Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60353, enero-diciembre 2025 (Publicado Mar. 27, 2025)

Effects of seedlac on soil bacterial diversity:

An indication of environmental safety

Thamilarasi Kandasamy1*; https://orcid.org/0000-0002-9646-4410

Mohammed F Ansari2; https://orcid.org/0000-0002-0402-8903

Sajiya Ekbal1; https://orcid.org/0000-0003-1691-8601

Kewal K Sharma1; https://orcid.org/0000-0002-9188-132X

1. Agri-bioresources Augmentation Division, ICAR-National Institute of Secondary Agriculture, Ranchi, Jharkhand,

India; k.thamilarasi@gmail.com (*Correspondence), sajiya.chand@gmail.com, kewalkks@gmail.com

2. Downstream Agro-Processing Division, ICAR-National Institute of Secondary Agriculture, Ranchi, Jharkhand, India;

mfansari@rediffmail.com

Received 07-VI-2024. Corrected 18-XII-2024. Accepted 13-III-2025.

ABSTRACT

Introduction: Lac resin, the only natural resin of animal origin, is exclusively produced by the lac insect (Kerria

spp.). It is non-toxic and considered biodegradable. However, the bacteria involved in biodegradation have not

been explored. Moreover, the fate of the soil bacteria during biodegradation has not been studied so far.

Objective: To explore the fate of soil bacteria due to the burial of seedlac in soil to ascertain whether seed-

lac is environmentally safe, and to explore the possibilities of identifying any bacterial flora involved in lac

biodegradation.

Methods: The study began in 2016 by burying seedlac samples (semi-refined lac resin product) in the field soil

and pot soil under replicated conditions. The seedlac samples were drawn from the soil in 2019, and the soil

adhering to the seedlac samples was used in further experiments. The bacterial diversity of these soils was docu-

mented by sequencing the V3-V4 region of 16S rRNA through the Illumina NGS platform.

Results: No significant variations were obtained in the soil bacterial diversity between samples except for the

marginal increase in the count of Actinobacteria, Myxococcales, Gemmatales, Gemmataceae, the WD2101-soil

group in seedlac buried pot soil, and Proteobacteria and Acidobacteria in field soil. Most of these bacterial groups

are known to degrade complex organic polymers.

Conclusions: Since there are no changes in the soil bacterial diversity due to seedlac burial, it may be concluded

that seedlac does not affect the soil microflora and is safe for the soil environment.

Key words: seedlac; shellac; microbiome; lac insect; soil bacteria; lac resin.

RESUMEN

Efectos de la resina de laca sobre la diversidad bacteriana del suelo:

Una indicación de la seguridad ambiental

Introducción: La resina de laca, la única resina natural de origen animal, producida exclusivamente por el insecto

laca (Kerria spp.) es no tóxica y se considera biodegradable. Sin embargo, hasta ahora, no se han explorado las

bacterias que están involucradas en la biodegradación. Además, hasta la fecha no se ha estudiado el destino de

las bacterias del suelo durante la biodegradación.

https://doi.org/10.15517/rev.biol.trop..v73i1.60353

BIOTHECHNOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e60353, enero-diciembre 2025 (Publicado Mar. 27, 2025)

INTRODUCTION

Lac resin is the unique and versatile resin of

insect origin secreted by the lac insects belong-

ing to Kerria spp. Uses of this unique animal

resin encompass many fields, such as food,

cosmetics, pharmaceutics, electrical appliances,

printing ink, machinery, leather, plastics, ethnic

jewelry, varnishes, paints, adhesives, perfumes,

etc. (Siddiqui, 2004). Lac resin is a polyester

complex comprising straight-chain fatty acids

and sesquiterpenic acids linked by ester and

lactide bonds (Sharma et al., 1983). Lac resin is

characterized by adhesiveness on a wide range

of surfaces; good electrical insulation prop-

erties; and resistance to moisture, corrosion,

ultraviolet radiation, oil, and acid. Besides these

characteristics, it is also a thermoplastic with a

thermosetting nature (Sharma et al., 2020).

The value of the global shellac market was

167.84 million USD in 2022 and will reach 191.8

million USD in 2028, with a CAGR of 2.25 %

during 2022-2028 (Industry Research, 2025).

United States Food and Drug Adminis-

tration recognizes shellac as GRAS (Gener-

ally Regarded as Safe) and approves it as a

food additive. European Food Safety Authority

(EFSA) has given E 904 to shellac. Since lac

resin is a natural product, it is assumed that it

is biodegradable. On the other hand, it is also

observed that shellac-coated objects are durable

and resist microbial attack. Hence, it is impera-

tive to study the rate of biodegradation of lac

resin and microbes involved in its biodegrada-

tion, and the fate of soil bacteria when lac is

disposed of in the soil.

Aleuritic acid is the most abundant fatty

acid present in lac resin which is used exten-

sively in cosmetics and pharmaceutical indus-

tries. Aleuritic acid is manufactured in the

industries from shellac by alkaline hydrolysis

method, by isolating its sodium salt by saponi-

fication, followed by decomposition of the salt

to obtain free acid (Agarwal et al., 1988). It

has been reported that out of 36 % of aleuritic

acid present in lac resin, only 25 % of it could

be recovered using alkaline hydrolysis method

(Anees, 2016). A large amount of aleuritic acid

remains unrecovered or degraded in this pro-

cess. Biological hydrolysis employing microbes

may help in increasing the yield of aleuritic acid

from lac resin, quantitatively as well as qualita-

tively. Biological hydrolysis will also contribute

to the release of other constituent acids such as

jalaric acid, a principle sesquiterpenic acid, and

other minor acids. Soil could be an efficient

source of microbes that can hydrolyze lac resin

and yield constituent acids.

A study of the soil metagenome of the con-

trol soil and lac sample buried soil sites would

give us an idea about the different groups of

bacteria present in each sample. Knowing their

Objetivo: Explorar el destino de las bacterias del suelo debido al entierro de resina de laca en el suelo para

determinar si es seguro para el medio ambiente o no y explorar las posibilidades de identificar cualquier flora

bacteriana involucrada en la biodegradación de laca.

Métodos: El estudio se inició en 2016 enterrando muestras de resina de laca (producto de resina de laca semirrefi-

nada) en el suelo a nivel de campo y en suelo de macetas, en réplicas. Las muestras de resina de laca se extrajeron

del suelo en 2019 y el suelo adherido a las muestras de resina de laca se utilizó en experimentos posteriores. La

diversidad bacteriana de estos suelos se documentó mediante la secuenciación de la región V3-V4 del ARNr 16S

a través de la plataforma NGS Illumina.

Resultados: No se obtuvo variación significativa en la diversidad bacteriana del suelo entre las muestras, excepto

por el aumento marginal en el recuento de Actinobacterias, Myxococcales, Gemmatales, Gemmataceae, grupo

WD2101-suelo en el suelo de maceta enterrada, y Proteobacterias y Acidobacterias en el suelo de campo. Se sabe

que la mayoría de estos grupos bacterianos degradan polímeros orgánicos complejos.

Conclusiones: Dado que no hay grandes cambios en la diversidad bacteriana del suelo debido al enterramiento de

resina de laca, se puede concluir que la resina de laca no afecta la microflora y es segura para el suelo.

Palabras clave: goma laca; microbioma; insecto laca; bacterias del suelo; resina de laca.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60353, enero-diciembre 2025 (Publicado Mar. 27, 2025)

identity is the foremost criterion that would

reveal the groups of bacteria having the poten-

tial to degrade lac resin. Metagenomics is the

power of genomic analysis that is applied to

entire communities of microbes, bypassing the

need to isolate and culture individual micro-

bial species (Handelsman, 2004). Metagenom-

ics employs the molecular-based taxonomic

investigation of bacteria by direct sequencing

of PCR-amplified small sequences of 16S rRNA

gene from extracted DNA. This approach

allows the identification of new species and the

investigation of low-abundance and uncultivat-

ed bacteria from a single analysis. In addition,

they are faster and more accurate compared

with classical identification methods.

No previous studies are available that focus

on the safety of lac resin in terms of soil bacte-

rial diversity. A study on the biodegradation of

lac resin by soil burial method was undertaken

by burying seedlac in the soil. Moreover, by

comparing the microbial diversity of control

and lac-buried soil the effect of lac on envi-

ronmental safety could be assessed. Hence, in

this study, seedlac buried soil samples were

subjected to metabarcoding using 16S rRNA

bacterial markers to find the fate of bacterial

diversity in the soil due to lac exposure to assess

the safety of lac resin to soil fauna, especially

the soil bacteria.

MATERIALS AND METHODS

Seedlac preparation, soil burial, and sam-

ple collection: Kusmi sticklac (phunki) was

procured from the Institute Research Farm

(IRF) of ICAR-National Institute of Secondary

Agriculture (ICAR-NISA), Ranchi, India. It

was converted to seedlac through soda washing

and cleaned thoroughly to remove any impuri-

ties from the samples. Seedlac samples were

covered in synthetic mesh sleeve and buried

in the soil (sandy loam soil with pH 4.5-5.0) at

1.5 ft depth on 17.6.2016 at IRF (23º19’51.2’’N

& 85º22’06.3’’E). Similarly, the seedlac samples

were also buried in the pot containing pot mix-

ture (soil: sand: farm yard manure in the ratio

2 : 1 : 1) for 3 years. Control and soil-buried

seedlac samples are shown in Fig. 1. Soil adher-

ing to the seedlac samples was collected care-

fully in the sterile tube and stored at -80 ºC till

further processing.

Genomic DNA and PCR amplification of

16S ribosomal V3-V4 region: Genomic DNA

was extracted from the soil samples using Qia-

gen DNeasy PowerSoil Kit (Qiagen, Germany)

according to the manufacturer’s instructions.

DNA concentration was estimated using Qubit

Fluorimeter (V.3.0). V3-V4 region of 16S rRNA

was amplified using specific V3 Forward prim-

er Pro341F- 5` CCTACGGGNBGCASCAG 3`

and V4 Reverse primer Pro805R- 5` GACTAC-

NVGGGTATCTAATCC 3` (Takahashi et al.,

2014). PCR was carried out in 25 μl reaction

volume containing 30 ng template DNA, 200

μM dNTPs, 2 U Taq DNA polymerase and 10

μM of the forward and reverse primers. PCR

conditions included initial denaturation at 95

°C for 30 s, followed by 35 cycles at 95 °C for

10 s, 56 °C for 15 s, 68 °C for 30 s, with a final

extension step at 68 °C for 5 min. The amplified

PCR products (~460 bp) from each sample were

run on 1 % agarose gel and were subjected to gel

extraction using GeneJet purification kit. The

purified samples were quantified using Nano-

drop spectrophotometer (Thermo Fisher).

Library preparation and 16S amplicon

sequencing: Five nanograms of amplified

products were used for library preparation

using Next Ultra DNA library preparation kit

(NEB) according to the manufacturer’s instruc-

tions. The library quantification and quality

estimation were done in Agilent 2 200 TapeSta-

tion. PCR products with unique indices from

each library were taken in equal (~2 ng) quan-

tities and subjected to 250 paired-end multi-

plex sequencing using Illumina HiSeq 2 500

platform (Agrigenome Labs Private Limited,

Cochin, India).

Initial processing and bioinformat-

ics analysis of sequence reads: Raw reads

obtained from the Illumina sequencing plat-

form after Demultiplexing were subjected to

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the FastQC program (Version 0.11.8) to check

the quality of the reads with default parameters.

Base quality (Phred Score; Q), base compo-

sition, GC content, ambiguous bases (other

than A, T, G, C), and adapter dimers were

thoroughly checked before the Bioinformatics

analysis. The forward V3 and reverse V4 spe-

cific primers were trimmed using the in-house

PERL script. Properly paired-end reads with

Phred score quality (Q > 20) were consid-

ered for V3-V4 consensus generation. Primer

trimmed, high-quality paired-end reads were

pair-wisely allowed to merge/stitch to get the

V3-V4 amplicon consensus FASTA sequences.

The reads were merged using FLASH program

(Version 1.2.11) with a minimum overlap of

10 bp to a maximum overlap of 240 bp with

zero mismatches. While making consensus of

V3-V4 sequences, all consensus reads formed

were with an average contig length of 350 to 450

bp. Chimeras were removed using the de-novo

chimera removal method UCHIME (Version

11) implemented in the tool VSEARCH.

Operational Taxonomic Units (OTUs)

picking and taxonomy classification were per-

formed using the pre-processed consensus

V3-V4 sequences. Pre-processed reads from all

samples were pooled and clustered into OTUs

based on their sequence similarity using Uclust

program (similarity cutoff = 0.97) available in

QIIME software. QIIME1 program (Version

1.9.1) was used for the entire downstream

analysis (Caporaso et al., 2010). Representa-

tive sequences from each clustered OTU were

picked and aligned against the SILVA core set of

sequences using the PyNAST program. Further,

Fig. 1. Seedlac samples after 3 years of burial. P: Buried in pot soil; F: Buried in field soil and C: control seedlac.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60353, enero-diciembre 2025 (Publicado Mar. 27, 2025)

taxonomy classification was performed using

an RDP classifier by mapping each representa-

tive sequence against the SILVA OTUs database.

The sequences that do not have any alignment

against the taxonomic database are categorized

as Unknown.

Statistical analysis: Rarefaction analysis

and alpha diversity index (Shannon, Chao1,

and observed species metrics) were used to

estimate the differences in microbial commu-

nities between different samples used in the

present study.

RESULTS

Soil sample collection, DNA isolation,

and PCR: Seedlac samples were buried in the

soil (field soil and in pot soil) at the Institute

Research Farm of ICAR-NISA, Ranchi, India.

Significant weight loss (23-29 %) was observed

for the buried seedlac samples after three years

(unpublished data from our lab). Besides weight

loss, the colour index of the buried samples was

increased. One of the important physicochemi-

cal properties, flow (fluidity) of the seedlac

samples became zero in just 12 months while

the control samples showed fluidity up to 33-36

months. These parameters pointed out that the

buried seedlac samples were undergoing bio-

degradation. Hence, the soil samples adhering

to the seedlac were used in this study to analyze

the bacterial metagenome.

DNA was isolated from different soil

samples (control and seedlac buried field soil

samples; control and seedlac buried pot soil

samples). A 460 bp fragment of the hypervari-

able V3-V4 region of 16S rRNA gene was PCR

amplified from isolated DNA using specific

universal primers. The amplified product was

checked on 2 % agarose gel and quantified

using Nanodrop and Qubit. To check their

quality, the OD 260 / 280 ratio was calculated

and found to be ranged from 1.68 to 1.89.

Analysis of Illumina-HiSeq raw reads:

Libraries (of V3-V4 region) prepared from

460 bp PCR-amplified fragments yielded raw

reads ranging from 340 729 to 558 260 in the

Illumina platform. Details about raw reads and

QC of various samples are given in Table 1.

In the pot burial experiment, Phred scores of

more than 30 and 20-30 were obtained for ~87

% and 4 % of the sequences, respectively. In the

field burial experiment, Phred scores of more

than 30 and 20-30 were obtained for ~86 % and

5 % of the sequences, respectively. Raw data

obtained from this study has been submitted to

the NCBI Sequence Read Archive (SRA) under

accession number PRJNA760653.

Pot Burial Experiment Results: After

removing primer containing sequences, pos-

sible adapter sequences, low-quality reads, and

unique dual index barcode sequences contain-

ing 5-7 nucleotides, duplicates, and chimeras,

from the raw reads, a total of 582 034 prepro-

cessed consensuses were obtained for control

pot soil and seedlac buried pot soil samples. A

total of 10118 OTUs at a confidence limit of 0.8

were obtained for the pot experiment samples.

The representative sequences from each clus-

tered OTUs were aligned and taxonomy clas-

sification was performed against the SILVA

database (Fig. 2).

Results showed that most of the phylum,

class, order, genus, and species OTUs were

unknown. Besides them, the most abundant

phylum OTUs were Proteobacteria and Acido-

bacteria in control and seedlac buried pot soil

samples. The count of Actinobacteria was mar-

ginally higher in seedlac-buried pot soil, where-

as the counts of Firmicutes and Chloroflexi

were marginally lesser in seedlac-buried pot

soil. The most abundant class OTUs belonged

to Alphaproteobacteria and Planctomycetacia

classes in both control and seedlac buried pot

soil samples. Planctomycetacia counts were

slightly higher in the seedlac buried pot soil

sample. The most abundant order OTUs were

Rhizobiales and uncultured bacterium in con-

trol whereas Rhizobiales and Gemmatales were

the most abundant order OTUs in control and

seedlac buried pot soil samples. Gemmatales

and Myxococcales were higher in seedlac bur-

ied pot soil when compared with control soil.

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Gemmataceae and WD2101-soil group fam-

ily counts were higher in seedlac buried pot

soil in comparison with control pot soil. The

most abundant genus OTUs included Candi-

datus-Solibacter and Candidatus-Udaeobacter.

In seedlac buried pot soil samples, the genus

Candidatus-Udaeobacter and Gemmatimonas

were slightly higher than the control. The

most abundant species OTUs were uncultured-

Acidobacteria-bacterium and uncultured-

archaeon in both samples.

Soil Burial Experiment Results: Results

showed that most of the phylum, class, order,

genus, and species OTUs were unknown.

Besides them, the most abundant phylum

OTUs were Proteobacteria and Acidobacteria

in control and seedlac buried soil samples. The

counts of Proteobacteria and Acidobacteria

were slightly higher in treated soil when com-

pared with the control soil. The most abun-

dant class OTUs belonged to Planctomycetacia

and Alphaproteobacteria classes in control and

seedlac buried field soil samples. The most

abundant order OTUs were Gemmatales and

Rhizobiales in control and seedlac buried soil

samples. The count of Betaproteobacteriales

was slightly higher in seedlac buried soil sam-

ples compared to the control. The Gemmati-

monadaceae family was slightly higher, and

the family Chthonobacteraceae was less in

the seedlac buried soil compared to the con-

trol. The most abundant genus OTUs included

Candidatus-Solibacter and Candidatus-Udaeo-

bacter. The count of Streptomyces was slightly

less in the seedlac buried soil compared to the

control soil. The most abundant species OTUs

were uncultured-Acidobacteria-bacterium and

uncultured-archaeon in both samples (Fig. 3).

The unique OTUs in all the seedlac exposed

soil samples including pot soils and field soils

are majorly either uncultured or unclassified

bacteria (Data not shown).

Table 1

Read particulars of all the samples used in the study.

Sl. no Sample Run Read

orientation

Mean read

quality

(phred

score)

Number

of reads % GC %Q < 10 %Q

10-20

% Q

20-30 % Q > 30

Number

of bases

(MB)

Mean read

length

(bp)

Pot Soil Burial Experiment

1 CONTROL-POT1 R1 36.75 543 784 56.85 0.04 4.82 4.01 91.13 135.95 250.0

R2 34.05 543 784 53.7 5.13 6.61 4.9 83.36 135.95 250.0

2 SEED-LAC-POT-1 R1 36.76 534 150 57.28 0.04 4.81 4.03 91.12 133.54 250.0

R2 32.71 534 150 54.14 5.23 9.88 6.92 77.96 133.54 250.0

3 SEED-LAC-POT-2 R1 36.81 471 047 57.27 0.04 4.58 3.98 91.4 117.76 250.0

R2 32.72 471 047 54.16 5.17 9.95 6.96 77.92 117.76 250.0

Field Soil Burial Experiment

1 CONTROL-FIELD-1 R1 36.72 558 260 57.56 0.04 4.75 4.17 91.05 139.56 250.0

R2 32.87 558 260 54.42 5.27 9.34 6.75 78.64 139.56 250.0

2 CONTROL-FIELD-2 R1 36.64 379 335 57.47 0.04 5.04 4.16 90.77 94.83 250.0

R2 32.57 379 335 54.41 5.04 10.54 7.26 77.16 94.83 250.0

3 SEED-LAC-FIELD-1 R1 36.7 470 104 57.1 0.04 4.91 4.08 90.97 117.53 250.0

R2 32.5 470 104 53.95 5.12 10.71 7.27 76.9 117.53 250.0

4 SEED-LAC-FIELD-2 R1 36.88 340 729 57.01 0.04 4.46 3.85 91.65 85.18 250.0

R2 32.8 340 729 53.81 5.25 9.7 6.73 78.32 85.18 250.0

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Diversity Indices: In both sets of experi-

ments, the microbial diversity within the

samples was analyzed by calculating Shannon,

Chao1, and observed species metrics (Fig. 4).

There is not much variation in the relative

diversity of the bacterial species in all the sam-

ples as revealed by the different metrics of

rarefaction. However, Shannon indices were

slightly higher for seedlac buried field soil

samples in comparison with control soil.

Fig. 2. Relative abundance of OTUs at different taxa levels for pot soil burial experiment A. phylum level; B. Class level; C.

Order level; D. Family level; E. Genus level; F. Species level.

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Fig. 3. Relative abundance of OTUs at different taxa levels for field soil burial experiment A. phylum level; B. Class level; C.

Order level; D. Family level; E. Genus level; F. Species level.

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DISCUSSION

Biodegradation is a process of degrading

any complex material by microbes and con-

verting it into its constituent parts which are

environmentally safe and acceptable products.

Biodegradation results in physical disintegra-

tion and weight loss of the material. Biode-

gradable polymers are used in various fields

such as agriculture, medicine, and packaging.

One such polymer is lac resin having applica-

tions in all these fields. Lac resin is degradable

in the sense that it undergoes degradation in

physico-chemical properties due to heat treat-

ment, improper storage, and UV irradiation but

degraded lac is not the same as biodegraded lac.

It has been reported that lac resin is resistant

to microbial and insect attacks and hence is

used in the preservation of perishable items.

Then, it becomes debatable whether lac resin

would undergo biodegradation due to micro-

bial activity or not. Therefore, this study was

taken up to explore lac biodegradation and the

bacterial players involved in it, and the safety of

Fig. 4. Rarefaction curves using different measures A, B, C. are for pot soil burial experiments; D, E, F. are for field soil burial

experiments.

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lac resin to soil microflora. Seedlac was used in

this study since it is a semi-refined form of lac

apparently without any particles from lac insect

or its host plants. Seedlac consists of 88.5 % lac

resin, 2.5 % dye, 4.5 % wax, 2.0 % gluten, and

2.5 % impurities (Sharma et al., 2020). Changes

in weight, colour, and fluidity were observed for

soil-buried seedlac, which suggested that seed-

lac underwent biodegradation in due course

of time. Nonetheless, the fate of soil bacterial

diversity due to soil burial of lac resin is the

foremost indicator in describing the safety of

lac resin to the environment. Hence, soil bac-

terial diversity in two sets of experiments was

explored by metabarcoding to assess the safety

of lac resin to the soil vis-a-vis the environment.

Pot burial experiment: Except for a few

bacterial taxa, the relative abundance of most of

the microbial communities present in control

and seedlac buried pot soil were found to be the

same (Fig. 2). The count of Actinobacteria in

seedlac buried pot soil is more suggesting a role

of seedlac decomposition for this phylum (Fig.

2A). Actinobacteria, the phylum of ubiquitous

saprophytes can degrade recalcitrant carbon

sources, and its role in the decomposition of

dead organic matter in the soil is a well-known

phenomenon (Anandan et al., 2016). Actino-

bacteria can thrive well even under oligotrophic

conditions and low soil moisture due to their

filamentous growth and can degrade poly lactic

acid type bioplastic (Butbunchu & Pathom-

Aree, 2019). On the other hand, the count of

Firmicutes and Chloroflexi were marginally

lesser in seedlac buried pot soil (Fig. 2A). In

an earlier study, nitrogen addition to the soil

also decreased the OTU richness of the phylum

Chloroflexi (Zhang et al., 2013), pointing to the

sensitive nature of this Phylum for extraneous

addition of foreign material to the soil.

The counts of Myxococcales, a soil-dwell-

ing bacterial group that feeds on insoluble

organic substances were found to be higher

in seedlac buried pot soil (Fig. 2C). The order

Gemmatales and the family Gemmataceae

within Gemmatales are strictly aerobic, neutro-

philic, or mildly acidophilic bacteria, capable

of growing on various sugars and polysaccha-

rides, and few of them are capable of degrading

chitin and cellulose (Dedysh, 2020). WD2101-

soil group family of bacteria under phylum

Planctomycetes are also capable of growing on

xylan, pectin, starch, lichenan, cellulose, chitin,

and polysaccharides of microbial origin due to

the presence of versatile hydrolytic capabilities

and repertoires of carbohydrate active enzymes

such as glycoside hydrolases (Dedysh & Iva-

nova, 2018). Since the bacterial groups such

as Actinobacteria, Myxococcales, Gemmatales,

Gemmataceae, WD2101-soil group which are

generally involved in the degradation of com-

plex carbohydrate polymers are increased in

the seedlac buried pot soil (Fig. 2A, Fig. 2C, Fig.

2D) it may be suggested that the same mecha-

nism of degrading or metabolizing complex

carbohydrates may be involved in the biodeg-

radation of lac resin as well.

Field burial experiment: Although the

count of Proteobacteria and Acidobacteria were

higher in all the soil samples studied, seedlac

buried soil had a slight increase in the counts

of both these phyla (Fig. 3A). Though the

bacterial species under the phylum Proteo-

bacteria and Acidobacteria are physiologically

and ecologically diverse, Proteobacteria are

important players in carbon, nitrogen, and sul-

phur cycles in the environment (Kersters et al.,

2006), whereas species under Acidobacteria are

involved in carbohydrate and nitrogen metabo-

lism (Kielak et al., 2016). We could observe a

marginal increase in the classes Gammaproteo-

bacteria (under Proteobacteria) and Subgroup

6 of Acidobacteria in the seedlac buried soil

samples (Fig. 3B). Poly (butylene adipate-co-

terephthalate) (PBAT) based blend film was

shown to be biodegraded by the genus Marino-

bacter within Gammaproteobacteria (Meyer-

Cifuentes et al., 2020). Betaproteobacteriales

class and Gemmatimonadaceae family were

marginally higher in seedlac-buried soil (Fig.

3B, Fig. 3D). It is intriguing to note that the

Gemmatimonadaceae family which are denitri-

fiers utilizing nitrite (Aanderud et al., 2018) is

higher in seedlac buried soil.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e60353, enero-diciembre 2025 (Publicado Mar. 27, 2025)

On the other hand, Chthoniobacteraceae

family and Streptomyces genus were slightly

less in the seedlac buried soil (Fig. 3D, Fig.

3E). The decreased count of Streptomyces bac-

teria having an important role in the turn-

over of organic material in the soil (Seipke et

al., 2012) would suggest that this genus may

not be directly involved in lac biodegradation.

Although the count of Chthoniobacteraceae at

family level is marginally reduced; one of its

genera, Candidatus Udaeobacter did not show

any difference between control and treated soil

(Fig. 3E).

Chao1 metric estimates the species rich-

ness while Shannon metric is the measure

to estimate observed OTU abundances, and

accounts for both richness and evenness. The

observed species metric is the count of unique

OTUs identified in the sample. The rarefaction

curves for each of the metrics reached near pla-

teau for all samples, showing that the sequenc-

ing depth and coverage were adequate for all

the samples studied (Fig. 4). Shannon indices

were slightly more for seedlac buried field soil

samples in comparison with control soil reveal-

ing a marginal increase in the OTU abundance

in the soil buried with seedlac (Fig. 4D).

Lac biodegradation in the soil is expect-

ed to start with depolymerization to release

oligomers and or monomers probably through

esterases or enzymatic hydrolysis. Once the

monomers, dimers and oligomers are released,

they can be transported into microorganisms

for the mineralization process to occur and

release water and carbon dioxide. The bacterial

groups that are relatively more in the seedlac

buried soil samples could be actively involved

in the hydrolysis and mineralization of seedlac.

Since there are no tremendous changes in the

diversity and structure of bacterial microbiome

in the soil buried with seedlac, it can be con-

cluded that the seedlac is not detrimental to the

soil ecosystem. Earlier studies also showed the

safety of shellac for consumption (Srivastava &

Thombare, 2017) and for cosmetic purposes

(Anonymous, 1986). This is the first report

stating that the lac resin is safer for soil bacterial

biodiversity and in turn environmental safety.

The scope of fungi in lac biodegrada-

tion may also be explored in the future to

get a holistic view of the biodegradation pro-

cess. Co-occurrence network analysis may

also prove useful in exploring the players of

lac biodegradation.

Since seedlac does not alter the soil bacte-

rial biodiversity it may be largely concluded

that lac resin specifically seedlac is safe to the

soil microflora vis a vis environment. Our study

also indicated that the bacterial groups such

as Actinobacteria, Proteobacteria, Acidobac-

teria, Myxococcales, Gemmatales, Gemmata-

ceae, WD2101-soil group would probably be

involved in the biodegradation of seedlac in

the soil.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

The authors wish to acknowledge the

Indian Council of Agricultural Research, New

Delhi, India for providing funds to carry out

this work under the Network Project on Con-

servation of Lac Insect Genetic Resources.

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