Economical production, optimization, and

characterization of bio-vanillin (4-hydroxy-3-

methoxybenzaldehyde) using lignocellulosic waste
Veena Paul
Banaras Hindu University
Aparna Agarwal
Delhi University
Abhishek Dutt Tripathi (  [email protected] )
Banaras Hindu University

Research Article

Keywords: bio-vanillin, coconut coir waste, lignocellulosic waste, ferulic acid, Taguchi, scale-up, ESI-MS-
QTOF

Posted Date: April 27th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2824027/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License

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Abstract
Coconut coir waste is a rich lignocellulosic biomass. The coconut coir waste generated from temples is
resistant to natural degradation, and its accumulation causes environmental pollution. Ferulic acid, a
vanillin precursor, was extracted from the coconut coir waste by hydro-distillation extraction. The
extracted ferulic acid was used for bio-vanillin synthesis by Bacillus aryabhattai NCIM 5503 under
submerged fermentation. The process variables were optimized using the Taguchi design of the
experiment. A 27 set of experimental trials was constructed with the seven most influential factors on bio-
vanillin biosynthesis at three levels for the proposed experimental design. The bio-vanillin yield obtained
was processed with Qualitek-4 software at bigger is better as a quality character and obtained a specific
combination of factors with a predicted bio-vanillin production of 644.61 ± 0.01 mg/L. The optimal
combinations of factors obtained from the proposed DOE methodology were further validated. The result
revealed an enhanced bio-vanillin yield of 29.23-fold (from 495.96 ± 0.01 to 640.96 ± 0.02 mg/L) from its
unoptimized condition. The extracted bio-vanillin was characterized using High-performance liquid
chromatography, Fourier transform infrared, and Electrospray Ionisation Mass Spectrometry Quadrupole
time-of-flight.

1. Introduction
The environment is a protective shelter for all living being. Every human action results in the creation of
waste, which eventually lowers the standard of human health and has an alarming impact on the
environment. Wastes are substances or things that are thrown away, intended to be thrown away, or
forced to be thrown out by the rules of national laws (Yadav et al., 2015). India is a nation that celebrates
a variety of festivities throughout the year, which eventually results in the production of solid waste. This
fraction of garbage is frequently disregarded and needs careful consideration. Many of us refrain from
disposing of flowers and other materials used for prayers in the trash because of our religious convictions;
instead, we wrap them in plastic bags and discard them into water bodies. For example, Varanasi, one of
the most sacred places in the nation, lacks a policy for disposing of the tons of waste generated by its
numerous temples. In the city of temples, waste is consistently left behind and weighs between 3.5 and 4
tonnes every day (Ranjan and Goel, 2022). The majority of temple waste is made up of organic materials,
including flowers, leaves, coconut coir, incense stick pieces, fruits, etc. Though these wastes are
biodegradable, they may lead to water and soil pollution (Singh et al., 2017). Varanasi, a city in Uttar
Pradesh, India, is considered the oldest living city in the world and one of the most religious cities in the
country. Approximately 23000 temples are there in Varanasi. Because the city is situated along the banks
of the River Ganges, a large amount of floral waste is discarded there, harming the river’s environment and
serving as a breeding ground for numerous microbial diseases (Singh et al., 2017). Coconut is one of the
extensively used offerings in the temples. After removing its edible portion, the coir is generally thrown
into the dustbins. This coconut coir ultimately finds its way into some water bodies or open areas/places,
causing environmental problems.

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There is a vast scope for using coconut coir as they are rich in lignocellulosic biomass (Lebedeva et al.,
2022). Previous reports describe the different approaches to converting coconut coir waste’s
lignocellulosic biomass to valorize into value-added aromatics (Mankar et al., 2022; Rejani and
Radhakrishnan, 2022). Lignocellulosic biomass is an inexpensive, zero-waste raw material (Harshvardhan
et al., 2017). Lignin is an aromatic phenolic compound comprised of highly stable phenylpropanoid units
(Xu et al., 2018). This insoluble polymer is known to be degraded by microorganisms that produce
aromatic aldehydes like vanillin (Reshmy et al., 2022). Ferulic acid can be obtained by hydrolysis of lignin
(Fache et al., 2016). Rice bran, wheat bran, rice straw, sugar beet pulp, and sugarcane bagasse are
common agro-waste abundant in lignin and ferulic acid, a crucial precursor for microbial synthesis of
vanillin (Chakraborty et al., 2017; Lesage-Meessen et al., 1999; Nurika et al., 2020; Pattnaik et al., 2022).
Ferulic acid is the most abundant hydroxycinnamic acid. Covalent bonds with lignin and polysaccharides
are primarily in the cell walls (dos Santos Barbosa et al., 2008; Rejani and Radhakrishnan, 2022). Ferulic
acid is an essential precursor for bio-vanillin production (Banerjee and Chattopadhyay, 2019; Paz et al.,
2016).

Taguchi design of experiment (DOE) approach facilitates identifying the influence of individual factors
and establishing the relationship between variables and operational conditions (Pignatiello Jr, 1988). The
application of Taguchi DOE methodology for microbial flavor production is superior to other statistical
approaches.

The present investigation deals with the methodological application of Taguchi DOE to optimize
submerged culture conditions for bio-vanillin production by Bacillus aryabhattai NCIM 5503 using ferulic
acid extracted from coconut coir.

2. Material and Methods

2.1 Microorganism and reagents
B. aryabhattai NCIM 5503 was procured from NCIM, NCL (Pune, Maharashtra, India). Coconut coir was
collected from outside of temples in Varanasi (U.P., India). Vanillin (99%) and ferulic acid were procured
from Sigma-Aldrich (USA), 2-thiobarbituric acid (99%), HPLC reagent, and HCl (35–38%) of analytical
grade was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India).

2.2 Ferulic acid extraction from coconut coir

Before extraction, the coconut coir was washed under running water, dried for 72 hours at 50 ℃ in a tray
dryer, and ground into fine powder. Hydro-distillation method was performed by mixing 50 g coconut coir
with 500 mL of distilled water and digesting at 100 ± 2 ℃ for 1 hour. The digest was then filtered and
acidified (pH 2). Then transferred to an Erlenmeyer flask, and precipitated lignin was removed.
Subsequently, the supernatant was transferred to a separating funnel and extracted with ethyl acetate. An
equal quantity of ethyl acetate was added to an equal volume of the supernatant. It was shaken and

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centrifuged for 15 min at 4000 rpm. All the organic fractions were collected and concentrated using a
rotary vacuum evaporator.
2.3 Bioconversion experiment
B. aryabhattai NCIM 5503 was maintained in nutrient broth in g/L (5 Peptone; 5 NaCl; 2 Yeast extract; 1
Beef extract) at 30 ℃ for 48 h. After two subcultures, 1% (v/v) inoculum was transferred in 250 mL
Erlenmeyer flask containing 50 mL nutrient broth, then cultivated overnight and used as the final inoculum
for the bioconversion process.

A hundred milliliters of production media were prepared by adding 1% (w/v) fructose and beef extract as
carbon and nitrogen sources. The production media was supplemented with 5% of extracted ferulic acid
and 1 mL trace metal solution in g/L (0.368 CaCl2.2H2O; 0.624 CuSO4.5H2O; 0.604 FeSO4.7H2O; 0.594
MnSO4.4H2O; 0.422 ZnSO4.7H2O; 0.788 CoCl2.6H2O; 0.696 NaMoO4). The pH of the media was
maintained at pH 7 ± 0.2. The inoculum 1% (v/v) B. aryabhattai NCIM 5503 was added as inoculum and
incubated at 30 ℃ for 96 h. The bio-vanillin yield was determined using the thiobarbituric acid (TBA)
method (He et al., 1999).

2.4 Statistical optimization of process variables using

Taguchi Design of Experiment (DOE)
The optimization methodology comprised four phases: design of experiments, performing experiments,
analysis, and validation. With the help of the design of experiments, Taguchi methodology generates
many experiments with reduced errors, referred to as orthogonal array (OA).

Phase I: Design of experiments (DOE): The initial stage in phase I was to decide the different parameters
to be optimized in the nutrient medium that significantly affects bio-vanillin production. The effect of
various carbon sources such as glucose, sucrose, fructose, sorbitol, lactose, xylitol, and mannitol was
studied on bio-vanillin yield (Paul et al., 2021b). In addition, the effect of various nitrogen sources, such as
ammonium sulfate, peptone, beef extract, yeast extract, and urea, on bio-vanillin yield was also studied
(Paul et al., 2021b). Based on the experimental results, the seven parameters significantly influencing the
bio-vanillin yield were chosen for the current Taguchi DOE study to optimize the medium components. The
significant influence of selected parameters, including fructose, beef extract, pH, temperature, agitation,
ferulic acid concentration, and trace metal solution, on bio-vanillin production, was studied. In the present
study, OAs of L27 (indicating 27 experimental trials) were constructed using DOE of selected parameters
with each of the three assigned levels, as represented in Table 1.

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 Table 1
– The variables and their levels used
SN Factor Level 1 Level 2 Level 3

1 Carbon (g) 0.5 0.75 1

2 Nitrogen (g) 0.5 0.75 1

3 pH 7 9 12

4 Temperature (℃) 25 30 35

5 Agitation (rpm) 100 150 200

6 Trace Metal (mL) 0.5 1 1

7 Ferulic Acid (mL) 1 1.5 2

Phase II: Submerged fermentation experiments: The pure culture of B. aryabhattai NCIM 5503 used as
inoculum of size 1% (v/v) grown for 96 h was inoculated into 100 mL medium of each designed trial and
kept for fermentation. Submerged fermentation experiments of 27 designed trials were performed for bio-
vanillin production in triplicate (mean ± standard deviation). Bio-vanillin production was measured by the
colorimetric method of TBA assay.

Phase III: Analysis of experimental data: The obtained experimental data regarding bio-vanillin yield was
analyzed using Qualitek-4 (Nutek Inc., MI, USA) with “bigger is better” performance quality. Analysis of
variance (ANOVA) study determines the influence of individual parameters on the bio-vanillin yield,
optimum contribution, and severity index (SI) of the experimental trials. The following equation was used
to measure the performance of the Taguchi method by calculating each experimental trial’s signal-to-
noise ratio (S/N).

27

2
S/N = −10log ∑(1/Y )/n

n=1

Where, Y is the experimental value obtained for each trial and n is the total number of trials.

Phase IV: Validation: From the results of bio-vanillin production, statistically significant optimum levels of
the selected parameters or process variables were determined by analysis of experimental data using
analysis of variance (ANOVA), severity index (SI), and main effects of individual parameters. Based on
optimized parameters and their assigned levels, further validation was performed in a 500 mL shake flask
containing a 100 mL optimized medium.

2.5 Scale-up study of optimized condition for bio-vanillin

production

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The optimized parameters were used for further scale-up and validation in the 7.5 L bioreactor
(BioFlo/Celligen 115, New Brunswick, USA) using a 3 L medium as a working volume in batch cultivation
mode. The optimum growth conditions of 30 ℃ and pH 9 were maintained by a pH-mV controller’s auto
control of acid or base addition (Mettler-Toledo, USA). The Dissolved oxygen (DO) at 40% saturation was
maintained by controlling the speed of the agitator and air flow rate and monitored by DO probe (Mettler-
Toledo, USA). The reactor was sterilized in an autoclave at 121°C for 20 min. The 10% (v/v) culture of B.
aryabhattai NCIM 5503 48 h old was inoculated to the medium in the bioreactor, and 20 mL samples were
withdrawn at regular interval of 24 h. The samples were centrifuged at 5009 g at 4°C for 20 min, and the
bio-vanillin yield was determined by TBA assay. The relationship between the bio-vanillin production and
growth of B. aryabhattai over the time period was described using the Luedeking-Piret model as expressed
by the following equation.

dp dX
= α + βX
dt dt

Where, p is product bio-vanillin concentration, X is cell concentration, and α and β are growth and non-
growth-related constants.

2.6 Extraction of bio-vanillin

The bio-vanillin from the fermented media was recovered by the solvent-extraction method (Chakraborty
et al., 2017). The fermented media was centrifuged for 10 min at 10,000 rpm at 10 ℃ to remove the
biomass. The supernatant was acidified to pH 2 using 6 N HCl to extract bio-vanillin. Phenolic
components were extracted by ethyl acetate. Then further concentrated using a rotary vacuum evaporator
(IKA RV-10 Digital, Karnataka, India).
2.7 Characterization of extracted bio-vanillin
Standard vanillin solutions for HPLC (High-performance liquid chromatography) analysis were prepared in
ranges of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL filtered using 0.2 µm membrane filters. The filtered extracts
were injected into the HPLC (Waters India, New Delhi, India) fitted with silica reversed-phase C-18 column
(25 cm × 4.6 mm × 5 µm) (Waters India, New Delhi, India). The mobile phase included Solvent A
(acetonitrile) and Solvent B (0.2% acetic acid) in a ratio of 60:40. The flow rate was 1 ml/min, injection
volume was 20 µL, and the maximum run pressure was 3000 psi. Moreover, UV detection was carried out
at 280 nm. Using Empower 2 Software (Waters India, New Delhi, India), computerized integration was
carried out to calculate specific compound peak areas at specific retention times. For the analysis of bio-
vanillin in broth, 500 µL of aliquots were centrifuged at 5009 g for 20 min. The supernatant was freeze-
dried, dissolved in 500 µL of methanol, and filtered using 0.2 µm filters before injecting into the HPLC
system. Bio-vanillin in the extracted broth was characterized by comparing sample retention time with the
standard solution (Paul et al., 2021b).

The Fourier Transform Infrared (FTIR) analysis to determine functional groups of extracted bio-vanillin
was carried out using a Perkin Elmer FTIR spectrometer equipped with a Universal KBr accessory. The

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sample was prepared by embedding 1% bio-vanillin in potassium bromide (KBr) pellets. The spectra were
recorded within the absorption band in a range of 4000 − 400 cm− 1 with a resolution of 4 cm− 1 at room
temperature.

The HPLC system Agilent 1200 was coupled to a hybrid mass spectrometer quadrupole-time of flight
Agilent Accurate Mass QTOF LC-MS via an electrospray ionization source (ESI) with JetStream
technology. Mass spectra were recorded in the negative ionization mode over a mass range from m/z 500
to 1000. Ultrahigh pure nitrogen (N2) was used as the collision gas and high-purity nitrogen (N2) as the
nebulizing gas. Mass spectra were recorded in the negative ionization mode. The scan source parameters
were capillary voltage, 3.5 kV, and fragmentor, 100. The ESI JetStream parameters were: nitrogen pressure
and flow rate on the nebulizer at 45 psi and 10 L/min, respectively, with a drying gas temperature of
350°C; sheath gas temperature, 250°C; sheath gas flow, 6 L/min; and with MS/MS collision energies set at
20 V. The metabolites were identified using Mass Data Bank.
2.8 Statistical analysis
Data from each experiment were statistically analyzed using SPSS software (Version 23). One-way
analysis of variance (ANOVA) methods were applied to analyze data. Analysis of Variance (ANOVA)
single-factor replication was performed for individual factors at different levels and compared at a
significance level of p < 0.05.

3. Results and Discussion

3.1 Statistical media optimization for bio-vanillin production
The bio-vanillin production utilizing ferulic acid extracted from coconut coir by B. aryabhattai NCIM 5503
under SMF was optimized using Taguchi DOE methodology. The designed experiments showed
significant variation in the bio-vanillin production from 406.06 ± 0.02 to 628.06 ± 0.01 mg/L, as shown in
Table 2. The average effect of parameters with the assigned levels and their interaction with bio-vanillin
yield are given in Table 3. The effect of individual parameters with the assigned level on bio-vanillin yield
was studied. Based on the ANOVA results, temperature and pH with level 2 have significantly influenced
the bio-vanillin yield. The relative effect of individual parameters on bio-vanillin yield was determined by
the difference between level 2 and level 1 (L2-L1), as represented in Table 3. The positive average value
shows an expansion underway as it moves from level 1 to level 2. At the same time, the negative esteem
demonstrates group decline amid the course from L1 to L2. The bigger the difference, the higher the effect
on bio-vanillin yield. It was deduced from Table 3 that among the studied factors, temperature showed a
stronger influence (3.05) on the bio-vanillin yield, followed by pH (0.243) and ferulic acid (0.192).

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 Table 2
×
– L27 (3 13) orthogonal array of designed experiments for vanillin production by Bacillus aryabhattai
Trial Carbon Nitrogen pH Temperature Agitation Trace Ferulic Vanillin S/N
No. (g) (g) (℃) (rpm) Metal Acid Yield Ratio
(mL) (mL) (mg/L)

1 0.5 0.5 7 25 100 0.5 1 416.03 52.381

± 0.01

2 0.5 0.5 7 25 150 1 1.5 426.07 52.588

± 0.01

3 0.5 0.5 7 25 200 2 2 428.07 52.628

± 0.02

4 0.5 0.75 9 30 100 0.5 1 610.06 55.706

± 0.01

5 0.5 0.75 9 30 150 1 1.5 615.05 55.777

± 0.03

6 0.5 0.75 9 30 200 2 2 628.06 55.959

± 0.01

7 0.5 1 12 35 100 0.5 1 456.05 53.179

± 0.02

8 0.5 1 12 35 150 1 1.5 460.06 53.255

± 0.02

9 0.5 1 12 35 200 2 2 462.04 53.292

± 0.01

10 0.75 0.5 9 35 100 1 2 529.02 54.469

± 0.01

11 0.75 0.5 9 35 150 2 1 470.04 53.441

± 0.02

12 0.75 0.5 9 35 200 0.5 1.5 505.04 54.065

± 0.03

13 0.75 0.75 12 25 100 1 2 410.05 52.255

± 0.03

14 0.75 0.75 12 25 150 2 1 413.02 52.319

± 0.01

15 0.75 0.75 12 25 200 0.5 1.5 406.06 52.17

± 0.02

16 0.75 1 7 30 100 1 2 620.04 55.847

± 0.01

(Values are mean ± standard deviation; n = 3) (Carbon: Fructose; Nitrogen: Beef Extract)
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Trial Carbon Nitrogen pH Temperature Agitation Trace Ferulic Vanillin S/N
No. (g) (g) (℃) (rpm) Metal Acid Yield Ratio
(mL) (mL) (mg/L)

17 0.75 1 7 30 150 2 1 607.01 55.663

± 0.01

18 0.75 1 7 30 200 0.5 1.5 613.01 55.749

± 0.02

19 1 0.5 12 30 100 2 1.5 596.02 55.504

± 0.03

20 1 0.5 12 30 150 0.5 2 556.02 54.901

± 0.04

21 1 0.5 12 30 200 1 1 571.03 55.132

± 0.01

22 1 0.75 7 35 100 2 1.5 477.06 53.57

± 0.01

23 1 0.75 7 35 150 0.5 2 492.01 53.839

± 0.01

24 1 0.75 7 35 200 1 1 468.01 53.404

± 0.02

25 1 1 9 25 100 2 1.5 448.01 53.025

± 0.02

26 1 1 9 25 150 0.5 2 430.02 52.669

± 0.03

27 1 1 9 25 200 1 1 434.02 52.749

± 0.04

(Values are mean ± standard deviation; n = 3) (Carbon: Fructose; Nitrogen: Beef Extract)

Table 3
– Main effects of selected factors.
Factor Level 1 Level 2 Level 3 L2 – L1

Carbon 53.863 53.998 53.866 0.134

Nitrogen 53.901 53.889 53.936 -0.013

pH 53.963 54.207 53.556 0.243

Temperature 52.532 55.582 53.613 3.05

Agitation 53.993 53.828 53.905 -0.165

Trace Metal 53.851 53.942 53.934 0.091

Ferulic Acid 53.775 53.967 53.984 0.192

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On the contrary, the agitation (-0.165) showed a less significant effect on bio-vanillin yield (Table 3). It was
observed that, from the selected parameters, the temperature showed a maximum effect on bio-vanillin
yield. The bio-vanillin production increased with the increase in temperature and pH up to level 2. But its
further increase reduces the bio-vanillin production, as shown in Table 2. The process analysis by Taguchi
DOE L27 OAs provided insights into the influence of interactions between the parameters and severity
index (SI) on bio-vanillin yield, as shown in Table 4. The highest interaction, SI 70.70%, was observed
between carbon and pH (level 1 × 2). However, the least interaction, SI 0.14%, was found between
temperature × trace metal solution. Figure 1 indicates the average effects of individual parameters and
levels on bio-vanillin yield under submerged fermentation. It was observed that bio-vanillin production
increased with the increase in temperature, showing a strong effect. Pictorial representation, Fig. 2 shows
temperature contributed maximally to bio-vanillin production, followed by pH. ANOVA results showed that
temperature (93.156%) has a strong effect on bio-vanillin yield, followed by pH (3.99%), ferulic acid
(0.317%), agitation (0.049%), and carbon source (0.014%). However, due to the experimental and
environmental conditions, other/errors (2.474%) occurred during the optimization of bio-vanillin
production under submerged fermentation by B. aryabhattai NCIM 5503 (Table 5).

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 Table 4
– Estimated interaction of the given factors depicted via severity index for different
factors.
S.N. Interacting Factor Reserved columns S.I (%) Column Level

1 Carbon × pH 1×3 70.70 2 1,2

2 Carbon × Nitrogen 1×2 70.46 3 1,2

3 Nitrogen × Trace Metal 2×6 66.55 4 1,2

4 Nitrogen × Agitation 2×5 64.15 7 1,1

5 Carbon × Agitation 1×5 57.51 4 2,1

6 Nitrogen × Ferulic Acid 2×7 46.34 5 1,2

7 Agitation × Trace Metal 5×6 41.92 3 1,2

8 pH × Agitation 3×5 29.41 6 2,1

9 Agitation × Ferulic Acid 5×7 24.40 2 1,3

10 Trace Metal × Ferulic Acid 6×7 14.72 1 2,3

11 Nitrogen × Temperature 2×4 12.88 6 2,2

12 pH × Trace Metal 3×6 12.45 5 2,2

13 Nitrogen × pH 2×3 10.51 1 2,2

14 pH × Ferulic Acid 3×7 9.69 4 2,3

15 Carbon × Trace Metal 1×6 8.96 7 2,2

16 Carbon × Ferulic Acid 1×7 7.96 6 2,3

17 Temperature × Agitation 4×5 3.31 1 2,1

18 Carbon × Temperature 1×4 3.13 5 1,2

19 pH × Temperature 3×4 3.1 7 2,2

20 Temperature × Ferulic Acid 4×7 1.01 3 2,2

21 Temperature × Trace Metal 4×6 0.14 2 2,3

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 Table 5
– Analysis of variance (ANOVA) of main effects of factors.
Factor DOF Sum of squares Variance F-Ratio Pure Sum Percent

Carbon (g) 2 0.105 0.520 1.066 0.006 0.014

Nitrogen (g) 2 0.009 0.004 0.099 0 0

pH 2 1.938 0.969 19.643 1.839 3.990

Temperature (℃) 2 43.053 21.526 436.249 42.954 93.156

Agitation (rpm) 2 0.121 0.060 1.231 0.022 0.049

Trace Metal (mL) 2 0.044 0.022 0.447 0 0

Ferulic Acid (mL) 2 0.245 0.122 2.485 0.146 0.317

Other Error 12 0.592 0.049 - - 2.474

Total 26 46.11 - - - 100

(Carbon: Fructose; Nitorgen: Beef Extract)

3.2 Optimized process parameters and validation

The optimized parameters with assigned levels and their contributions are presented in Table 6. The
optimized parameters for maximum bio-vanillin production in mg/L were carbon source 0.75 g, nitrogen
source 1 g, pH 9, temperature 30 ℃, agitation 100 rpm, trace metal solution 1 mL, and ferulic acid 2%. The
total contribution from all the parameters was 2.277. The performance distribution of current and
improved conditions is shown in Fig. 3. The temperature was found to be more effective in enhancing bio-
vanillin yield. The experiments were performed in a 100 mL medium to validate the optimized parameters
with optimum levels. The experimental results with the optimized process parameters increased bio-
vanillin production from 495.96 ± 0.01 to 640.96 ± 0.02 mg/L giving 29.23% enhancement, which was
significant and nearly validated the Taguchi methodology (Ashengroph et al., 2010; Tilay et al., 2010).
Lesage-Meessen et al. (1999) reported producing 253 mg/L bio-vanillin using sugar beet pulp. In another
study, Lesage-Meessen et al. (2002) reported a bio-vanillin yield of 767 mg/L using maize in a two-step
process by Aspergillus niger and Pycnoporus cinnabarinus.

Page 12/23
 Table 6
– The optimum levels of selected parameters and their contribution to
vanillin production
Factor Level Description Level Contribution

Carbon (g) 0.75 2 0.088

Nitrogen (g) 1 3 0.027

pH 9 2 0.297

Temperature (℃) 30 2 1.673

Agitation (rpm) 100 1 0.084

Trace Metal (mL) 1 2 0.033

Ferulic Acid (mL) 2 3 0.075

Total contributions from all factors 2.277

Current grand average of performance 495.96 ± 0.01 mg/L

Expected result at optimum condition 644.61 ± 0.01 mg/L

Experimental result at optimum condition 640.96 ± 0.02 mg/L

3.3 Kinetics of bio-vanillin production

The scale-up and validation of bio-vanillin production were performed in batch cultivation mode using
optimized parameters, optimum temperature, pH, controlled agitation, and aeration using a 7.5 L
bioreactor. Figure 4 shows the kinetics of bio-vanillin production under optimized conditions. The dry cell
mass increased from an initial 0.8 g/L to 14.5 g/L after 60 h of fermentation. The Luedeking-Piret model
classified the bio-vanillin yield from B. aryabhattai as mixed-growth associated. Figure 4 represents the
impact of the incubation period on bio-vanillin yield. Under batch cultivation maximum prodigiosin yield
of 736 ± 0.04 mg/L from B. aryabhattai was observed after 72 h. Bio-vanillin yield enhanced up to 72 h. A
further decrease in yield was observed. This may be due to the expression of bio-vanillin-degrading
enzymes in later phases. The specific growth rate (µ), Yp/x, Yp/s, and productivity were 0.28/hr, 0.10 g/L/hr,
0.20 g/L/hr, and 0.008 g/L/hr, respectively. Bio-vanillin production was initiated in the late exponential
phase. It was maximum in the stationary phase as a secondary metabolite (Paul et al., 2021a). The
current finding suggests that bio-vanillin production was mixed growth associated.

3.4 Characterization of extracted bio-vanillin

The HPLC chromatogram of extracted bio-vanillin shows a retention time of 1.44 minutes (Fig. 5). The
purity of vanillin was also assessed. Comparing the sample and standard, a purity of 97% was obtained.
Our study agrees with our previous report (Paul et al., 2021b). In another study, Rana et al. (2013)
investigated the bio-vanillin produced from Bacillus subtilis at a retention time of 2.76 minutes.
Page 13/23
FTIR was used to confirm the presence of extracted flavoring compound as vanillin. The FTIR spectrum of
extracted bio-vanillin is depicted in Fig. 6. The spectra show an absorption band at 2985.50 cm− 1 (C–H)
due to the stretching of the aldehyde group. The fingerprint region of the flavoring compound was
characterized by bands of medium intensity: 1738.91 cm− 1 (C = O), 1373.07, and 1236.27 cm− 1 (C-O-
CH3). The C-C-CHO band was evident at 784.36 cm− 1. The peaks around 848.15 cm− 1 are attributed to the
O-CH3 group. The FTIR spectrum was comparable to vanillin, as given in previous reports (Saeed et al.,
2022).

Figure 7a depicts the ESI-MS chromatogram of standard vanillin. The ESI-MS-QTOF (Electrospray
Ionisation Mass Spectrometry Quadrupole time-of-flight) chromatogram confirms the presence of vanillin
and 4-vinyl phenol due to bioconversion using B. aryabhattai NCIM 5503 (Fig. 7b). Thus, Fig. 7. depicts
that the m/z value of the extracted bio-vanillin was 152.14. This was similar to the m/z value of standard
vanillin.

4. Conclusion
Optimization of bio-vanillin production under submerged fermentation by B. aryabhattai NCIM 5503 was
performed via the Taguchi method of DOE using Qualitek-4. The study aided in the understanding of
individual factor contribution and interaction among factors. Eliminating unwanted factors significantly
reduces the loss during the process, which needs to be met otherwise. The Taguchi (DOE) method has
been employed, and this statistical model predicts a maximum bio-vanillin yield of 644.61 ± 0.01 g/L.
Experiments to verify the model prediction showed a maximum bio-vanillin yield of 640.96 ± 0.02 g/L,
29.23-fold higher than the unoptimized condition. Validation of optimized parameters provides an
optimum set of conditions insensitive to noise factors that can be used in large-scale bioprocess.

Declarations
Acknowledgments

The authors thank DST-INSPIRE, Govt. of India, for providing financial assistance (DST/INSPIRE
Fellowship/2018/IF180477) to this work. The authors gratefully acknowledge R. K. Roy, Nutek, Inc.,
Michigan, USA, for making available the Qualitek-4 software.

Authors Contributions: Veena Paul: Experiment conduction, data compilation and manuscript writing.
Aparna Agarwal: Analytical study, data interpretation and manuscript editing. Abhishek Dutt
Tripathi:Experiment design, data interpretation, manuscript editing andformatting.

Funding Declaration: The funding for present investigation was provided by DST-INSPIRE, Govt. of India,
(DST/INSPIRE Fellowship/2018/IF180477).

Data Availability Statement: The data presented in the manuscript are original and no data has been
copied from any other source.

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Conflict of Interest: There is no conflict of interest among the authors and all the authors mutually agree
to submit the manuscript.

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Figures

Figure 1

Individual factors performance at different levels: (a) carbon; (b) nitrogen; (c) pH; (d) temperature; (e)
agitation; (f) trace metal; (g) ferulic acid.

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Figure 2

ANOVA results showing the percent contribution of each individual factor on vanillin yield using B.
aryabhattai NCIM 5503.

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Figure 3

Variation reduction plot based on current and improved conditions; Normal performance distribution
profiles for vanillin yield with higher improved frequency.

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Figure 4

Scale-up of vanillin yield using B. aryabhattai NCIM 5503: variation of ferulic acid concentration and dry
cell mass with time during batch fermentation.

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Figure 5

HPLC chromatogram of extracted bio-vanillin

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Figure 6

FTIR spectra of extracted bio-vanillin

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Figure 7

ESI-MS chromatogram (a) Standard Vanillin; (b) Extracted Bio-vanillin

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