Microbial conversion of vanillin from ferulic acid extracted from raw coir pith

Article abstract of DOI:10.1080/14786419.2020.1849194

Coir pith, an agro-industrial residue, is resistant to natural degradation, and its accumulation causes environmental pollution. Ferulic acid, a precursor of vanillin, was extracted from the raw coir pith by chemical pre-treatment such as alkaline hydrolysis, acidification, and liquid–liquid extraction method. The obtained ferulic acid (1.2 g/50 g) was analysed using high-performance liquid chromatography (HPLC) and used as a substrate for biotransformation by Aspergillus niger to vanillic acid, which, in turn, was fermented by using Phanerochaete chrysosporium to vanillin. The quantity of vanillic acid detected by HPLC on the third day of incubation was 0.773 g/L, while the optimal yield of vanillin on the subsequent third day of incubation was 0.628 g/L. Thus, the chemical extraction of ferulic acid from coir pith ensued bioconversion into vanillin. These products are highly valuable and economical to be used in industries such as pharmaceuticals, health, cosmetics, and neutraceuticals.

Full text of DOI:10.1080/14786419.2020.1849194

Natural Product Research
Formerly Natural Product Letters
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Microbial conversion of vanillin from ferulic acid
extracted from raw coir pith
Chalikkaran Thilakan Rejani & Sivaramapillai Radhakrishnan
To cite this article: Chalikkaran Thilakan Rejani & Sivaramapillai Radhakrishnan (2020): Microbial
conversion of vanillin from ferulic acid extracted from raw coir pith, Natural Product Research, DOI:
10.1080/14786419.2020.1849194
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1849194
Microbial conversion of vanillin from ferulic acid
extracted from raw coir pith
a,b
b
Chalikkaran Thilakan Rejani and Sivaramapillai Radhakrishnan
a
Department of Biotechnology and Research, KVM College of Engineering and IT, Alappuzha, Kerala,
b
India; Department of Microbiology, Central Coir Research Institute (CCRI), Alappuzha, Kerala, India
ABSTRACT
ARTICLE HISTORY
Coir pith, an agro-industrial residue, is resistant to natural degrad-
ation, and its accumulation causes environmental pollution.
Ferulic acid, a precursor of vanillin, was extracted from the raw
coir pith by chemical pre-treatment such as alkaline hydrolysis,
acidification, and liquid–liquid extraction method. The obtained
ferulic acid (1.2 g/50 g) was analysed using high-performance
liquid chromatography (HPLC) and used as a substrate for bio-
transformation by Aspergillus niger to vanillic acid, which, in turn,
was fermented by using Phanerochaete chrysosporium to vanillin.
The quantity of vanillic acid detected by HPLC on the third day of
incubation was 0.773 g/L, while the optimal yield of vanillin on
the subsequent third day of incubation was 0.628 g/L. Thus, the
chemical extraction of ferulic acid from coir pith ensued biocon-
version into vanillin. These products are highly valuable and eco-
nomical to be used in industries such as pharmaceuticals, health,
cosmetics, and neutraceuticals.
Received 1 June 2020
Accepted 6 November 2020
KEYWORDS
Coir pith; ferulic acid;
vanillic acid; vanillin;
aspergillus niger;
phanerochaete
chrysosporium
1. Introduction
Coir pith, a fluffy thrown out lignocellulosic material obtained during the separation
process of coir fibres, amasses together to form coir pith hillocks (Figure S1). It is a
concerned agricultural waste and environmental pollutant which decomposes
CONTACT Chalikkaran Thilakan Rejani
rejanict.unom@gmail.com
Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2020.1849194.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
C. T. REJANI AND S. RADHAKRISHNAN
gradually in the soil as it has a lower pentosan/lignin ratio 1:0.30 (w/w) (Joachim
929). Coir pith pollutes water bodies by changing the physico-chemical properties,
1
whereas air pollution results because of its light weight dust in air (Prabhu and
George 2002). Traditionally, the coir pith is disposed of by burning which ensues vari-
ous environmental problems such as carbon deposition and atmospheric warming.
Coir pith contains many phenolic compounds such as resorcinol, 4-nitrophenol, cat-
echol, 2-aminophenol, quinol, O-cresol, phenol and 2-chlorophenol (Namasivayam and
Sangeetha 2006). Tannins and phenols of the coir pith leach out into the soil and
make agricultural lands unproductive (Ghosh et al. 2007). It also poses a fire hazard,
health hazard, space problem and disposal problem (Ronald et al. 2010).
Ferulic acid is a phenylpropanoic acid, naturally occurring in plants, which confers
rigidity to the cell wall by cross-linking (Figure S2) with polysaccharides and lignin
(Oliveira et al. 2015; Boz 2016). It is a strong antioxidant and used in numerous indus-
tries especially food, health, cosmetics, and pharmaceuticals. The feasibility to use
agro-industrial wastes rich in ferulic acid is a valuable renewable source for bio-vanillin
production (Banerjee and Chattopadhyaya 2019). Vanillin (4-hydroxy-3-methoxy ben-
zaldehyde) is widely used in foods, beverages, perfumes, and pharmaceuticals (Kaur
and Chakraborty 2013). Numerous reports have established the biotransformation of
vanillin using natural abundant substrates including lignin, phenolic stilbenes, isoeuge-
nol, eugenol, ferulic acid, vanillic acid, sugars, aromatic amino acids creosol, vanillyl
amine, and waste residues (Kaur and Chakraborty 2013). Among various substrates,
ferulic acid is the most excellent precursor of vanillin production (Yan et al. 2016). As
per the Transparency Market Research (2017), global bio-vanillin has been expected to
rise 7.4% from 2017 to 2025, which indicates a great opportunity for the production
of bio-vanillin from natural substrates and agro-residual waste such as coir pith.
Microbes such as Aspergillus niger (A. niger) and Phanerochaete chrysosporium (P.
chrysosporium) possess the potential to convert lignocellulosic biomass into vanillin
(Salleh et al. 2011). Though Ghosh et al. (2007) only indicated the utilization of coir
pith for industrial applications but comprehensive investigation on biotransformation
of coir pith to value added products like ferulic acid and bio-vanillin is still elusive.
Thus, the present study is an attempt to approve coir pith as a cheap and suitable
substrate for ferulic acid and vanillin biosynthesis with the adoption of biotechno-
logical approach.
2. Results and discussion
2.1. Pre-treatment of coir pith and extraction of ferulic acid
Lignocellulosic biomasses are resistant to chemical and biological breakdown, which is
attributed to several factors such as the crystalline structure of cellulose, degree of lig-
nification and structural heterogeneity, and complexity of cell-wall constituents
(Guerriero et al. 2016). To overcome these issues various targeted pre-treatment steps
have been suggested that enhance digestibility and availability of cellulosic and hemi-
cellulosic fractions and remove lignin fraction as well (Kumari and Singh 2018), one of
them is alkaline hydrolysis using NaOH. In the present study, coir pith was introduced
to NaOH to extract ferulic acid, which is linked to the lignin and cellulose. It causes

NATURAL PRODUCT RESEARCH
3
swelling, leading to the increased internal surface area, diminished degree of polymer-
ization and crystallinity, separation of structural linkages between lignin and carbohy-
drates, and disruption of the lignin structure (Fan et al. 1987). Thus, lignin dissolves
out from the raw material, being separated in the form of a liquor rich in phenolic
compounds (Fengel and Wegener 1989; Mussatto et al. 2007). Moreover, hemicellulose
solubilization and hydrolysis can be achieved by either high temperature and short
ꢀ
residence time (270 C, 1 min) or lower temperature and longer residence time (Wright
ꢀ
1
998). The refluxing of hydrolysate in the digestion port at 185 C for 20 min breaks
the ester links between ferulic acid and polysaccharides. After filtration, the filtrated
solution is acidified (pH 2.5) using concentrated HCl to precipitate out lignin (Mussatto
et al. 2007).
Ferulic acid is recovered from the aqueous phase using ethyl acetate (organic solv-
ent) by liquid–liquid extraction method, and amounted to be 78% ferulic acid recov-
ery, and also removal of 95% sugars (Di Gioia et al. 2007). Removal of ethyl acetate is
carried out by rotar vacuum evaporation, and crystallized ferulic acid is obtained. In
the present study, a similar complex pre-treatment process was adopted to extract
ferulic acid from raw coir pith. The amount of ferulic acid extracted from coir pith
(50 g) was 1.2 g. high-performance liquid chromatography (HPLC) chromatogram
clearly showed retention time (Table S1) of the ferulic acid sample obtained from coir
pith (4.460) (Figure S3(b)) is in accordance with the standard ferulic acid (4.484) (Cas
No: 1135–24–6, Sisco Research Laboratories Pvt. Ltd., India) (Figure S3(a)). The amount
of ferulic acid obtained from coir pith (1.2 g/50 g) in the present study is higher than
the quantity acquired (0.5–1.0 g/100 g) by using raw wheat straw and sugar beet pulp
(
Thibault et al. 1998). The content of ferulic acid was 1.0–1.2 g/100 g produced from
raw palm oil, wheat straw and wheat bran involving the enzymatic procedures
Lesage-Meessen et al. 1996), and 13.6 g/100 g from rice bran oil (Zheng et al. 2007).
(
The differences of extracted ferulic acid from various lignocellulosic sources could be
attributed to the different extraction conditions of their raw materials.
2.2. Microbial conversion of ferulic acid to vanillic acid
The optimum production of crude vanillic acid from ferulic acid recorded 0.7726 g/L
on the 3rd day of incubation, though the yield decreased on the subsequent days.
The retention time (Table S2) in HPLC chromatogram for sample (4.238) is in accord-
ance with standard vanillic acid (4.207) (Cas No: 121–34–6, Sisco Research Laboratories
Pvt. Ltd, India) (Figure S4(a,b)) which confirmed the presence of vanillic acid. The
amount of crude vanillic acid obtained from ferulic acid is 77.26% which is more than
the amount of vanillic acid (63.5%) produced from rice bran oil using A. niger (Zheng
et al. 2007) and approximately 60.5% produced from sugar beet pulp (Lesage et al.
1999). The comparative analysis performed using data obtained from different sources
of various microorganisms are known to possess the capacity to degrade ferulic acid
to vanillin (Lesage-Meessen et al. 1996; Hansen et al. 2009; Di Gioia et al. 2011). In
addition, microbially-produced vanillin, also considered as natural vanillin, carries
supremacy over synthetic vanillin and thus the followed pathway is of utmost import-
ance in the biosynthesis of vanillin (Yang et al. 2013).

4
C. T. REJANI AND S. RADHAKRISHNAN
In the present work, we selected micro florae A. niger and P. chrysosporium, both
are useful in the bioconversion process of lignocellulosic materials (Ruggeri and Sassi
003). A. niger utilizes ferulic acid as a sole source of carbon and energy for its growth
2
and proliferation (Gunnarsson and Palmqvist 2006), whereas P. chrysosporium produces
lignin-degrading enzymes (Bosco et al. 1999). During the biotransformation of ferulic
acid by A. niger, the biosynthesis of vanillin was nil which indicates the occurrence of
the reaction via a propenoic chain degradation to vanillic acid (Lesage-Meessen et al.
1996; Motedayen et al. 2013). A similar route for ferulic acid degradation has been
reported using A. niger (Baqueiro et al. 2010). Vanillic acid and vanillin are catabolic
products of ferulic acid, which in turn, have been extracted from coir pith. It is inter-
esting to notice that both these filamentous fungi catalyze aerobic bioconversion to
form vanillin. Moreover, it has been validated that these organisms decarboxylate
ferulic acid (C H O ) to 4-vinyl guaiacol (Huang et al. 1993) which is immediately
1
0 10 4
converted to vanillic acid, the reduction of which leads to vanillin production (Li and
Rosazza 2000). The aforementioned studies justify our selection of microbes (A. niger
and P. chrysosporium), essentially needed to degrade ferulic acid during the bioconver-
sion process.
2.3. Microbial conversion of vanillic acid to vanillin
The bioconversion of vanillic acid produced 0.628 g/L crude vanillin. HPLC chromato-
gram of vanillin obtained from vanillic acid at retention time 5.505 min accorded with
standard vanillin (5.549) (Cas No: 121–33–5, Sisco Research Laboratories Pvt. Ltd, India)
(Figure S5(a,b) and Table S3). The conversion of vanillin was detected on day 6, which
was after 3 days of the introduction of P. chrysosporium along with 0.5 g/L of cello-
biose. The formation of vanillin was slowly reduced as fermentation proceeded to day
8
(Figure S6). The obtained amount of vanillin was higher as compared to vanillin
obtained from sugar beet pulp (0.357 g/L) (Lesage et al. 1999) but lower than the
quantity obtained from maize bran (0.767 g/L) (Lesage-Meessen et al. 2002) and rice
bran oil (1.09 g/L) (Zheng et al. 2007) (Table S4).
Numerous pathways exist that degrade vanillic acid, passing through various inter-
mediate stages, to form vanillin. It has been experimentally established that vanillic
acid catabolism occurs through demethylation to form protocatechuate involving aro-
matic ring cleavage (Milstein et al. 1983), oxidative decarboxylation to methoxy-p-
hydroquinone (MHQ) (Yajima et al. 1979), and reduction to vanillin and vanillyl alcohol
(
Ander and Eriksson 1978). Moreover, direct conversion of ferulic acid into vanillic acid
and vanillin is also known (Henderson and Farmer 1955). Furthermore, Tilay et al.
2008) reported vanillic acid as an intermediate in the production of vanillin. It is ardu-
(
ous to choose a common pathway in the microbial conversion of vanillic acid and van-
illin, and this could be attributed to relative amounts of these two compounds and
their reversible conversion.
P. chrysosporium is known to metabolize vanillic acid using two different pathways:
(
(
a) reduction pathway that leads to the formation of vanillin (Salleh et al. 2011) and
b) oxidative decarboxylation pathway leading to the formation of methoxy hydro-
quinone (Falconnier et al. 1994). Interestingly, the addition of cellobiose in the culture

NATURAL PRODUCT RESEARCH
5
medium has been noted to augment vanillin production (Bonnin et al. 1999) as it
inhibits the production of methoxy-hydroquinone from vanillic acid (Lesage-Meessen
et al. 1996). Besides, cellobiose also acts as an energy source required for the expres-
sion of the reductive pathway, or as an inducer of quinone oxidoreductase which
inhibits vanillic acid decarboxylation (Bonnin et al. 2000). In the present study, the
metabolism of ferulic acid to vanillin by A. niger and P.chrysosporium along with cello-
biose may have followed a similar pathway.
3. Experimental
Raw coir pith was collected from coir fiber extraction units located in and around the
central coir research institute (CCRI), Kalavoor, Alappuzha, Kerala (India). The
Aspergillus niger NCIM 1245 and Phanerochaete chrysosporium NCIM 1106 cultures
were obtained from the Microbiology Laboratory of CCRI Kalavoor, Alappuzha,
Kerala (India).
3.1. Extraction of ferulic acid from coir pith
3.1.1. Alkaline hydrolysis of coir pith
Alkaline hydrolysis was carried out by mixing 50 g raw coir pith with 1500 mL of 1 N
ꢀ
NaOH (1:30, w/v) and digested at 185 C for 20 min. The digest was filtrated, acidified
(pH 2.5), and transferred to a separating funnel, and precipitated lignin was removed.
Subsequently, the supernatant obtained was extracted with ethyl acetate.
3
.1.2. Liquid–liquid extraction with ethyl acetate
For liquid–liquid extraction, an equal quantity of ethyl acetate was added to an equal
volume of the supernatant obtained from acidification. It was shaken and centrifuged
for 15 min at 3000 rpm; the organic phase was taken out and transferred to a 50 mL
bottle. Furthermore, an equal volume of ethyl acetate was added again to the aque-
ous phase, centrifuged, and the two phases were collected. Ethyl acetate in the
organic phase was removed by rotor vacuum evaporation. The obtained compound
(crystalline form) was analysed in HPLC (supplementary material) and identified as
ferulic acid. The detailed extraction of ferulic acid from coir pith has been shown in
the schematic diagram (Figure S7).
3.2. Microbial conversion of ferulic acid to vanillin
3.2.1. Preparation of medium and culture condition for A. niger and P.
chrysosporium
Preparation of potato dextrose broth: To prepare potato dextrose broth, 300 g of
sliced potato (unpeeled and washed) was boiled in 1 L water for 30 min and filtered
broth using cheesecloth. The total volume of suspension was made up to 1 L using
distilled water and subsequently added 20 g of dextrose. The pH of the medium was
adjusted to 3.5. Thereafter, the medium was sterilized by autoclaving at 15 lbs
for 15 min.

6
C. T. REJANI AND S. RADHAKRISHNAN
Preparation of basal medium: Maltose (20 g/L), as the carbon source; and ammo-
nium tartrate (1.82 g/L), as the nitrogen source, were used as the major ingredients to pre-
pare basal medium. Moreover, other essential nutrients needed for the medium such as
KH PO (0.2 g/L), CaCl (0.132 g/L), MgSO (0.5 g/L), and 5 g/L yeast extract were also intro-
2
4
2
4
duced. The pH of the medium was adjusted to 5.5 and autoclaved. Maltose was sterilized
separately to be mixed to the culture medium during inoculation.
6
7
Fungal culture: The inoculation was carried out with 1 Â 10 À10 spores/mL of coni-
diospores in 0.9% NaCl. The cultivation of fungal culture (A. niger and P. chrysosporium)
was carried out in a conical flask containing 100 mL potato dextrose broth for 3 days
ꢀ
at 30 C: After 3 days, the cultured A. niger and P. chrysosporium were transferred to
ꢀ
their respective basal medium and incubated at 30 C with 150 rpm agitation.
3.2.2. Bioconversion of ferulic acid to vanillic acid
In the first step of the fermentation process, ferulic acid, obtained from alkaline
hydrolysis of raw coir pith, was added to the culture as an inducer cum substrate for
A. niger to transform it into vanillic acid. 500 mL of basal medium was inoculated with
ꢀ
A. niger along with 1 g of ferulic acid and, kept for incubation at 30 C for 3 days, with
an agitation of 150 rpm. HPLC analysis was performed on each day of incubation.
3.2.3. Biotransformation of vanillic acid to vanillin
In the second step, vanillic acid obtained after the first part of fermentation was again fer-
mented using P. chrysosporium to obtain vanillin. Subsequently, another 500 mL of basal
ꢀ
medium was inoculated with P. chrysosporium and incubated at 30 C for 3 days. The formed
crude vanillic acid-enriched culture medium of A. niger in the fermentation process was sup-
plemented in basal medium containing P. chrysosporium along with 0.5 g of sterilized cello-
biose. This mixture was also analysed using HPLC during the incubation period.
4. Conclusion
The study unveils coir pith can be used as a cheap substrate for the higher yield of
ferulic acid and bio-vanillin with the adoption of suggested biotechnological approach
for industrial applications in biomedicals, pharmaceuticals, neutraceuticals and cosmet-
ics, and simultaneously endorses the sustainable concept of waste management. This
study further confirms that the selected strains (A. niger and P. chrysosporium) are cap-
able to grow on phenolic compound having a potential for its application in bio-
remediation, especially in detoxification of phenolic compound released in the
vicinities of coir fibre extraction units. Further, we expect our future strategy to extract
and purify vanillin from the medium and characterization of intermediate products to
understand the pathway followed towards the biosynthesis of vanillin.
Acknowledgement
The authors are thankful to immense contribution of Dr. U.S. Sharma, former Director of Central
Coir Research Institute, Kalavoor and Dr. Anita Das Ravindranath, Director, Central Coir Research
Institute, Kalavoor for providing guidance and the facility to carry out the work. The technical
support provided by Mr. Jayaraj is also acknowledged.

NATURAL PRODUCT RESEARCH
7
Disclosure statement
No potential conflict of interest was reported by the authors.
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