PLOS ONE

RESEARCH ARTICLE

Bacterial diversity dynamics in microbial

consortia selected for lignin utilization
Isis Viana Mendes1,2, Mariana Botelho Garcia1,3, Ana Carolina Araújo Bitencourt1,2,
Renata Henrique Santana4, Philippe de Castro Lins1,2, Rafaella Silveira2, Blake
A. Simmons5, John M. Gladden5,6, Ricardo Henrique Kruger2, Betania
Ferraz Quirino ID1,2,3*
1 Embrapa Agroenergia, Parque Estação Biológica (PqEB), PqEB s/n, Brası́lia, DF, Brazil, 2 Universidade
de Brası́lia, Brası́lia, DF, Brazil, 3 Universidade Católica de Brası́lia, Brası́lia, DF, Brazil, 4 Instituto Federal
de Brası́lia, Brası́lia, DF, Brazil, 5 Deconstruction Division, Joint BioEnergy Institute, Emeryville, California,
a1111111111 United States of America, 6 Department of Biomass Science and Conversion Technology, Sandia National
a1111111111 Laboratories, Livermore, California, United States of America
a1111111111 * betania.quirino@embrapa.br, bfq@uwalumni.com
a1111111111
a1111111111

Abstract
Lignin is nature’s largest source of phenolic compounds. Its recalcitrance to enzymatic con-
OPEN ACCESS version is still a limiting step to increase the value of lignin. Although bacteria are able to
Citation: Mendes IV, Garcia MB, Bitencourt ACA, degrade lignin in nature, most studies have focused on lignin degradation by fungi. To
Santana RH, Lins PdC, Silveira R, et al. (2021) understand which bacteria are able to use lignin as the sole carbon source, natural selection
Bacterial diversity dynamics in microbial consortia over time was used to obtain enriched microbial consortia over a 12-week period. The
selected for lignin utilization. PLoS ONE 16(9):
e0255083. https://doi.org/10.1371/journal.
source of microorganisms to establish these microbial consortia were commercial and back-
pone.0255083 yard compost soils. Cultivation occurred at two different temperatures, 30˚C and 37˚C, in
Editor: Erika Kothe, Friedrich Schiller University,
defined culture media containing either Kraft lignin or alkaline-extracted lignin as carbon
GERMANY source. iTag DNA sequencing of bacterial 16S rDNA gene was performed for each of the
Received: August 20, 2020
consortia at six timepoints (passages). The initial bacterial richness and diversity of back-
yard compost soil consortia was greater than that of commercial soil consortia, and both
Accepted: July 10, 2021
parameters decreased after the enrichment protocol, corroborating that selection was
Published: September 13, 2021 occurring. Bacterial consortia composition tended to stabilize from the fourth passage on.
Copyright: This is an open access article, free of all After the enrichment protocol, Firmicutes phylum bacteria were predominant when lignin
copyright, and may be freely reproduced, extracted by alkaline method was used as a carbon source, whereas Proteobacteria were
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
predominant when Kraft lignin was used. Bray-Curtis dissimilarity calculations at genus
The work is made available under the Creative level, visualized using NMDS plots, showed that the type of lignin used as a carbon source
Commons CC0 public domain dedication. contributed more to differentiate the bacterial consortia than the variable temperature. The
Data Availability Statement: The data is publicly main known bacterial genera selected to use lignin as a carbon source were Altererythro-
available at the DOE Joint Genome Institute IMG bacter, Aminobacter, Bacillus, Burkholderia, Lysinibacillus, Microvirga, Mycobacterium,
database (Project ID: 1126255, Proposal ID:
Ochrobactrum, Paenibacillus, Pseudomonas, Pseudoxanthomonas, Rhizobiales and
503004).
Sphingobium. These selected bacterial genera can be of particular interest for studying lig-
Funding: This work was supported by grants from
nin degradation and utilization, as well as for lignin-related biotechnology applications.
FAP-DF (B.F.Q.), CNPq (B.F.Q.), CAPES (I.V.M., M.
B.G., A.C.A.B. and P.C.L.) and DOE-JBEI DE-AC02-
05CH11231 (J.M.G. and B.A.S.).

Competing interests: The authors have declared

that no competing interests exist.

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PLOS ONE Lignin-adapted consortia

1. Introduction
The world is looking for petroleum substitutes, both for energy and for making products that
are clean and renewable [1–4]. Lignocellulosic biomass is already being successfully used
industrially for these purposes, but its full potential has not been reached [5, 6]. Plant biomass
has cellulose, hemicellulose and lignin as its main components. Lignin is the second most
abundant biopolymer in nature, accounting for 5–35% of plant biomass composition [7, 8].
While there are a number of industrial applications for cellulose and hemicellulose that range
from the production of the biofuel ethanol to the sugar substitute xylitol, applications for lignin
are more restricted. In fact, it is quite common to burn lignin for energy, a low technology
application that does not explore its potential as a natural source of phenolic compounds of
industrial interest [9]. In a biorefinery model, lignin can be depolymerized and used to make
value-added compounds, such as guaiacol, syringaldehyde, vanillin and ferulic acid [10].
These monomers can be used as the building blocks for bioproducts, phenolic resins, carbon
fibers and nanomaterials [10–17].
Chemically, lignin is a phenolic heteropolymer formed by the dehydrogenation polymeriza-
tion of p-hydroxyphenyl (H), guaiacil (G) and syringyl (S) units, derived from the monolignols
coumaryl, coniferyl and synaphyl alcohol, respectively [18]. Lignin’s monomers are intercon-
nected by ester, ether, C-C and β-O,4 bonds [18–20]. Coupling of these units by different
bonds is what confers lignin its recalcitrance to enzymatic degradation. Despite lignin’s struc-
tural complexity, fungi and bacteria are capable of degrading it using the oxyreductase
enzymes laccase, manganese peroxidase, versatile peroxidase and DyP peroxidase [20, 21]. Lig-
nocellulosic biomass degradation by biological models, whether by microorganisms or
enzymes alone, is of great interest as it occurs in mild conditions of temperature, pressure and
pH [22]. Fungi from the Ascomycota and Basidiomycota phyla have been classified as lignin
degrading [23]. Bacteria can also use lignin as carbon source, yet fewer ligninolytic bacterial
species are known [24]. Studies show that when compared to fungi, bacteria ligninolytic
enzymes can depolymerize lignin into a more restricted number of aromatic compounds,
which would facilitate their use in biotechnological applications [9, 25].
Bacteria from the phyla Phylobacteria, Firmicutes and Bacterioidetes are capable of produc-
ing enzymes to breakdown lignin [24]. From a biotechnology perspective, bacterial diversity
can be explored in search of new and better ligninolytic enzymes [26]. Previous studies have
shown that ligninolytic bacterial species were successfully identified in soil-enriched microbial
consortia for lignin degradation [10, 27–29]. Soil has a complex composition which includes
microbial communities that perform multiple activities such as lignin degradation [27, 30].
However, the complexity of soil’s microbial communities can be overwhelming. A strategy to
bypass this problem is to selectively cultivate specific microorganisms, such as those that can
live using lignin as a sole carbon source. The process of enrichment of microbial consortia
decreases functional complexity by converging the microbial community to similar biochemi-
cal functions facilitating identification of ligninolytic groups [10, 31].
In this work, an enrichment strategy was applied to establish microbial consortia selected to
use lignin as carbon source. To this end, two sources of microorganisms (i.e., commercial soil
and backyard compost soil), two cultivation temperatures (i.e., 30˚C and 37˚C) and two sources
of lignin (i.e., alkaline-extracted and Kraft lignin) were used to establish eight microbial consor-
tia in defined laboratory medium. Cultivation occurred over a period of 12 weeks, in which an
aliquot of the consortia was inoculated into fresh medium every two weeks, totaling 6 subcultur-
ing cycles or passages. To understand the dynamics of microbial selection for the ability to uti-
lize lignin as carbon source, we analyzed Illumina 16S rDNA gene sequence (iTag) phylogenetic
profiling of bacteria data from these eight microbial consortia over six passages.

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Fig 1. Schematic of enrichment experiment strategy. Eight microbial consortia enriched for microorganisms able to
utilize lignin (i.e., base-extracted or Kraft) as carbon source. Four of the consortia had back yard soil and other four had
MG soil as the as the original source of microorganisms. The enrichment experiments were conducted in aerobic
conditions at 30˚C and 37˚C. Every two weeks, an aliquot of the growth medium containing microorganisms was
transferred to fresh culture medium. The consortia were enriched over six cycles (passages), totaling 12-weeks. Samples
were taken for each passage, DNA was extracted and the ribosomal gene 16S rDNA was amplified and sequenced.
https://doi.org/10.1371/journal.pone.0255083.g001

2. Materials and methods

2.1. Establishment of microbial consortia
For the establishment of microbial consortia, two sources of microorganisms were used: Miracle
Growth commercial substrate and backyard compost soil which was obtained from Alameda
County (California, USA). Commercial Kraft lignin and lignin from switchgrass extracted by
alkaline method were used as carbon sources. The alkaline method for lignin extraction con-
sisted in grinding 12.5 g of biomass and adding to it 250 mL of 0.5 M NaOH were added to 12.5
g of biomass and the solution was sterilized by autoclaving for 30 min at 121˚C. Subsequently,
H2SO4 5 M was added until the pH of 7 was obtained. The solution was kept for 24 hours at
4˚C, and then centrifuged at 10,000 x g for 30 min at 4˚C. The supernatant was transferred to a
second flask and centrifuged at 18,514 x g for 1 hour at 4˚C; and the new supernatant was steril-
ized with 0.45 μm pore filter. A total of 50 mL of M9 minimal medium with trace elements [32]
was added to eight 200 mL Erlenmeyer flasks (Fig 1). To each flask, 0.5 g of either Miracle
Growth or backyard soil was inoculated. As carbon source, either 0.5 g/L of Kraft lignin (Sigma-
Aldrich 471003) or 10% (w/w) of alkaline extract lignin was added. The cultures were kept at
aerobic conditions with shaking at 200 rpm, either at 37˚C or 30˚C. For the enrichment process,
a 2 mL aliquot of the microbial consortia was transferred to a new flask under the same condi-
tions every two weeks, in a total of six passages [33]. At different cultivation times, 20 mL of
each enriched culture were collected and then centrifuged at 20,000 g for 10 min at 4˚C. The
obtained microbial pellet was stored at -80˚C until DNA extraction [33].

2.2. Illumina sequencing of 16S rDNA region

To identify the bacteria present in each consortium at each timepoint, DNA extraction was
performed using All Prep DNA/RNA kit (QIagen) following the manufacturer’s instructions.

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PLOS ONE Lignin-adapted consortia

DNA was then used as template in polymerase chain reactions (PCR) to amplify the copies of
the 16S rDNA V4-V5 region. For this the FW (515F): 5’GTG CCA GCM GCC GCG GTAA 3’
and RV (805R): 5’ GGA CTA CHV GGG TWT CTA AT 3’ primers were used [34]. The
fragments were analyzed using Bioanalyzer for quality control. Sequencing was according to
JGI guidelines (Joint Genome Institute, CA, personal communication) [34] on the Miseq plat-
form (Illumina) by pair end (2 x 150 bp) sequencing by the Joint Genome Institute (Walnut
Creek, CA, USA) [34].

2.3. Bacterial diversity analyses

The 16S rDNA amplicons were grouped and classified into their respective operating taxo-
nomic units (OTUs) using iTagger 2.0 program with the Usearch tool using 97% similarity by
the Joint Genome institute (JGI) [35]. Silva SSU database was used to make taxonomic notes
for 16S rRNA and Silva LSU for 18S rRNA [36, 37]. iTag data from cultures inoculated with
Miracle Growth or Backyard soil were subjected to diversity analysis. Operational taxonomic
units (OTUs) were analyzed using Quantitative Insights in Microbial Ecology (QIIME) soft-
ware [38]. To calculate the alpha diversity of communities, the following scripts were
employed: “alpha_rarefaction.py” with a sample of 150,000 bacterial OTUs. For richness and
diversity analysis, the following metrics were calculated: observed OTUs, Chao1, Shannon,
1-Simpson, Good’s coverage and PD whole tree. To calculate the absolute abundance of each
community, the script “summarize_taxa.py” was used. In Excel (Microsoft Office), the abso-
lute abundance data for each community was calculated. For the consortia inoculated with
Miracle Growth soil, only the main bacterial OTUs with relative abundance above 1.0% were
used to generate taxonomy graphs. For the consortia established from backyard compost soil,
bacterial OTUs with relative abundance above 1.0% were used to generate taxonomy graphs.
The relative abundance data was used to create bar graphs with the main bacterial families
present in the consortia. To analyze consortia dissimilarity, genus data from different passages
was used in Bray-Curtis based non-metric multidimensional scaling (NMDS) [39]. Bray Curtis
matrix PERMANOVA testing using the adonis function of vegan package in R [39]. Addition-
ally, vectors of taxa driving clusters were found using the envfit function of vegan package in R
performing 10,000 permutations [39]. The data of the most abundant genera with relative
abundance above 1.0% that were present in the sixth passage of each microbial consortium
were used for construction of a Venn diagram [40].

3. Results
3.1. Alpha diversity of microbial consortia
As shown in Fig 1, eight microbial consortia enriched for the ability to utilize lignin as a carbon
source were obtained. Four of the consortia were obtained using Miracle growth (MG) com-
mercial soil as a source of microorganisms, and four others used backyard (BY) compost soil.
Two types of carbon source were used, base-extracted lignin (BE Lig) and Kraft lignin. Growth
under aerobic conditions occurred at 30˚C and 37˚C.
16S rDNA sequencing data from the original soil (control) were used to estimate the diver-
sity present in each consortium prior to the enrichment process for lignin utilization. As
shown on Table 1, Good’s coverage of 99% and 100% for all passages of all consortia indicate
that the number of sequences sampled was sufficient to represent the diversity of the consortia
studied (Table 1 and S1 and S2 Tables). The original soil sample, MG or BY, shows a higher
number of bacterial operating taxonomic units (OTUs) than that found for the sixth passage
for both types of lignin substrate and for both temperatures. Chao 1 index shows that richness
decreased both for MG and BY derived consortia at both temperatures, when one compares

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Table 1. Diversity indexes for the original substrate (i.e., MG and BY compost soils) and at the sixth passage of enriched bacterial consortia, for base-extracted and
Kraft lignin utilization cultivated at 30˚C and 37˚C.
Sample Sample OTU Goods’ Chao1 PD wholetree Shannon 1-Simpson
coverage
MG_BE_30˚C 307 100% 346.723 32.327 4.091 0.861
MG_BE_37˚C 308 100% 340.453 32.375 4.093 0.862
MG_Kraft_30˚C 309 100% 346.310 32.568 4.094 0.862
Original MG_Kraft_37˚C 309 100% 349.022 32.390 4.093 0.862
Soil BY_BE_30˚C 1,061 99% 1,175.026 72.810 7.991 0.990
BY_BE_37˚C 1,061 99% 1,186.272 72.873 7.991 0.990
BY_Kraft_30˚C 1,061 99% 1,173.724 72.804 7.993 0.990
BY_Kraft_37˚C 1,059 100% 1,172.735 72.871 7.992 0.990
MG_BE_30˚C 209 100% 227.927 22.425 3.467 0.803
MG_BE_37˚C 171 100% 158.112 12.750 3.428 0.766
MG_Kraft_30˚C 144 100% 184.670 18.653 2.662 0.682
th
6 Passage MG_Kraft_37˚C 107 100% 114.618 10.081 4.104 0.904
BY_BE_30˚C 230 100% 248.303 22.722 4.075 0.885
BY_BE_37˚C 185 99% 185.470 15.421 4.930 0.934
BY_Kraft_30˚C 171 100% 173.545 18.517 3.770 0.839
BY_Kraft_37˚C 101 100% 109.451 13.352 3.500 0.806
https://doi.org/10.1371/journal.pone.0255083.t001

numbers for the bacterial consortia found in the original soil samples to those found in the
sixth passage. Diversity indexes (i.e., PD whole tree, 1-Simpson and Shannon) show a decrease
in diversity in the bacterial consortia present in the sixth passage compared to the original
ones, both for MG and BY derived consortia and for both temperatures. It is also noticeable
that the indexes obtained show that bacterial richness and diversity is greater in backyard-
derived samples in comparison to MG-derived samples.

3.2. Dynamics of taxonomic profiles during enrichment

To understand the dynamics during the enrichment process, taxonomic affiliation at family
level of 16S rDNA amplicons to operational taxonomic units (OTUS) was performed using
97% sequence similarity [34]. Figs 2 and 3 show family-level taxonomy data of bacterial con-
sortia over the six passages.
The main bacterial families with relative abundance above 1% in any of the passages were
represented in taxonomy graphs (Figs 2 and 3 and S4 and S5 Tables). At the end of the enrich-
ment process on the sixth passage, the families present in the microbial consortium derived
from MG soil enriched with lignin extracted by alkaline method and grown at 30˚C (i.e.,
BE-Lig at 30˚ C) represent more than 95% of the abundance total relative abundance, the most
abundant families present being: Planococcaceae (37.8%), Paenibacillaceae (17.6%), Flavobac-
teriaceae (13.4%), Bacillaceae (8.6%), Other 20 (Order: Planctomycetales) (2.9%), Other 22
(Order: Sphingobacteriales) (3.5%) and Other 59 (Order: Burkholderiales) (2.4%) (Fig 2A).
For the BE-Lig consortium at 37˚ C (i.e., MG BE-Lig at 37˚ C), the families present in the sixth
passage represent more than 93% of the total relative abundance, with the most abundant fam-
ilies being: Planococcaceae (46.59%), Bacillaceae (32.65%), Paenibacillaceae (3.89%), Other 59
(Order: Burkholderiales) (3.2%), and Sphingomonadaceae (1.51%) (Fig 2B). For consortia
derived from Miracle Growth soil, enriched with Kraft lignin and grown at 30˚ C (i.e. Kraft at
30˚C), the families present in the sixth passage represent more than 96% of the total relative
abundance, the main ones are: Pseudomonadaceae (46.35%), Sphingomonadaceae (10.60%),

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Fig 2. Family level taxonomic profile of bacteria at present in consortia obtained from MG soil over successive
passages in enrichment experiment. M9 medium containing either base-extracted lignin, BE Lig, (panels A and B) or
Kraft lignin (panels C and D) was initially inoculated with MG soil (MG) as the source of microorganisms.
Experiments were performed at two temperatures. 30˚C (panels A and C) and 37˚C (panels B and D). Every 2 weeks
an aliquot was transferred to fresh growth medium for enrichment in successive passages (0, 1, 2, 3, 4, 5 and 6).
Bacterial families are depicted by different colors. For the families classified as Other and Unknown, we were unable to
obtain the taxonomic affiliation at the family level, so the closest previous hierarchical level is provided.
https://doi.org/10.1371/journal.pone.0255083.g002

Other 59 (Order: Burkholderiales) (8.84%), Other 56 (Order: Rhodospirillales) (6.22%), Bru-

cellaceae (3.93%), Erythrobacteraceae (2.87%), Burkholderiaceae (3.59%) and Chitinophaga-
ceae (2.34%) (Fig 2C). For consortia derived from Miracle Growth soil, enriched with Kraft
lignin and grown at 37˚C (i.e. Kraft at 37˚C) the families present in the sixth passage represent
more than 92% of the total relative abundance, the main ones being: Other 59—Order: Bur-
kholderiales (18, 0224%), Brucellaceae (17.3061%), Pseudomonadaceae (11.0626%), Phyllo-
bacteriaceae (10.3328%), Microbacteriaceae (6.3061%), Other 58—Order: Sphingomonadales
(5.3817%), Sphingomonadaceae (5.1807%), Promicromonosporaceae (5.0468%), Erythrobac-
teraceae and (4.9365%) and Hyphomicrobiaceae (3.8272%) (Fig 2D).
A similar analysis was performed for backyard compost derived consortia (Fig 3 and S5
Table). At the end of the enrichment process at the sixth passage, bacterial families that showed
relative abundance greater than 1% accounted for 81%, 81%, 84% and 87% of the total abun-
dance present in the consortia that used base-extracted lignin at 30˚C and at 37˚C (i.e., BE-Lig
at 30˚C, BE-Lig at 37˚C) and those that used Kraft lignin at 30˚C and at 37˚C (i.e., Kraft at

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Fig 3. Family level taxonomic profile of bacteria at present in consortia obtained from backyard (BY) soil over
successive passages in enrichment experiment. M9 medium containing either base-extracted lignin, BE Lig, (panels
A and B) or Kraft lignin (panels C and D) was initially inoculated with backyard (BY) as the source of microorganisms.
Experiments were performed at two temperatures. 30˚C (panels A and C) and 37˚C (panels B and D). Every 2 weeks
an aliquot was transferred to fresh growth medium for enrichment in successive passages (0, 1, 2, 3, 4, 5 and 6).
Bacterial families are depicted by different colors. For the families classified as Other and Unknown, we were unable to
obtain the taxonomic affiliation at the family level, so the closest previous hierarchical level is provided.
https://doi.org/10.1371/journal.pone.0255083.g003

30˚C, and Kraft at 37˚C), respectively. Predominant bacterial families at the sixth passage for
consortia that used base-extracted lignin as carbon source at temperatures 30˚C and 37˚C
were the same and were: Other 59 (Order: Burkholderiales) (29.651%), Bacillaceae (28.628%),
Paenibacillaceae (10.402%), Sphingomonadaceae (6.908%), Xanthobacteraceae (2.944%) and
Brucellaceae (2.837%). For backyard compost derived consortia that used Kraft lignin as car-
bon source cultivated at 30˚C, the predominant bacterial families at the sixth passage were:
Methylobacteriaceae (22.516%), Sphingomonadaceae (11.565%), Other 59 (Order: Burkhol-
deriales) (9.760%), Hyphomicrobiaceae (6.466%), Pseudomonadaceae (4.486%), Other 55
(Order: Rhizobiales) (4.357%), Rhizobiaceae (3.560%), Burkholderiaceae (3.434%), Other 49
(Order: Planctomycetales) (3.210%), Xanthomonadaceae (2.924%), Microbacteriaceae
(2.919%), Erythrobacteraceae (2.421%), Bradyrhizobiaceae (2.341%), Chitinophagaceae
(2.176%) and Beijerinckiaceae (2.041%). Finally, for backyard compost derived consortia that
used Kraft lignin as carbon source cultivated at 37˚C, the predominant bacterial families at the

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Fig 4. Venn diagram of bacterial genera present at the 6th passage of enrichment in consortia obtained using
either base-extracted (BE-Lig) or Kraft lignin (Kraft) as carbon source at cultivation temperatures of 30˚C and
37˚C. Shown in parenthesis is the number of genera for each growth condition with relative abundance above 1%. (A)
Bacterial consortia derived from MG compost. (B) Bacterial consortia derived from backyard (BY) compost.
https://doi.org/10.1371/journal.pone.0255083.g004

sixth passage were: Phyllobacteriaceae (44.365%), Mycobacteriaceae (8.472%), Nocardiaceae

(7.829%), Hyphomicrobiaceae (5.818%), Sphingomonadaceae (4.966%), Promicromonospora-
ceae (4.291%), Burkholderiaceae (3.903%), Erythrobacteraceae (2.673%), Brucellaceae
(2.540%), Other 59 (Order: Burkholderiales) (2.314%).
The Venn diagram in Fig 4A (also see S1 Data) was constructed from genera in the sixth
passage with relative abundance above 1%. It shows the 41 most abundant bacterial genera
(i.e., from 498 MG-derived consortia present at the sixth passage). A total of 9 genera were
present in the consortium that used base-extracted lignin as carbon source at 30˚C (BE-Lig
30˚C); 6 genera were present in the consortium that used base-extracted lignin as carbon
source at 37˚C (BE-Lig 37˚C); 14 genera were present in the consortium that used Kraft lignin
as carbon source at 30˚C (Kraft 30˚C), and 12 genera were present in the consortium that used
Kraft lignin as carbon source at 37˚C (Kraft 37˚C). Only one genus is shared by the four con-
sortia, Other 120, an unknown genus of the Burkholderiales order. The consortia that used
base-extracted lignin as carbon source (i.e., BE-Lig 30˚C and BE-Lig 37˚C) share three genera:
Bacillus, Lysinibacillus and Paenibacillus; meanwhile, the consortia that used Kraft lignin (i.e.,
MG Kraft 30˚C and MG Kraft 37˚C) share four genera: Altererythrobacter, Camelimonas,
Ochrobactrum and Pseudomonas. The genera Sphingobium was shared by the three consortia
BE 37˚C, Kraft 30˚C and 37˚C. The genera present exclusively at BE 30˚C were Other 38
(Family: Flavobacteriaceae), Other 44 (Order: Sphingobacteriales), Other 86 (Phylum: Other),
Other 92 (Order: Planctomycetales) and Other 109 (Family: Rhodobacteraceae). The genera
present exclusively at BE Lig 37˚C were: Microvirga. The genera that appeared exclusively in
Kraft 30˚C were: Burkholderia, Devosia, Other 45 (Family: Sphingobacteriaceae), Other 100
(Class: Alphaproteobacteria), Other 111 (Ordes: Rhodospirillales), Other 124 (Class: Betapro-
teobacteria), Sphingopyxis, and Taibaiella. The genera that appeared exclusively in Kraft 37˚C
were: Aminobacter, Inquilinus, Isoptericola, Microbacterium, Roseomonas and Uncul-
tured_2490 (Family: Hyphomicrobiaceae).
The Venn diagram in Fig 4B (also see S1 Data) shows the 59 most abundant bacterial gen-
era (i.e., from a total of 427) BY derived consortia present at the sixth passage. The diagram
was constructed from genera in the sixth passage with relative abundance above 1%. A total of
10 genera were present in the consortium that used base-extracted lignin as carbon source at
30˚C (BE 30˚C); 15 genera were present in the consortium that used base-extracted lignin as
carbon source at 37˚C (BE 37˚C); 19 genera were present in the consortium that used Kraft

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PLOS ONE Lignin-adapted consortia

lignin as carbon source at 30˚C (Kraft 30˚C), and 15 genera were present in the consortium that
used Kraft lignin as carbon source at 37˚C (Kraft 37˚C). The genera present exclusively at BE
30˚C were Lysinibacillus, Other 42 (Family: Chitinophagaceae), Other 120 (Order: Burkholder-
iales), Unknown_Genus_1000421 (Class: Thermomicrobia), Unknown_Genus_1001041
(Order: Chthoniobacterales). The genera present exclusively at BE Lig 37˚C were: Brevibacillus,
Mesorhizobium, Other 52 (Class: Thermomicrobia), Other 53 (Class: Thermomicrobia), Other
86 (Phylum: Other), Pseudoxanthomonas, Sphingopyxis, Unknown_Genus_1000489 (Family:
Paenibacillaceae). The genera that appeared exclusively in Kraft 30˚C were Azospirillum, Bur-
kholderia, Dokdonella, Kaistia, Microvirga, Terrimonas, uncultured_2499 (Family: Methylobac-
teriaceae). The genera that appeared exclusively in Kraft 37˚C were Hyphomicrobium,
Isoptericola, Mycobacterium, Other 106 (Family: Phyllobacteriaceae), Other 118 (Order: Bur-
kholderiales), and Rhodococcus. There was no genus present in common for the four conditions
(i.e., BE Lig 30˚C, BE Lig 37˚C, Kraft 30˚C and Kraft 37˚C). Only 2 genera were present when
base-extracted lignin was used as carbon source at both cultivation temperatures (Bacillus and
Paenibacillus). On the other hand, 8 genera were present when Kraft lignin was used as carbon
source at both cultivation temperatures (Afipia, Altererythrobacter, Aminobacter, Microbacter-
ium and Other 105 (Order: Rhizobiales).

3.3. Influence of temperature and type of lignin in the selection of bacteria

To evaluate how temperature and type of lignin (i.e., base-extracted or Kraft lignin) influenced
the composition of microbial consortia during the enrichment process, genus level data were
used for Bray-Curtis Non-Metric Multidimensional Scaling (NMDS) matrix dissimilarity anal-
ysis (Fig 4). NMDS graphs present coordinates that represent adjusted distances, therefore, it
is possible to observe the dissimilarity between the taxonomic composition of samples.
Fig 5 shows a stress number of 0.157 for the NMDS grouping of MG- and backyard (BY)
compost-derived consortia, indicating that the representation (i.e., NMDS ordination) of the
data is a good fit. Bacterial consortia present in the original composts, P0_MG and P0_BY, are
highly dissimilar to each other, P0_MG being located in the lower right region of the graph,
and P0_BY in the upper left region of the graph. Furthermore, these consortia are separated
from all other ones shown in the graph. It is also interesting that consortia are separated in the
graph according to the type of carbon source used during the selection process. Regarding the
variable temperature, a pattern of dissimilarity between the samples cannot be observed, indi-
cating that this variable does not appear to influence the composition of the consortia as
strongly as with the type of lignin available as carbon source. To determine the statistical sig-
nificance of this, PERMANOVA testing was used for the individually analyzing variables (Soil/
MG and BY, Passage/0 to 6, Substrate/base extracted and Kraft lignin, Temperature/30˚C and
37˚C). As shown in the S1 File and Fig 5, the variable that best explained variation (~ 32%) of
distances was Substrate (i.e., lignin type: base-extracted or Kraft lignin) for which an R2 of
0.32559 was obtained (p-value = 9.999e-05). Furthermore, temperature, passage and soil pre-
sented R2 values of 0.12246, 0.08914 and 0.05114, respectively. Fig 5 also shows vectors for the
most statistically significant taxa driving the red and green clusters (based extracted lignin and
Kraft lignin, respectively). At P � 0.0001, taxa driving the red cluster were the genera Bacillus
and Lysinibacillus, the family Paenibacillaceae, and order Gemmatimonadales, (see S2 File).
Similarly, at P � 0.0001, the taxa driving the green cluster are the genera Pseudomonas, Afipia,
Mycobacterium, Methylobacterium, Ancylobacter, Aminobacter, Hyphomicrobium, Variibacter,
Sphingobium, Isoptericola, Altererythrobacter and Microbacterium; the order Burkholderiales,
and the family Phyllobacteriaceae (see S2 File).

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PLOS ONE Lignin-adapted consortia

Fig 5. Bray-Curtis analysis of dissimilarity in the non-metric multidimensional scaling matrix (NMDS) to consortia composition at genus level in relation to
the variables type of lignin used as carbon source and temperature. MG- and backyard (BY) compost derived consortia were analyzed. P0 refers to the original
compost; “P1” to “P6” refer to the six passages (selection cycles); The stress number is given at the top. � Stress <0.05 excellent, <0.1 great, <0.2 good and<0.3 bad.
https://doi.org/10.1371/journal.pone.0255083.g005

4. Discussion
4.1. Consortia alpha diversity
Lignin is highly resistant to chemical as well as biological degradation [41]. However,
some groups of fungi and bacteria have the functional machinery for lignin degradation.
Although few bacterial ligninolytic enzymes are known, bacteria are an attractive option for
studying because they can be grown to large scale and be genetically engineered easily [24]. In
addition, prokaryotes are the most abundant group when it comes to microbial soil communi-
ties [42–44].
In this study, two sources of microorganisms (i.e., commercial MG soil and backyard com-
post soil), two different temperatures (i.e., 30˚C and 37˚C) and two types of lignin (i.e., base-
extracted and Kraft) were used to maximize the different kinds of bacteria selected during
enrichment. At the end of the protocol, eight microbial consortia enriched for the ability to
degrade lignin were obtained. The use of enriched microbial consortia has been considered as
a promising approach to identify microorganisms involved in lignin degradation and

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conversion [10, 27, 30]. The enrichment process is expected to select for microorganisms that
carry enzymatic machinery to degrade lignin and use it as carbon source. In our study, there
was an approximate 6- and 2- fold reduction in the number of OTUs present in the original
soil samples and at the sixth passage of enrichment of MG derived and backyard compost
derived consortia, respectively. Comparative analysis of richness and diversity indexes between
the different consortia passages (i.e., number of OTUs observed, Chao 1, PD whole tree, Shan-
non and 1-Simpson) showed that there is an overall pattern of decreasing richness and diver-
sity, similar to what was described by Yu et al. (2015) [27]. Nevertheless, there were small
fluctuations in the values found for expected richness and diversity indexes when microbial
consortia of successive passages are analyzed. It is important to point out that these consortia
are composed of living microorganisms that interact in an intraspecific and interspecific man-
ner; thus, the dynamics of the consortia tend to be constantly changing.
Similar results of decreased diversity were also observed by Moraes et al. (2018) [10]. when
using an enrichment approach to obtain a microbial consortium (LigMet) from sugarcane
plantation soil, using lignin as carbon source for 50 weeks. This decrease in diversity is an
expected response as in each subculturing cycle there is a progressive selection of microorgan-
isms that display adaptive characteristics. During the enrichment process, the different lignin
substrates, temperatures and cultivation time used favor selection of families that have the
enzymatic machinery necessary to degrade lignin, and resist toxicity of the compounds gener-
ated by this process, therefore some bacteria become dominant at the expense of others
[10, 30].
Consortia started from backyard compost soil showed higher initial richness and diversity
in comparison to consortia derived from MG soil, a commercial product. Furthermore, there
is a legacy effect for the initial inoculum as the higher bacterial richness and diversity for back-
yard compost derived consortia persisted at the sixth passage after successive cycles of selec-
tion. Therefore, in future similar experiments if the intention is to maximize different taxa at
the end of the selection process, it may be important to choose a highly diverse source of
microorganisms for the initial inoculum.
Our data also show influence of the type of carbon source on richness and diversity indexes.
At the end of the enrichment process, consortia that used lignin extracted by alkaline method
as carbon source had a higher number of observed OTUs, a higher PD whole tree index and a
higher Chao 1 richness index, when compared to the sixth passage of the consortia that used
Kraft lignin. It is likely that the lignin extracted by the alkaline method from plant biomass pre-
sented some contamination with carbohydrates (i.e., hemicellulose), which would act as an
alternative carbon source. This alternative carbon source may have allowed the presence of
bacterial species that do not use lignin in the environment as an exclusive carbon source, and
thus may have contributed to a higher richness and diversity.

4.2. Dynamics of taxonomic profiles during enrichment

In this study, sequencing data were analyzed down to the genus level, as it was not possible to
perform taxonomic affiliation at the level of species. Analysis of the consortia bacterial compo-
sition based on the amplification and sequencing of 16S rDNA are dependent on the sequences
already deposited in public databases [45]. At the end of successive sub culturing cycles,
although with reduced richness and diversity, the consortia obtained are still highly complex,
being composed of dominant as well as rare species. Despite the fact that rare microorganisms
are important to ecosystem function, in our work we focused on the abundant taxa [46].
Taxonomic profile graphs at family level revealed the bacterial composition and dynamics
of consortia across the six passages (Figs 2 and 3). Bacterial consortia tended to stabilize in

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terms of family composition after the fourth passage. A similar result was obtained by Yu et al.
(2015) [27] in a lignin degrading microbial consortium enrichment study, where the consor-
tium reached phylum-level stability after the third passage. In the work by Fang et al. (2018)
[29], the microbial community of the ligninolytic consortia inoculated with samples of decay-
ing tree trunks and soil close to the trunks reached stability in their genera composition after 6
days of cultivation. These data suggest that the taxonomic profile of consortia at different hier-
archical levels may reach stability in terms of the composition at different times.
When observing the family composition of each consortium, it is noticeable that each con-
sortium tends to acquire its own specific profile. The divergence in bacterial family composi-
tion among consortia indicates that the variables used during the enrichment process, such as
temperature and substrate, changed the composition of initial bacteria in the consortia. Our
results indicate that the type of lignin used as a carbon source in the enrichment experiment,
Kraft lignin or lignin extracted by alkaline method, favored distinct groups of bacteria. At pas-
sage sixth, Kraft lignin favored families of the Proteobacteria phylum, while base-extracted lig-
nin favored the Firmicutes phylum. Kraft lignin is a highly pure commercial lignin. As
previously mentioned, during the manual process of lignin extraction by alkaline method,
small amounts of carbohydrates may remain, and act as an alternative source, acting as a
“start-up” carbon source for the degradation of polymers [47]. Certain groups of microorgan-
isms require an easily metabolizable co-substrate for degradation of aromatic compounds, as
in the case of lignin biodegradation [48, 49]. Alternative sources of carbon may be needed to
promote Kraft lignin degradation due to its high molecular weight [50]. The literature points
to some studies in which the genera Lysinibacillus, Paenibacillus and Bacillus from the phylum
Firmicutes act on the degradation of Kraft lignin in the presence of 1% glucose and 0.5% pep-
tone as an alternative source of carbon and nitrogen [50–52]. Therefore, the favoring of differ-
ent families of Proteobacteria or Firmicutes according to the type of lignin used may be related
to the need of the phylum Firmicutes for an alternative carbon source to start the process of
lignin degradation. It is also possible that representatives of the Proteobacteria phylum are
strong competitors favored in the presence of Kraft lignin, not allowing the colonization of the
environment by species of the phylum Firmicutes. Also note that, although not predominant,
bacteria from the Firmicutes phylum were present in the consortia fed with Kraft lignin, so it
cannot be ruled out that the genera of the phylum Firmicutes present in our experiments can
act on Kraft lignin in the absence of alternative carbon sources.
The phyla Bacteroidetes and Actinobacteria have also been identified in microbial consortia
enriched to degrade lignin [10, 27, 30, 53]. However, only one representative of the phylum
Actinobacteria was identified in the sixth passage of the consortia inoculated with Kraft lignin:
Microbacteriaceae. The Microbacteriaceae family was also present in the enriched Lig Met
consortium that used Kraft lignin as carbon source reported by Moraes et al. (2018) [10].
Some of the genera and families found in this work have previously been identified by their
ligninolytic activity. In the enriched Lig Met consortium that used Kraft lignin isolated from
black liquor obtained from delignification of sugarcane bagass, reported by Moraes et al.
(2018) [10], it was possible to identify the classes Betaproteobacteria, Alphaproteobacteria,
Gammaproteobacteria and members of the phyla Actinobacteria and Firmicutes. Similar to
our results, Moraes et al. (2018) found that representatives of the phylum Proteobacteria were
the most abundant in the consortium grown with Kraft lignin. Within the phylum Firmicutes
that was also reported in this work, Paenibacillus from the phylum Firmicutes was one of the
main genera identified. In our work, the genus Paenibacillus is one of the most abundant in
the sixth passage of the enriched consortia that used lignin extracted by alkaline method as car-
bon source, but its relative abundance was less than 0.5% in the sixth passage of consortia
enriched using Kraft lignin.

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Another interesting fact is that the Flavobacteriaceae family of the order Flavobacteriales,
Pseudomonadaceae of the order Pseudomonadales, Sphingobacteriaceae of the order Sphingo-
bacteriales also have a great representation in the sixth passage of consortia inoculated with
both soils. The Sphingobacteriales and Pseudomonadaceae were present in the sixth passage in
three of four consortia inoculated with Miracle Growth (i.e., MG BE LIG 30˚C, MG Kraft
30˚C and 37˚C (Fig 2A, 2C and 2D). In the consortia inoculated with backyard compost soil,
the Pseudomonadaceae was present in two of four consortia (i.e., BY BE LIG 30˚C and BY BE
LIG 37˚C (Fig 3A and 3B). The Flavobacteriaceae family was present in MG BE LIG 30˚C, MG
Kraft 30˚C and BY Kraft 30˚C (Figs 2A, 2C and 3C). These orders were also identified by Jimé-
nez et al. (2016) [28] in the microbial consortium SG-M enriched to degrade maize straw that
showed activity to degrade lignin.
Bacterial families of the classes Betaproteobacteria, Alphaproteobacteria, Gammaproteo-
bacteria, Bacilli, Planococaceae were found in all consortia derived from MG and backyard
compost soils, and these classes are frequently studied for presenting species capable of degrad-
ing lignin and its aromatic compounds [10]. Some genera of these classes have been reported
as ligninolytic bacteria, namely Ochrbactrum, Rhizobiales, Sphingobium, Lysinibacillus and
Bacillus [10, 54].
Analyzing the taxonomy data of the bacterial families of the microbial consortia MG BE lig
30˚C and MG Kraft 30˚C (Fig 2), the third passage proved to be different in terms of the com-
position of families from the rest of the passages. Note that in the third passage of the consor-
tium MG BE lig 30˚C, the families Pseudomonadaceae and Sphingomonadaceae of the
phylum Protebacteria become dominant, while the families Bacillaceae and Planococcaceae
decrease in terms of abundance (Fig 2A). The opposite occurs in the third passage of the con-
sortium MG Kraft 30˚C, where the families Bacillaceae and Planococcaceae become dominant
while the families Pseudomonadaceae and Sphingomonadaceae decrease in terms of abun-
dance (Fig 2C). It is also noticeable a change that occurs in the BE lig 37˚C consortium, where
the Bacillaceae family suddenly increases its relative abundance in the third pass (from 19% to
54%) and decreases the abundance in the fourth pass again to 21% (Fig 2B). The same
occurred in relation to the taxonomy data of the bacterial families of the microbial consortia
inoculated with backyard compost soil. In consortium BY BE lig 30˚C, BY BE lig 37˚C and BY
Kraft 30˚C in the third passage, the family Paenibacillaceae is high in abundance and then in
the fourth passage it decreases significantly (Fig 3A, 3B and 3C). There are different hypothesis
to explain this atypical data found in both experiments with MG and backyard compost soil: 1.
this dynamic is due to the multiple interactions among the microbial community members
including competition between species; 2. poor sample homogenization, which could lead to
poor representation of the families present; 3. presence of spores of microorganisms that ger-
minated during the enrichment. Some genera of the phylum Firmicutes have been studied
extensively for their ability to form endospores [55–58]; and 4. presence of ciliated protozoa
detected by sequencing the ITS region (S3 Table), which can feed on bacteria [59]. The activity
of ciliates in microbial consortia can affect the dynamics of the bacterial community [10].
Venn diagrams (Fig 4) were useful to show information at the genus level, and provided
insight about which genera were exclusive to certain conditions and which were the common-
alities. Venn diagrams showed that the genus Sphingobium was present in three of the four
consortia inoculated with MG soil and in the two of four consortia inoculated with backyard
compost soil. This genus showed highest abundance in the enriched consortia that used Kraft
lignin as carbon source (i.e., MG Kraft 30˚C and BY Kraft 30˚C). Previous works demonstrate
that species of this genus present ligninolytic enzymes and pathways to catabolize the com-
pounds generated in the degradation of lignin [60, 61]. In this study, this genus managed to

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PLOS ONE Lignin-adapted consortia

persisted despite the difference in temperature and lignin variables, confirming what previous
studies pointed out about its well-studied ligninolytic capacity.
The genus Microvirga represents 1% of the abundance of the MG-soil derived bacterial con-
sortium in the sixth passage of that used base-extracted lignin and was grown at 37˚C (i.e., MG
BE lig 37˚C) and this genus is also present in the sixth passage of the backyard compost soil
derived consortia that used Kraft lignin and was grown at 30˚C (i.e., Kraft Lig 30˚C). A species
in this genus isolated from nodules of the plant Vicia alpestris, Microvirga ossetia, had its
genome completely sequenced and this allowed identification of a laccase [62]. This enzyme is
part of the group of oxidoreductases, enzymes that are capable of degrading lignin [20, 21].
The Aminobacter genus of the Phyllobacteriaceae family is present in the microbial consor-
tia MG Kraft 37˚C and BY Kraft 37˚C in the sixth passage (Figs 2, 3 and 4). Although no previ-
ous studies of enriched microbial consortia for lignin degradation have identified species of
the genus Aminobacter, sequencing of the genome from a member of this genus indicates the
presence of a ligninolytic enzyme, a laccase [63]. This representative of the genus in our experi-
ments may have a role on the degradation of lignin, indicating the need for further characteri-
zation of the activities by bacteria of this genus.
For OTUs classified as Other or Unknown throughout this work, we were unable to assign
the sequence to a taxon [45]. This result may indicate the lack of sequences in databases to
compare the results obtained or it may indicate that for these OTUs the sequence presents
errors and it was not possible to find homologous sequences in the databases. Nevertheless,
OTUs classified in Other and Unknown should not be disregarded as, most likely, they repre-
sent groups not yet classified with ligninolytic activity.
The Venn diagram shows that the OTU at the genus level Other 120 (family Other 59) of
the order Burkholderiales appeared in all MG soil derived consortia having the greatest abun-
dance in the consortium enriched to degrade Kraft lignin and grown at 37˚C (Figs 2D and
4A). This genus (Other 120) appeared in all BY soil derived consortia as well, and had the high-
est abundance in the consortium BE Lig 37˚C (Figs 2B and 4B). Despite the lack of taxonomic
classification at the genus level, this OTU should not have its ligninolytic activity disregarded
since species of this order have already been described in the literature acting on lignin degra-
dation. As an example, the species Burkholderia sp. strain CCA53 of the order Burkholderiales
was identified based on 16S rDNA gene sequencing and it could grow in culture medium with
lignin monomers or alkali lignin as the only carbon source proved that this species expresses
ligninolytic enzymes that allow lignin degradation [64]. This strain, in addition to degrading
lignin, uses the released aromatic compounds as a carbon source.
For the genera Other 86 present in the MG BE Lig 30˚C and BY BE Lig 37˚C consortia (Fig
4), we were unable to assign the taxonomic classification at any hierarchical level (Figs 2 and
3). It can be inferred for this genus that the type of lignin used in the enrichment process was
an important variable since this genus remains abundant only in consortia enriched with lig-
nin extracted by alkaline method. On the other hand, this genus seems to support temperature
variation. Despite appearing with relative abundance higher than 1% in the sixth passage of
the referred consortia, its identity remains unknown.

4.3. Influence of temperature and type of lignin in the selection of bacteria

When compared to fungi, bacterial ligninolytic enzymes have a lower redox potential, so struc-
tural differences in lignin would lead to the need for different enzymes to degrade different
parts of lignin [65]. It is believed that for the microbial consortia analyzed in this work, the
bacteria would be acting in cooperation to degrade different fractions of lignin. It is known
that different types of pre-treatment can give rise to different three-dimensional structures of

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PLOS ONE Lignin-adapted consortia

lignin and it is known that the process of biodegradation of lignin by microorganisms depends
on the type of lignin structure [19, 65–68]. Therefore, the structural differences between Kraft
lignin and lignin extracted by alkaline method may be sufficient to select different groups of
microorganisms capable of degrading them.
In this work, Bray-Curtis dissimilarities were visualized with NMDS. Fig 5 showed group-
ing according to the type of carbon source, as well as grouping of MG- and backyard (BY)
compost-derived consortia using Kraft lignin as carbon source. To test the significance of
these results, PERMANOVA testing was performed and the amount of variance correlated
with each variable studied (type of soil, passage, type of lignin and temperature) was obtained.
PERMANOVA is a method that uses permutation for non-parametric multivariate analysis of
variance. The type of lignin, base-extracted or Kraft lignin, was able to best explain variation
and corresponded to approximately 32% of the variation observed. Therefore, despite the tem-
perature being one of the important physical factors that affects microbial growth, the varia-
tion of 7˚C between the cultivation temperatures was not as important as the type of carbon
source to differentiate the consortia. Although microorganisms have an optimal growth tem-
perature, but they can also grow at a range of temperatures [69]. As mentioned previously,
base-extracted lignin may have been less pure, and therefore may be metabolized by bacteria
that are able to use lignin as well as other alternative contaminating carbon source. On the
other hand, Kraft lignin would select for groups of bacteria that can specifically metabolize this
type of lignin. Therefore, it would be expected that the taxa driving the Kraft lignin cluster in
Fig 5 would be more stringent in their ability to use lignin as a carbon source.

5. Conclusions
Most works about degradation of lignin focus on fungi. To increase our knowledge on lignino-
lytic bacteria, we used the powerful method of culture enrichment to select from highly diverse
soil microorganisms the ones most adapted to using lignin as a carbon source. To the best of
our knowledge our work has used the most variables to obtain the consortia (two sources of
inoculum, two cultivation temperatures and two types of lignin). High-throughput 16S rRNA
gene sequencing was used to assess the bacterial community structure during the enrichment
process. Consistent with a selection process, diversity indexes showed a decrease in bacterial
richness as well as diversity in consortia at the end of six enrichment cycles. Analysis of bacte-
rial composition over 2-week enrichment cycles indicated that stability was achieved beyond
the fourth cycle. Although the focus of the present work was bacteria, the presence of ciliates
in the consortia may have influenced the dynamics of the bacterial community in the micro-
bial consortia. The main known bacterial genera selected to use lignin as a carbon source were
Altererythrobacter, Aminobacter, Bacillus, Burkholderia, Lysinibacillus, Microvirga, Mycobacte-
rium, Ochrobactrum, Paenibacillus, Pseudomonas, Pseudoxanthomonas, Rhizobiales and.
Sphingobium. There were also OTUs classified as Other and Unknown indicating the presence
of bacteria not yet resolved taxonomically that may play a direct or indirect role in lignin deg-
radation. These bacterial genera can be of particular interest for studying lignin degradation
and utilization as well as for the development of lignin-related biotechnology applications.

Supporting information
S1 Table. Diversity indexes for the consortia in the original MG compost soil (0P) and six
enrichment cycles or passages (1P, 2P, 3P, 4P, 5P and 6P) using either base-extracted of
Kraft lignin as carbon source and cultivated either at 30˚C or 37˚C.
(DOCX)

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PLOS ONE Lignin-adapted consortia

S2 Table. Diversity indexes for the consortia in the original backyard (BY) compost soil
(0P) and six enrichment cycles or passages (1P, 2P, 3P, 4P, 5P and 6P) using either base-
extracted of Kraft lignin as carbon source and cultivated either at 30˚C or 37˚C.
(DOCX)
S3 Table. Relative abundance (%) of phylum ciliophora for the consortia in the original
backyard (BY) compost soil (0P) and six enrichment cycles or passages (1P, 2P, 3P, 4P, 5P
and 6P) using either base-extracted of Kraft lignin as carbon source and cultivated either
at 30˚C or 37˚C. Data based on sequencing of the ITS region� . � Primers used for amplification
were FW (ITS9): 5’ GAA CGC AGC RAA IIG YGA 3’ and RV (ITS4): 5’ TCC TCC
GCT TAT TGA TAT GC 3’ [34].
(DOCX)
S4 Table. Percentage of each bacterial group at family level depicted in Fig 2 present in
consortia obtained from MG soil (MG) over successive passages (0, 1, 2, 3, 4, 5, 6) in
enrichment experiment using M9 medium containing either base-extracted lignin, BE, or
Kraft lignin, at two temperatures. 30˚C and 37˚C. For the families classified as Other and
Unknown, we were unable to obtain the taxonomic affiliation at the family level, so the closest
previous hierarchical level is provided.
(DOCX)
S5 Table. Percentage of each bacterial group at family level depicted in Fig 2 present in
consortia obtained from backyard soil (BY) over successive passages (0, 1, 2, 3, 4, 5, 6) in
enrichment experiment using M9 medium containing either base-extracted lignin, BE, or
Kraft lignin, at two temperatures. 30˚C and 37˚C. For the families classified as Other and
Unknown, we were unable to obtain the taxonomic affiliation at the family level, so the closest
previous hierarchical level is provided.
(DOCX)
S1 File. Variance of the Bray Curtis dissimilarities matrix associated with substrate (base
extracted and Kraft lignin), temperature (30˚C and 37˚C), Passage (0 to 6) and soil (MG
and BY). Bray Curtis matrix PERMANOVA analysis was performed using the adonis function
of vegan package in R.
(DOCX)
S2 File. Taxa driving clusters (p-value < = 0.0001) based on substrates (lignin type: base
extracted and Kraft lignin) observed in NMDS plot (Fig 5). Vectors were found using the
envfit function of vegan package in R performing 10,000 permutations.
(XLSX)
S1 Data. Supplementary data for Fig 4.
(DOCX)

Acknowledgments
The authors would like to thank CLP from Embrapa-Agroenergy for insightful comments.

Author Contributions
Conceptualization: Blake A. Simmons, John M. Gladden, Betania Ferraz Quirino.
Data curation: Renata Henrique Santana.

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PLOS ONE Lignin-adapted consortia

Formal analysis: Isis Viana Mendes, Mariana Botelho Garcia, Renata Henrique Santana, Phi-
lippe de Castro Lins, Rafaella Silveira.
Funding acquisition: Blake A. Simmons, John M. Gladden, Ricardo Henrique Kruger, Betania
Ferraz Quirino.
Investigation: Isis Viana Mendes, Mariana Botelho Garcia, Renata Henrique Santana, Betania
Ferraz Quirino.
Methodology: Renata Henrique Santana.
Resources: John M. Gladden, Ricardo Henrique Kruger.
Supervision: Renata Henrique Santana, Betania Ferraz Quirino.
Writing – original draft: Isis Viana Mendes, Mariana Botelho Garcia, Ana Carolina Araújo
Bitencourt, Philippe de Castro Lins.
Writing – review & editing: Ana Carolina Araújo Bitencourt, Philippe de Castro Lins, Rafaella
Silveira, John M. Gladden, Ricardo Henrique Kruger, Betania Ferraz Quirino.

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