢁ1 hꢁ1. From bolic performances of the used fungus R. oryzae 1526, was not
24–36 h of SmF, the FA product yields for CCMPs and CCNPs inuenced by the replacement of CCMPs with CCNPs. It also
were almost similar (around 12.33 and 13.11 g Lꢁ1, respec- ruled out any associated nano-toxicity of CCNPs on the fungus
tively). However, the most productive phase of FA was in or in other words, application of CCNPs was safe for this fungus
between 36 and 48 h that resulted in around 31.55 and 30.65 g mediated FA production through SmF technology. Nanoparticle
Lꢁ1 of FA for CCMPs and CCNPs, respectively. Overall, FA concentrations such as 60 g Lꢁ1 used in the form of CCNPs, was
production was found to be higher from 12–60 h of SmF with an exceptionally higher concentration to be tolerated by any
CCNPs. The application of CCNPs as neutralizing agent in the microorganisms including R. oryzae 1526. From the FA
SmF production of FA has not been carried out earlier and thus production proles obtained with CCMPs and CCNPs, it was
the discussion needs to be made for higher FA yield with CCNPs concluded that volumetric productivity of FA was enhanced by
can only be referred to the other supporting experimental CCNPs, but not the nal FA concentration.
results obtained during the present investigation. As shown
earlier, CCNPs slurries were found to be less viscous to CCMPs
slurries at same applied concentration (60 g Lꢁ1) and temper-
3.6 MWI vs. conventional heating in FA downstream
processing
ature (25 ꢂC). As FA production is inuenced by the broth
viscosity during the entire time frame of SmF, there was high
possibility that FA production was favoured by the use of
CCNPs. Moreover, as the highest FA concentrations (67.34 ꢀ 2
and 66.92 ꢀ 2.7 g Lꢁ1, respectively) obtained with CCMPs and
The two well-known FA recovery methods proposed by Gang
et al. (1990) and Dang et al. (2009) make use of heat energy and
CC neutralizing properties of mineral acids (such as HCl or
H2SO4).32,45 Acidication neutralizes unused CC, while heating
increases the solubility of both FA and CaC4H2O4. Technically,
both methodologies facilitate the separation of soluble FA from
the solid mass (fungal biomass and CaSO4). However, due to the
technical advantages such as no need of special heating
equipment and lower consumption of heat energy, the FA
recovery method of Dang et al. (2009) is mostly followed.32 In the
present study, MWI was intended for application in FA recovery.
For a comparative study, fermented broth samples were pro-
cessed through conventional method and studied parameters
were recorded.32 The results are presented in Table 4. SmF broth
samples from both CCMPs and CCNPs neutralizing agents
mediated SmF of FA production were considered for this
investigation. The broth samples were also heated under MWI
and required time was monitored. For both conventional and
MWI heating, acidication and pH adjustment were similar.
For MWI heating, an experiment was carried out on the opti-
mization of minimum time required for maximum FA recovery
from the broth. A time range of 2–20 min was applied and
correlated with the recovered FA concentration (g Lꢁ1) (data not
shown). From this optimization study, it was concluded that
under MWI heating conditions, a minimum of 10 ꢀ 1 minute
was required for maximum FA recovery. Although, the
Fig. 5 Production profile of fumaric acid under submerged fermen-
tation conditions of 25 ꢂC, 200 rpm and 84 h with CaCO3 micro and
nanoparticles as neutralizing agent.
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Table 4 Comparison of the conventional and microwave irradiation methods applied for fumaric acid downstream processinga
Conventional method
Broth with CCMPs
Microwave irradiation
Broth with CCMPs
Parameter studied
Broth with CCNPs
Broth with CCNPs
Heating temperature (ꢂC)
H2SO4 concentration
Final pH
80 ꢀ 1 ꢂ
C
80 ꢀ 1 ꢂ
C
95 ꢀ 1 ꢂ
C
95 ꢀ 1 ꢂ
C
5 N
5 N
5 N
5 N
1.0 ꢀ 0.1
1.0 ꢀ 0.1
1.0 ꢀ 0.1
1.0 ꢀ 0.1
Heating time required (min)
28 ꢀ 2 (until clear)
67.55 ꢀ 2
27 ꢀ 2 (until clear)
66.45 ꢀ 2.7
10 ꢀ 1
10 ꢀ 1
FA (g Lꢁ1) recovered
65 ꢀ 2.56
68.5 ꢀ 1.7
a
CCMPs, calcium carbonate microparticles; CCNPs, calcium carbonate nanoparticles.
minimum MWI heating time required for the visual manifes- carbonate nanoparticles enhanced the volumetric productivity
tation of broth clarity was around 6–7 min, this resulted in lower (from 0.47 g Lꢁ1 hꢁ1 to 0.74 g Lꢁ1 hꢁ1) of fumaric acid in the
FA recovery for both CCMPs and CCNPs (data not shown). Thus, rst 12–24 h of fermentation. Nanoformulation of calcium
on an average, 10 min MWI heating time was found to be carbonate did not exhibit toxicity towards the fungus Rhizopus
optimum for maximum recovery of FA (65 ꢀ 2.56 g Lꢁ1 and 68.5 oryzae 1526. Microwave irradiation shortened the time
ꢀ 1.7 g Lꢁ1 for CCMP and CCNPs, respectively). The compara- required for the maximum recovery of fumaric acid from 28 to
tive outcome of this study conrmed the lower time consuming 10 min.
FA recovery through MWI. The heating time was almost lowered
by 2.8 folds without affecting the recovery of FA. This was
conclusive for both CCMPs and CCNPs. The results also sug-
gested that application of CCNPs in FA production did not
Acknowledgements
Financial support from the Natural Sciences and Engineering
Research Council of Canada (NSERC, Discovery Grant 355254),
inuence the downstream processing of FA under conventional
or MWI heating conditions. With higher or lower broth
volumes, heating time will be changed accordingly. It was
concluded that irrespective of CCMPs and CCNPs, the well-
known technical advantages of MWI over conventional heat-
ing resulted in FA recovery in lesser time. A rapid and homog-
enous heating of the broth and dissolution enhancement of FA
under MWI, shortened the FA recovery time by almost 2.8
folds.26–28 MWI energy heated the broth samples directly, while
under conventional heating condition, heat energy rst had to
conduct through the walls of the Erlenmeyer asks containing
the broth samples. It delayed the heating of the broth samples.
In general, MWI heating takes place through ionic conduction
and dipole rotation.27 In the present study, presence of polar
groups such as –COOH and pC]O (in FA) and –OH (in water) in
the broth samples, accelerated (rotational alignment) the
conversion of MWI into heat energy. Moreover, the high
dielectric constant value (80.37, at 25 ꢂC) of water enhanced the
MWI efficacy of heating the fermented broth in a shortened
time.
`
MAPAQ (No. 809051) and Ministere des Relations Inter-
´
nationales du Quebec (Quebec-Vietnam 2012–2015) are
sincerely acknowledged. FQRNT merit scholarship program for
foreign student (MELS) is also acknowledged for nancial
assistance to Mr Ratul Kumar Das.
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