Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

Bacteria associated with the alga Ulva lactuca (Ulvaceae)

from the Colombian Caribbean and their laccase activity

Nohora Millán-Aldana1; https://orcid.org/0009-0006-8551-9689

Natalia Comba1; https://orcid.org/0000-0001-6359-8474

Johanna Santamaría1*; https://orcid.org/0000-0001-5345-6683

1. Área de Ciencias Biológicas y Ambientales, Universidad de Bogotá Jorge Tadeo Lozano, Carrera 4ta #22-61, Bogotá,

D.C., Colombia; nohoraa.millana@utadeo.edu.co, natalia.comba@gmail.com, johanna.santamaria@utadeo.edu.co

(*Correspondence)

Received 03-IX-2024. Corrected 19-I-2025. Accepted 08-X-2025.

ABSTRACT

Introduction: Marine macroalgae, their associated bacteria and the environment, interact to produce com-

pounds that aid the holobiont in adapting to biotic and abiotic challenges. These compounds include several

novel enzymes with industrial applications and with less environmental impact than industrial chemical reac-

tions. Laccases are an example of enzymes that are of interest due to their wide range applications, and their ver-

satility is a subject of research and exploration. Despite the abundance of macroalgal holobionts in the Caribbean

region of Colombia, little is known about the microorganisms associated with these hosts and their potential for

biotechnology.

Objective: To evaluate the epibiont and endobiont bacteria associated with the macroalga Ulva lactuca present in

Santa Marta, Colombian Caribbean, and to search for laccase producers among them.

Methods: Culture techniques were used to isolate bacteria from U. lactuca collected on February 27, 2023.

The 16S rRNA region was sequenced to determine the identity of the different isolates. Lacase production was

screened by inoculating the isolates in guaiacol medium, which was later confirmed in nutrient agar with 0.2 %

dimethoxyphenol.

Results: 118 isolates were obtained, of which 64 were epibionts and 54 were endobionts. 75 % were identified

to genus and species level. The predominant epibiont isolates were Proteobacteria, especially Vibrio, while

Firmicutes, with Bacillus, had a higher representation in the endobiont isolates. Laccase activity was found in 42

isolates including Enterobacter, Halomonas, Paenibacillus, Priestia, Pseudomonas, Shewanella, and Vibrio. Among

them, endobionts related to Bacillus had the highest number of isolates positive for laccase.

Conclusions: Proteobacteria and Firmicutes dominated the culturable bacterial community of U. lactuca. This

study indicates that several bacterial genera associated with U. lactuca in the Colombian Caribbean are positive

for laccase activity. Further research is needed to explore the potential industrial applications of these enzymes.

Key words: seaweed; culturable marine bacterial diversity; biological substance of interest; laccase screening.

RESUMEN

Bacterias asociadas con el alga Ulva lactuca (Ulvaceae) del Caribe colombiano y su actividad lacasa

Introducción: Las macroalgas marinas, sus bacterias asociadas y el medio ambiente interactúan para producir

compuestos que ayudan al holobionte a adaptarse a desafíos bióticos y abióticos. Estos compuestos incluyen

enzimas novedosas con aplicaciones industriales y con un menor impacto ambiental que las reacciones químicas

https://doi.org/10.15517/8pkmas90

OTHER

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INTRODUCTION

Macroorganisms provide a habitat for

microorganisms, which can live on the surface

of the host as epibionts, and within their tis-

sues as endobionts (Kumar et al., 2017; Kumar

et al., 2019; Tadych & White, 2009). Together,

a macroorganism and its associated microbial

community, along with their interactions, form

a holobiont. The microorganisms involved in

this association have the potential to be either

latent pathogens or mutualistic symbionts that

form a mutually beneficial ecological relation-

ship with the host organism (Bordenstein &

Theis, 2015; Tadych & White, 2009; van der

Loos et al., 2019).

The symbiotic microorganisms in the

holobiont produce metabolites that promote

the growth and overall health of the host by

enhancing its nutrient uptake and strengthen-

ing its defenses against pathogen attack (Aze-

vedo et al., 2000; Armstrong et al., 2001; Chen

et al., 2022; Saha & Weinberger, 2019). They

also enhance host resistance to abiotic stressors,

such as drought and salinity (Arnold et al., 2003;

Devarajan et al., 2021; Hardoim et al., 2015;

Kannadan & Rudgers, 2008; Waller et al., 2005).

Notably, within these communities, micro-

organisms produce enzymes and other com-

pounds that enhance microbial resilience to

host defenses against colonizing microorgan-

isms (Egan et al., 2013; Kumar et al., 2017) and

substances that prevent biofouling and preda-

tion by inhibiting the growth of other microor-

ganisms (Sánchez-Rodríguez et al., 2018). They

also play an active role in decomposing senes-

cent host biomass (Kumar et al., 2017; Kumar

et al., 2019). Furthermore, ecological studies

have documented substantial genetic variability

among microorganisms in holobionts (Guyo-

mar et al., 2018), thereby reflecting their exten-

sive biochemical capabilities and remarkable

involvement in ecosystem processes through

their participation in the decomposition of

senescing biomass. These microbial communi-

ties play a crucial role in the biogeochemical

cycles of carbon, nitrogen, and phosphorus

(Abril et al., 2005; Bell, 2008; Fürnkranz et al.,

2008; Moyes et al., 2016; Pita et al., 2018; Pura-

hong et al., 2010; Sun et al., 2011; Voříšková &

Baldrian, 2012).

Understanding the ecological relationships

between hosts, microbial epibionts, and endo-

bionts is crucial for understanding ecosystem

industriales. Las lacasas son enzimas que suscitan interés debido a sus aplicaciones y versatilidad, por lo que son

objeto de investigación y exploración. A pesar de la gama de holobiontes macroalgales en el Caribe colombiano,

poco se conoce sobre los microorganismos asociados a estos hospederos y su potencial biotecnológico.

Objetivo: Evaluar las bacterias epibiontes y endobiontes asociadas a la macroalga Ulva lactuca presente en Santa

Marta, Caribe colombiano, y buscar productores de lacasas.

Métodos: Se emplearon técnicas de cultivo para aislar bacterias de U. lactuca recolectadas el 27 de febrero de

2023. Se secuenció la región 16S ARNr para determinar la identidad de los aislamientos. La actividad lacasa se

comprobó inoculando los aislamientos en medio suplementado con guayacol y posteriormente se confirmó en

agar nutritivo con 0.2 % de dimetoxifenol.

Resultados: Se obtuvieron 118 aislamientos, siendo 64 bacterias epibiontes y 54 endobiontes. El 75 % de

ellas se identificaron a nivel de género y especie. Los aislamientos epibiontes predominantes pertenecen a las

Proteobacterias, en particular Vibrio, mientras que los Firmicutes, con Bacillus, tuvieron una mayor represen-

tación en los aislamientos endobiontes. Se encontró actividad lacasa en 42 aislamientos, incluidos Enterobacter,

Halomonas, Paenibacillus, Priestia, Pseudomonas, Shewanella, y Vibrio. Entre ellos, los endobiontes afiliados a

Bacillus presentaron el mayor número de aislamientos positivos para lacasas.

Conclusiones: Proteobacterias y Firmicutes predominaron en la comunidad bacteriana cultivable de U. lactuca.

Este estudio indica que varios géneros de bacterias asociadas a U. lactuca en el Caribe colombiano son positivas

para actividad lacasa. Se requiere investigación adicional para explorar los potenciales usos de estas enzimas.

Palabras clave: algas marinas; diversidad bacteriana marina cultivable; sustancia biológica de interés; cribado de

lacasas.

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functioning and for discovering novel second-

ary metabolites, antimicrobials, and enzymes

with biotechnological potential (Newman &

Cragg, 2015; War Nongkhla & Joshi, 2014).

The biotechnological potential of epibionts and

endobionts communities has been evaluated in

marine organisms (Pita et al., 2018, Sánchez-

Rodríguez et al., 2018). However, the coexis-

tence of these two communities within a single

host (i.e., from a holobiont perspective) has not

yet been thoroughly investigated. The study

of the entire microbial community associated

with a host is necessary, as it lays a foundation

for understanding holobiont interactions and

allows the relationship between these microbial

communities and the physiological function of

the host to be assessed. Furthermore, holobiont

interactions are important in bioprospecting, as

many substances of interest to industry are part

of the traits that result from such interactions

(Pita et al., 2018).

An ideal holobiont for the study of its asso-

ciated microbial diversity and biotechnological

potential is the marine macroalga Ulva lactuca.

U. lactuca is a sessile organism that lives in envi-

ronments with wide variations in temperature,

radiation, salinity, and osmotic stress, such as

rocky coastlines. Due to these environmen-

tal conditions, the microorganisms associated

with U. lactuca produce substances with a wide

range of biochemical properties and functions

(Beygmoradi & Homaei, 2017; Busetti et al.,

2017). Furthermore, the microbial communi-

ties associated with macroalgae, which reach

average densities between 106 and 109 bacteria

cm-2 (Egan et al., 2013), represent microbial

diversity hotspots that are a promising source

of new molecules.

Most reports of bacteria associated with

Ulva sp., have come from studies conducted

in Australia, the Baltic and North Seas in Ger-

many, and the North Atlantic in the United

Kingdom, Portugal, and Spain (Singh & Reddy,

2014). Until recently, there were no published

studies on the ecology and diversity of micro-

organisms associated with marine macroalgae

in the Caribbean, nor on the potential biotech-

nological applications of these communities.

A first study by Comba-González et al. (2018)

investigated the epibiont bacterial community

associated with the macroalga U. lactuca off the

coast of Santa Marta, Colombia, and showed

that these epibiont bacteria synthesize amy-

lases, lipases, cellulases, agarases, and sidero-

phores. However, as this pioneering study only

addressed epibiont characteristics, the biologi-

cal aspects (such as phylogenetics, functional

diversity and biotechnological potential) of the

joint assessment of epibionts and endobionts

of macroalgal hosts in the Caribbean remain

to be investigated.

Currently, fungal, and bacterial enzymes

with industrial applications are in high demand

because their use has a lower environmental

impact than industrial oxidation reactions that

lead to undesirable side reactions and the pro-

duction of hazardous pollutants (Fernández &

Gómez-Dégano, 2017). Enzyme types current-

ly in demand include amylases, glycosidases,

pullulanases, agarases, lyases, galactosidases,

cellulases, xylanases, chitinases, cresolases, pro-

teases, lipases, hydrogenases, and laccases

(Kennedy et al., 2008). Laccases are polyphe-

nol oxidases (PPOs) that belong to the family

of multicopper blue oxidases (MCOs). These

enzymes perform substrate oxidation, reducing

molecular oxygen to water without producing

harmful by-products. Current applications of

laccases include dye degradation and detoxifi-

cation, biobleaching of paper pulp, wastewater

pretreatment, biodegradation of environmen-

tal xenobiotics, ethanol production, biosensor

production, and drug synthesis (Agarwal et al.,

2022; Harris, 2017).

Bacterial laccases have gained interest due

to their sustained activity at elevated tempera-

tures, stability at extreme pH, and exceptional

stability in the presence of inhibitors such as

halides, organic solvents, and at high salt con-

centrations (Agarwal et al., 2022). Nevertheless,

despite these encouraging properties, bacterial

laccases have not been adequately studied and

produced (Maharsiwi et al., 2020). Only lac-

cases of fungal origin have been produced on a

large scale (Agarwal et al., 2022). However, their

applications have been limited due to the slow

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growth rate of fungi, low tolerance to adverse

conditions, unsuitable growth in liquid media,

preference for low pH ranges, and long fermen-

tation times (Chandra & Chowdhary, 2015).

Laccase bioprospecting in bacteria associ-

ated with marine macroorganisms is a prom-

ising endeavor because enzymes are most

likely to catalyze reactions under alkaline pH

and fluctuating temperature conditions in the

marine environment. Applications in the textile

industry (Subash & Muthiah, 2021), biofu-

els (Ragauskas et al., 2006), and degradation

of residual crop biomass (Galić et al., 2021)

require laccases that are stable under harsh con-

ditions such as ionic liquids, extreme pH, and

high temperatures. Such conditions occur, for

example, in the degumming of high lignocellu-

lose natural fibers (Barber-Zucker et al., 2022).

Therefore, the objectives of the present

study were, first, to evaluate the microbial

community associated with the macroalgae U.

lactuca present in the Colombian Caribbean,

assessing the composition of its epibiont and

endobiont bacteria based on a culture approach,

and second, to continue the bioprospecting

work looking for laccase-producing bacteria.

MATERIALS AND METHODS

Sampling site: Samples of U. lactuca were

collected on January 27, 2023, from the site “La

Punta de la Loma” (11°07’00.9’’ N & 74°14’01.3’’

W) in Santa Marta, Colombia (Fig. 1). The

sampling site is primarily a fossil coral reef

with sand cover and scattered batholith rocks

from the nearby Sierra Nevada de Santa Marta

(Márquez & Patiño, 1986). The climate in this

area is determined by the prevailing patterns

that affect the Colombian Atlantic coast. The

Intertropical Convergence Zone (ITCZ) causes

a dry season, typically between December and

April, followed by a rainy season from May

to December (García-Hoyos et al., 2016). The

rocky platform found in this location provides

ideal conditions to support a rich diversity of

macroalgae, among which the U. lactuca species

is dominant (Diaz-Pulido & Díaz-Ruíz, 2003;

García & Diaz-Pulido, 2006).

Sample collection: Sterile tweezers were

used to detach 36 whole macroalgal thalli from

rocks in the constant wave intertidal zone

(Fig. 2). These thalli were washed with sterile

sea water to remove surface solids and macro-

organisms (Tujula et al., 2010). They were then

Fig. 1. Study area “Punta de la Loma” (Colombian

Caribbean), adapted from Camacho and Montaña-

Fernández (2012).

Fig. 2. Morphological characteristics of Ulva lactuca (Ismail

& Mohamed, 2017).

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placed in 200 ml sterilized containers, which

were transported to the laboratory in a portable

cooler at 4 °C. Once in the laboratory, each of

the collected thalli was rinsed with a sterile 0.85

% saline solution to remove any remaining sand

or embedded macroorganisms.

Isolation of epibiont bacteria: Three

methods were used to isolate epibiont bacte-

ria from U. lactuca to maximize the number

of isolates: i) Epibiont resuspension. This was

performed by obtaining three 1 g wet-weight

fragments from the three macroalgal samples.

These fragments were then placed in 50 ml

of a sterile saline solution (0.85 % concentra-

tion) and shaken at 130 rpm for 25 min. Then,

100 µl of the resulting solution was added in

triplicate to a Petri dish containing nutrient

agar with NaCl (1 % w/v). ii) Swabbing. The

macroalgal surface was scraped with a sterile

swab, then nutrient agar with NaCl (1 % w/v)

was inoculated by exhaustion with it. iii) Direct

inoculation. Three 1 g fragments of macroalgal

thallus were placed in the center of Petri dishes

containing Zobell marine agar.

After inoculation, all cultures were incu-

bated at 29 °C, like the temperature recorded

at the collection site. The different isolates were

reinoculated on nutrient agar with NaCl (1 %

w/v) until pure isolates were obtained.

Isolation of endobiont bacteria: Two pro-

tocols were followed to obtain endobiont bacte-

ria from U. lactuca thalli: i) Bacterial extraction.

A 5 g wet-weight macroalgal thallus sample was

immersed in 70 % ethanol for 50 s. This sample

was then immersed in 0.85 % sterile NaCl solu-

tion to wash off residual ethanol. Each thallus

was then homogenized in 20 ml of deionized

water for 10 min using a sterile blender. The

resulting homogenate was then inoculated onto

Zobell marine agar in triplicate, using 100 µl of

each sample. This procedure was performed on

six macroalgal samples. ii) Petri dish isolation.

32 fragments from the thallus were immersed

in 70 % ethanol for 50 s, then immersed in 0.85

% sterile NaCl solution (Flewelling et al., 2013)

and finally placed on Zobell marine agar. This

procedure was performed on three macroalgal

samples. A total of 96 fragments were placed on

Zobell marine agar.

Endophytic bacterial isolates were grown

in the above media and incubated at 29 °C, then

reinoculated on nutrient agar plus NaCl (1 %

w/v) until pure isolates were obtained.

For cryopreservation of isolated pure

strains, each isolate was cultured at 30 °C for

24 h in 5 ml Zobell medium. Each culture was

then centrifuged at 6 000 rpm, the superna-

tant discarded, and the pellet resuspended in

5 ml Zobell medium supplemented with 20 %

glycerol. Aliquots of 1 ml were placed in sterile

cryovials and stored at -80 °C.

Qualitative screening of laccase activity

in Ulva lactuca epibiont and endobiont bacte-

rial isolates: Laccase production capacity was

qualitatively assessed in each of the U. lactuca

bacterial isolates by inoculating the isolated

colonies in culture medium supplemented with

guaiacol (5 mM) and NaCl (1 %) w/v (Ali et al.,

2022). Isolates positive for laccase activity were

identified by the appearance of a brown color-

ation in the colonies approximately eight days

after the start of incubation. A confirmatory

test for laccase activity was then performed by

inoculating the strains preliminarily identified

as laccase producers onto nutrient agar supple-

mented with 0.2 % dimethoxyphenol (DMP).

If the colony turned brown orange after at least

eight days of incubation, it was confirmed as a

laccase producer (Neifar et al., 2016). Bacillus

subtilis was used as a positive control as it is

a confirmed laccase producer (Muthukuma-

rasamy et al., 2015).

Identification of epibiont and endobiont

bacteria: Identification of isolates began with

Gram staining followed by amplification and

sequence analysis of the 16S rRNA region of

each isolated bacterium. PCR amplification

was performed using the universal primers 8F

5’-AGA GTT TGA TCC TGG CTC AG-3’- and

1541R 5’-AAG GAG GTG ATC CAG CCG

CA-3’. Inoculum from a colony of all isolated

bacteria was used as the source of template

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DNA for the reaction. Each inoculum was

suspended in 10 µl of molecular grade water.

Amplification reactions were performed in a

thermal cycler with a final reaction volume of

50 µl. The reaction mixtures contained 1X PCR

reaction buffer, 2 mM MgCl2, 0.2 mM of each

dNTP, 250 nM of forward and reverse primers,

1U Taq polymerase (Promega, Madison, WI,

USA), 400 ng/µl BSA (Bioline, Taunton, MA,

USA) and 1 µl of bacterial suspension. The

following thermocycling program was used

for the PCR reactions with the 8F and 1541R

primers: initial denaturation at 94 °C for 4 min;

followed by 30 cycles at 94 °C for 50 s, 55 °C for

45 s, and 72 °C for 50 s; with one final extension

step at 72 °C for 5 min.

Amplicons were sequenced at the Genecore

Sequencing Center of the Universidad de Los

Andes using the Sanger method. The result-

ing sequence files were evaluated and edited

using the FinchTV 1.4.0 software (Geospiza

Inc.; Seattle, Washington). After editing the

sequence files and curation procedures, result-

ing sequences were compared with those avail-

able in the National Center for Biotechnology

Information (NCBI) and the EzTaXon platform

for taxonomic classification of the isolated bac-

terial strains (Chun et al., 2007).

The 16S rRNA sequences were aligned

using Clustal W to determine the relationship

of the epibiont and endobiont bacterial isolates.

To construct the distance tree of the aligned

sequences, the maximum parsimony method

was used (Nei & Kumar, 2000), implemented in

MEGA (Tamura et al., 2021). Bootstrap analysis

was performed on 1 000 resamplings to evalu-

ate the tree topologies, as described by Tamura

et al. (2021).

Finally, three subgroups were defined for

the studied culturable marine bacterial com-

munity: (i) genera present as epibionts and

endobionts; (ii) genera present exclusively as

epibionts; and (iii) genera present exclusively as

endobionts. These subgroups were represented

in a Venn diagram generated by the online tool

Venny (Oliveros, 2007).

Sequence submission to gene Bank:

Nucleotide sequences were deposited in NCBI’s

GenBank and accession numbers were retrieved.

RESULTS

Epibiont and endobiont bacterial iso-

lates: A total of 118 pure isolates were obtained,

of which 64 were epibionts bacteria and 54 were

endobionts. Several of these isolates had similar

morphotypes and colors. Gram-negative bacilli

with non-pigmented colonies predominated

among the epibionts (52 isolates) and gram-

positive bacilli with white colonies among the

endobionts (32 isolates). A gram-positive coc-

cus was observed in both endobiont and epibi-

ont isolates.

Identity of the epibiont and endobiont

bacterial isolates: Based on 16S rRNA analy-

sis, we were able to identify 41 epibiont and

48 endobiont isolates. However, 29 of the iso-

lates did not give any amplification product

within the 16S rRNA region. It is likely that

the unidentified bacterial isolates associated

with U. lactuca belong to taxa whose 16S rRNA

regions cannot be amplified with the prim-

ers used in this study. Amplification of the

16S rRNA gene via PCR primers shows a bias

towards certain taxa, resulting in the inability

to detect others that could be identified using

a metagenomic approach (Brown et al., 2015;

Eloe-Fadrosh et al., 2016).

It should be noted that although most of

the sequences analyzed showed identity simi-

larities higher than 98 % (Table 1 and Table 2),

the limitations associated with the low resolu-

tion of 16S rRNA sequences (González et al.,

2013; Mulet et al., 2010; Rodicio & Mendoza,

2004) make it impossible to obtain a reliable

identification at the species level. For this rea-

son, it is necessary to be cautious with the

taxonomic identification of isolates at the spe-

cies level, and therefore we focus the discussion

of the results mainly on the higher taxonomic

levels. The genera and species assigned to

the identified isolates are detailed in Table 1

and Table 2. Sequences were submitted to the

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Table 1

Identity of Ulva lactuca epibiont and endobiont isolates (Phyla Proteobacteria and Actinobacteria).

Phylum Class Order Genus

Epibionts Endobionts

Specie Percent

Identity Specie Percent

Identity

Proteobateria Gamma Vibrionales VibrioVibrio halioticoli (2) 99.53 Vibrio sp. (66) 99.72

Vibrio sp. (3) 99.22 Vibrio rumoiensis (91) 100

Vibrio sp. (4) 94.44

Vibrio sp. (11) 94.02

Vibrio sp. (12) 99.86

Vibrio alginolyticus (13) 89.41

Vibrio alginolyticus (14) 99.26

Vibrio sp. (16) 99.85

Vibrio sp. (19) 99.45

Vibrio sp. (21) 99.94

Vibrio sp. (24) 98.96

Vibrio sp. (28) 99.86

Vibrio sp. (29) 99.06

Vibrio brasiliensis (30) 99.86

Vibrio brasiliensis (31) 100

Vibrio Ponticus (32) 99.72

Vibrio tubiashii (35) 99.10

Vibrio sp. (45) 99.58

Vibrio sp. (56) 99.3

Vibrio parahaemolyticus (62) 98.01

Vibrio alginolyticus (63) 99.81

Vibrio alginolyticus (64) 99.18

Photobacterium Photobacterium sp. (9) 99.86

Pseudomonadales Pseudomona Pseudomonas putida (42) 100 Pseudomonas sp. (74) 100

Pseudomonas sp. (52) 100 Pseudomonas sp. (79) 100

Pseudomonas sp. (60) 100

Psychrobacter Psychrobacter sp. (96) 100

Alteromonadales Shewanella Shewanella baltica (17) 99.74 Shewanella baltica (77) 98.68

Shewanella algae (58) 100 Shewanella baltica (89) 100

Shewanella baltica (90) 100

Shewanella baltica (92) 100

Oceanospirillales Halomonas Halomonas sp. (57) 99.7 Halomonas sp. (101) 100

Xanthomonadales Stenotrophomonas Stenotrophomonas sp. (72) 99.71

Stenotrophomonas sp. (73) 99.03

Stenotrophomonas sp. (75) 99.85

Enterobacterales Pantoea Pantoea eucrina (80) 97.55

Beta Urkholderiales Ralstonia Ralstonia sp. (67) 100

Ralstonia sp. (68) 99.86

Actinobacteria Actinomycetia Micrococcales Kocuria Kocuria gwangalliensis (5) 99.86

Pseuduclavibacter Pseudoclavibacter sp. (18) 100

Numbers in parentheses are isolate identification numbers.

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NCBI database and assigned accession num-

bers OR863796-OR863885.

Most of the epiphytic bacterial isolates

belonged to the phylum Proteobacteria, repre-

senting 70.7 % of all epibiont isolates (Table 1).

These isolates were all from the class Gamma-

proteobacteria with the order Vibrionales (53.6

%) being the most abundant. The phylum Fir-

micutes (24.4 %) contained only members of the

class Bacilli and the order Bacillales (Table 2).

Epibiont isolates also included Actinobacteria

with members of the class Actinomycetia and

the order Micrococcales (4.9 %) (Table 1). The

most abundant endobiont bacteria were from

Table 2

Identity of Ulva lactuca epibiont and endobiont isolates (Phylum Firmicutes).

Phylum Class Order Genus

Enpibionts isolates Endobionts isolates

Specie Percent

Identity Specie Percent

Identity

Firmicutes Bacilli Bacillales Bacillus Bacillus altitudinis (38) 100 Bacillus xiamenensis (65) 100

Bacillus sp. (39) 100 Bacillus sp. (69) 100

Bacillus altitudinis (40) 100 Bacillus sp. (70) 100

Bacillus sp. (43) 100 Bacillus xiamenensis (71) 100

Bacillus licheniformis (44) 100 Bacillus sp. (82) 100

Bacillus sp. (48) 99.87 Bacillus subtilis (85) 100

Bacillus cereus (53) 100 Bacillus sp. (93) 98.88

Bacillus sp. (94) 97.77

Bacillus pumilus (95) 94.27

Bacillus altitudinis (97) 100

Bacillus sp. (98) 100

Bacillus tequilensis (99) 99.43

Bacillus sp. (100) 99

Bacillus sp. (102) 99.28

Bacillus xiamenensis (103) 100

Bacillus altitudinis (104) 100

Bacillus sp. (106) 99.4

Bacillus sp. (107) 100

Bacillus glycinifermentans (108) 98.1

Bacillus altitudinis (109) 100

Bacillus toyonensis (110) 100

Bacillus velezensis (112) 100

Bacillus sp. (115) 100

Bacillus sp. (116) 100

Bacillus sp. (117) 95.38

Bacillus paramycoides (118) 100

Bacillus paramycoides (119) 100

Priestia Priestia megaterium (46) 100 Priestia flexa (81) 100

Priestia megaterium (105) 100

Cytobacillus Cytobacillus oceanisediminis (49) 99.87

Paenibacillus Paenibacillus illinoisensis (84) 99.71

Paenibacillus sp. (84) 100

Staphylococcus Staphylococcus warneri (83) 100

Uncultured bacteria (47) 90.11

Numbers in parentheses are isolate identification numbers.

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the phylum Firmicutes, accounting for 66.7 %

of all endobiont isolates. These bacteria were

exclusively from the class Bacilli and the order

Bacilliales. The second most common phylum

was Proteobacteria, which represented 33.3 %

of the isolates. The majority of Proteobacte-

ria were from the class Gammaproteobacteria

(29.2 %), dominated by isolates from the orders

Alteromonadales and Pseudomonadales. The

class Betaproteobacteria represented less than

4.1 % of the isolates.

The relationships studied among the bac-

terial isolates, based on 16S rRNA gene analy-

sis, showed that both epibiont and endobiont

isolates were present in each of the two main

branches corresponding to the Proteobacteria

and Firmicutes (Fig. 3). Only epibionts were

present in the Actinobacteria. The results also

show that the 16S rDNA region did not dis-

criminate between bacteria isolated from the

macroalgal surface and those present in the

macroalgal tissue. A grouping of epibiont and

endobiont isolates belonging to the same genus

was observed.

The most common bacterial genus in

the community of cultivable epibionts was

Vibrio, followed by Bacillus, Pseudomonas,

Shewanella, Pseudoclavibacter, Kokuria, Photo-

bacterium, Priestia, Cytobacyllus, Halomonas

and the uncultured bacteria. The genera Pseu-

doclavibacter, Kocuria, Photobacterium, and

Cytobacyllus and the uncultured bacteria, were

isolated only as epibionts; the presence of these

genera was not evidenced in the taxonomic

identifications of the isolated endobionts. On

the other hand, the most common genus in the

community of cultivable endobionts was Bacil-

lus, followed by Shewanella, Stenotrophomonas,

Priestia, Pseudomonas, Ralstonia, Vibrio, Pae-

nibacillus, Pantoea, Psychrobacter, Halomonas

and Staphylococcus. The genera Ralstonia, Pae-

nibacillus, Pantoea, Staphylococcus, Psychrobac-

ter, and Stenotrophomonas were isolated only as

endobionts (Fig. 4).

The genera Vibrio, Bacillus, Pseudomo-

nas, Shewanella, Priestia, and Halomonas were

among the bacterial isolates living equally

inside and on the algae. However, there was a

higher proportion of Vibrio isolates among the

epibionts, and more Bacillus isolates among the

endobionts (Fig. 4).

The genus Vibrio accounted for the highest

percentage of gram-negative epibionts (53 %)

among the identified Gram-isolates, while the

highest percentage of gram-positive isolates

belonged to Bacillus (57 %).

Laccase production by bacterial isolates:

42 bacterial isolates associated with U. lactuca

were found to be laccase producers, the major-

ity of which belonged to the genus Bacillus. 17

epiphytes and 25 endophytes showed positive

laccase activity (Table 3 and Fig. 5).

DISCUSSION

Epibiont and endobiont bacterial com-

position: In this study, we characterized the

culturable epiphytic and endophytic bacteria

isolated from Ulva lactuca, a marine macroalga

found in the Caribbean region of Colombia.

We also determined the laccase activity of these

bacteria. The culturable microbial community

associated with U. lactuca was dominated by

Proteobacteria and Firmicutes. Isolates collect-

ed from the macroalgal surface indicated that

Gammaproteobacteria were the most abundant

taxon. This finding is consistent with previous

reports of Ulva sp. from Germany, Austra-

lia, Spain, Portugal, and the United Kingdom,

which also showed a significant number of

Gammaproteobacteria.

Although Gammaproteobacteria are found

at high frequencies in other global locations,

the dominant microbial groups in these other

locations are Alphaproteobacteria, Deltapro-

teobacteria, Bacteroidetes, Actinobacteria, and

Planctomyces (Singh & Reddy, 2014). While

there is evidence suggesting a specificity for the

microorganisms and their algae host (Egan et

al., 2013), it is not unexpected that the domi-

nant microbiota of the Caribbean holobiont

may differ from those documented in other

regions. Invasive macroalgae, such as those in

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

Fig. 3. Distance tree of the identified bacterial isolates from Ulva lactuca based on 16S rRNA. Green labels = epibiont isolates,

red labels= endobiont isolates.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

the family Ulvaceae (Williams & Smith, 2007),

have been shown to have a strategy of colonizing

new habitats without relying on specific taxa.

Instead, they have the ability to reconfigure

their associated microorganisms using a wide

variety of microorganisms from the immediate

environment (Bonthond et al., 2021). Func-

tional diversity, rather than taxonomic diver-

sity, has recently been emphasized in holobiont

relationships. A metagenomic study by Burke et

al. (2011) showed that microbial communities

of Ulva australis from nearby sites, although

differing in taxonomic composition, shared a

core of functional genes common to all hosts.

Consequently, although the composition of the

dominant bacterial community in the Carib-

bean differs from that in other regions, the eco-

logical functions provided to the host by these

microbial communities may be maintained. It is

important to note that the discrepancy between

microbial community data for the Caribbean

and other areas may also be a result of the

methodology used to assess microbial com-

position. Previous studies have used the 16S

RNA gene metabarcoding technique to identify

microbial diversity, including taxa that cannot

be grown in culture media (Steen et al., 2019;

Theron & Cloete, 2000). In contrast, we used a

cultural approximation method, which may be

biased towards certain communities (Montalvo

et al., 2014; Wang et al., 2020).

Due to insufficient data on the endobiotic

microbiota of Ulva sp. from other latitudes, we

are unable to make any comparisons with

our results.

The genus Vibrio, which is commonly

found in marine, estuarine, and freshwater

environments (Sampaio et al., 2022), accounted

Fig. 5. Shewanella baltica, an example of an endobiont

isolate positive for laccase activity. A. Growth in nutrient

agar supplemented with guaiacol (5 mM) and NaCl (1 %

w/v). B. Growth on nutrient agar is supplemented with

NaCl (1 % w/v) without adding guaiacol.

Fig. 4. The Venn diagram illustrates the distribution of genera both within (Endobionts) and on the algae (Epibionts). The

number of isolates associated with each genus is shown in the bar graph.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

for the majority of the culturable epibiont bac-

teria isolated from U. lactuca. Vibrio species

can either grow as free-living organisms in the

water column or be associated with organic

particles and macroorganisms (Froelich et al.,

2013; Lyons et al., 2007; Sorroza-Ochoa et al.,

2017; Thompson et al., 2004) such as corals,

fish, mollusks, seagrasses, sponges, shrimp,

and zooplankton. This genus can thrive in

the water column, showing dominance in its

microbial communities (Gilbert et al., 2012)

and increasing in abundance with temperature

and salinity (Takemura et al., 2014). Vibrio

species are opportunistic and can exploit a

wide range of resources. They have the ability

to attach to surfaces, form biofilms (Hood &

Winter, 1997), and readily colonize the surfaces

of macroorganism, resulting in the develop-

ment of symbiotic relationships with their hosts

(Wahl et al., 2012). Several studies have shown

that members of the Vibrionaceae family living

as epiphytes on macroalgae can play multiple

roles, such as inducing macroalgal morphogen-

esis, promoting macroalgal spore colonization,

exerting antimicrobial activity, and degrading

algal compounds (Florez et al., 2017).

Therefore, it is common to observe Vibrio

as the primary culturable genus among bacte-

rial epibionts in algae. In our research, Vibrio

accounted for 53.6 % of U. lactuca epibiont iso-

lates in the Caribbean region of Colombia. This

finding is consistent with previous research

showing Vibrio as the dominant cultivable group

found in epibiont isolates collected from brown

macroalgae such as Ascophyllum nodosum,

Laminaria spp. (Takemura et al., 2014), Saccha-

rina japonica (Wang et al., 2008), and Splach-

nidium rugosum (Albakosh et al., 2016), as well

as from the red macroalgae Hypnea spp. and

Polysiphonia lanosa, and the green macroalgae

Table 3

Ulva lactuca epibiont and endobiont bacterial genera positive for laccase activity.

Bacterial isolates positive for laccase activity.

Number of isolates Genus Species (Isolates per species)

Epibionts 17 Bacillus Bacillus altitudinis (2)

Bacillus sp. (3)

Bacillus licheniformis (1)

Bacillus cereus (1)

Halomonas Halomonas sp. (1)

Priestia Priestia megaterium (1)

Pseudomonas Pseudomonas putida (1)

Pseudomonas sp. (2)

Vibrio Vibrio sp. (3)

Vibrio parahaemolyticus (1)

Unidentified (1)

Endobionts 25 Bacillus Bacillus sp. (9)

Bacillus xiamenensis (1)

Bacillus subtilis (1)

Bacillus altitudinis (3)

Bacillus velezensis (1)

Bacillus paramycoides (2)

Enterobacter Enterobacter sp. (1)

Halomonas Halomonas sp. (1)

Paenibacillus Paenibacillus illinoisensis (1)

Priestia Priestia megaterium (1)

Pseudomonas Pseudomonas sp. (1)

Shewanella Shewanella baltica (3)

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Chaetomorpha spp., Enteromorpha intestinalis,

and U. lactuca (Takemura et al., 2014).

Interestingly, Vibrio does not appear to be

as successful in colonizing U. lactuca tissues

as it is in living as an epibiont, with its relative

abundance as an endobiont being lower (8 %).

While many studies have documented the pres-

ence of Vibrio on macroalgal surfaces, there

are only a few reports indicating the presence

of this genus within macroalgae. Singh et al.

(2015) reported a low occurrence of Vibrio as

an endobiont of the red macroalga Gracilaria

corticata on the coast of India. This reduced

incidence of Vibrio as an endobiont could be

attributed to the inhibitory effect of bioactive

compounds produced by the macroalgae and

its associated epibionts and endobionts. Mac-

roalgal lipid extracts from the red macroalga

Gracilariopsis longissima have shown inhibitory

activity against various Vibrio species (Cavallo

et al., 2013). In addition, chlorophyte marine

macroalgae, including U. lactuca, produce bio-

active molecules such as steroids, alkaloids,

and terpenes, that exhibit antimicrobial activity

(Shah et al., 2020). Microorganisms with inhib-

itory activity against various Vibrio species have

been discovered, such as Bacillus amyloliquefa-

ciens, an epibiont of the red macroalgae Lau-

renciae papillosa and Padyna gymnospermaha

(Chakraborty et al., 2017; Chakraborty et al.,

2018), Bacillus subtillis associated with the

brown macroalga Anthofolphycius, the endo-

biotic fungus Aspergillus terreus isolated from

the marine macroalga Laurencia okamurai (Li

et al., 2019), and Penicillium citrinum, another

endobiotic fungus obtained from the mangrove

Bruguiera sexagula var. rhynchopetala (He et

al., 2017). Polyketides, terpenes, and nitro-

gen-containing compounds are the three main

structural types of active molecules produced

by microorganisms to inhibit aquatic bacteria

(Guo et al., 2022). There is limited literature on

the effect of U. lactuca on Vibrio growth, but

studies examining pathogenic Vibrio species

have found that incorporation of U. lactuca

as a feed supplement and water bioremedia-

tion agent in shrimp farms effectively reduces

Vibrio alginolyticus levels in water (Mangott

et al., 2020). Additionally, research has shown

that residues of Ulva prolifera increase disease

resistance to Vibrio parahaemolyticus in white

shrimp (Litopenaeus vannamei) (Ge et al., 2019).

Our distance analysis showed that one of

the endobiont Vibrio isolates clustered with

epibiont isolates of the same genus and Vibrio

rumoiensis, the other endophytic vibrio, is dis-

tant from the grouping formed by this genus

(Fig. 3). Genetic variation within the genus

Vibrio may allow certain species to inhab-

it macroalgal tissues, which warrants further

investigation through genome sequencing and

subsequent whole-genome sequence compari-

sons between epibiont and endobiont Vibrios

in future studies. This method may help to

identify specific and common functions of

the endobiotic Vibrio isolates discovered in

this research.

Bacillus was the most common genus

(56.3 %) among the endobiont bacterial isolates

in our study. This genus has also been com-

monly isolated as an endobiont from Sargassum

sabrepandum, a brown macroalga (Ahmed et

al., 2016), as well as from several macroalgal

species in different water sources in Israel

(Deutsch et al., 2021). One of the rare studies

that assessed both epibionts and endobionts

also reported Bacillus as the most common

endobiont bacterium in Sargassum honeyri

from the Yellow Sea, China (Mei et al., 2019).

The diverse association of Bacillus with differ-

ent macroorganism taxa (Saxena et al., 2020)

can be attributed to its rapid growth and abil-

ity to thrive under challenging environmental

conditions, including temperature, salinity, pH,

and nutrient levels (Xiao et al., 2022). The pres-

ence of Bacillus bacteria as endobiont micro-

organisms in marine macroalgae is associated

with their ability to enhance the colonization

and growth of macroalgal zoospores (Singh &

Reddy, 2014; War Nongkhla & Joshi, 2014).

In contrast, Bacillus was a less common

macroalgal epibiont in our study, with only a

few epibiont isolates (17 %) belonging to this

genus. Similarly, Mei et al. (2019) evaluated

both endobionts and epibionts and did not find

Bacillus in the epibiont microbial community.

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

However, Kumar et al. (2022) found Bacillus

to be one of the most abundant epibionts in

several macroalgal species on the central West

coast of India. Although this genus has been

reported in numerous marine and terrestrial

macroorganisms (Gao et al., 2021; Korsten et

al., 1995; Xiong et al., 2018), it is difficult to

determine whether Bacillus species are prefer-

entially endobionts because most studies are

limited to assessing the presence of microor-

ganisms in one of two niches, the host surface,

or internal tissues.

The genera Pseudomonas, Halomonas, She-

wanella, and Priestia were rare in our work,

but were also present as U. lactuca endobi-

onts and epibionts. Pseudomonas has shown

a wide range of biotechnological potential in

the production of antimicrobials and industri-

ally valuable enzymes (Bollinger et al., 2020).

Species of this genus form biofilms in water,

which allows them to exist as epibionts (Millas

& France, 2020) in both aquatic and terrestrial

environments (Krimm et al., 2005; War Nong-

khla & Joshi, 2014). Pseudomonas species are

known endobiotic in terrestrial plants (Chen et

al., 2014; Fu et al., 2018). However, their pres-

ence in macroalgae has been poorly reported.

In one such work, Pseudomonas stutzeri was

found in the macroalga Ulva prolifera (Fu et

al., 2018). In addition, Halomonas and She-

wanella are commonly found in macroalgae

as well as marine and terrestrial organisms.

The genus Halomonas has been identified in

epibiont bacterial communities associated with

macroalgae, specifically Saccharina japonica

(Zhang et al., 2020), Laminaria japonica (Wang

et al., 2008), and Ulva sp. In this last macroalga,

Halomonas has an important function in the

induction to the normal morphological devel-

opment (Kaur et al., 2023). This species has also

been found as an endobiont of the terrestrial

plant Mesembryanhemum crystallinum (Zhang

et al., 2018; Zhang et al., 2020). Shewanella has

been observed as an epibiont of marine organ-

isms including sponges, invertebrates, and

brown macroalgae such as Bifurcaria bifurcata

(Horta et al., 2014). In addition, strains isolated

from the surfaces of Ulva sp. were reported

to stimulate the zoospore settlement of this

macroalgae (Kaur et al., 2023). Shewanella spe-

cies have also been discovered as endobiont in

Indonesian seagrasses (Fitri et al., 2017). While

the genus Priestia has been documented as an

epibiont on several macroalgae from the central

west coast of India (Kumar et al., 2022), there

are few reports of this genus as an endobiont.

Although our study detected Pseudoclavi-

bacter, Kocuria, Photobacterium, and Cytobacil-

lus exclusively on the surface of U. lactuca and

Ralstonia, and Paenibacillus, Pantoea, Staphylo-

coccus, Enterobacter, Psychrobacter, and Steno-

trophomonas exclusively in macroalgal tissues,

these genera are not restricted to either niche.

Previous studies indicate that they are also

found worldwide as both epibionts and endobi-

onts in marine and/or terrestrial hosts (Table 4).

Exceptions are Photobacterium, which is mainly

found in marine environments (Fuertes-Perez

et al., 2021), and Ralstonia, which has not been

reported as an epibiont or endobiont of mac-

roalgae. However, Sarr et al. (2010) were able to

isolate Ralstonia from the nodules of cowpea, a

type of bean.

Interestingly, Kocuria, a common epibi-

ont of marine hosts, especially macroalgae,

has shown antibacterial activity against other

bacteria associated with macroalgae (Leiva et

al., 2015). Furthermore, it has been found to

produce an extracellular polymer against the

colonization of macroalgal surfaces by bacteria

and barnacle larvae (Ba-Akdah & Satheesh,

2021). Stenotrophomonas is an opportunistic

pathogen in corals, reported by Meyer et al.

(2014). However, certain species of this genus

found in terrestrial plants have shown the abil-

ity to produce chitinases that can inhibit the

growth of the pathogenic Fusarium oxysporum

and Leptinotarsa decemlineata, both of which

pose a threat to agricultural production (Aktas,

2022). Paenobacillus, an endobiont of Lonicera

japonica, can inhibit the growth of phytopatho-

genic fungi and promote the growth of its host

plant by producing siderophores and solubiliz-

ing phosphorus (Zhao et al., 2015).

The results of this preliminary study show

that most of the bacterial genera obtained

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Table 4

Bacterial genera associated with marine and terrestrial hosts as epibionts or endobionts.

Genus Habitat Host Niche World region Source

Pseudoclavibacter Marine Mnemiopsis leidyi (Comb jelly) Epi Kiel Bight, Baltic Sea Weiland-Bräuer et al., 2020

Terrestrial Panax ginseng C. A. Meyer

(Ginseng)

End Korea Cho et al., 2007

Terrestrial Glycyrrhiza uralensis

(Perennial herb)

End North-West China Li et al., 2016

Kocuria Marine Ulva lactuca (Macroalgae) Epi Central Red Sea, S.

Arabia

Ba-Akdah & Satheesh, 2021

Marine Antarctic marine macroalgae Epi South Shet- land

Islands, Antarctica

Peninsula

Leiva et al., 2015

Marine Marine macroalgae (Sargassum,

Ulva, Padina, Dictyota and

Pterocladia sp.)

Epi Central West coast of

India

Kumar et al., 2022

Terrestrial Coffea arabica LEnd Colombia Vega et al., 2005

Terrestrial Ficus carica L End Southern Bulgaria Lozanova et al., 2022

Brevundimonas Marine Marine macroalgae (Sargassum,

Ulva, Padina, Dictyota and

Pterocladia sp.)

Epi Central West coast of

India

Kumar et al., 2022

Terestrial Olea europaea L (Olive trees) End Mediterranean Basin Mina et al., 2020

Terrestrial Buxus sempervirens (Bush) End North Carolina, U.S.A. Li et al., 2023

Photobacterium Marine Fucus serratus (Seaweed) Epi Kiel Bight, Western

Baltic Sea

Nasrolahi et al., 2012

Marine Cyanea capillata (Jelly Fish)

Tubularia indivisa (Hydrozoa)

Sagartia elegans (Sea anemone)

End Orkney Islands,

Northeastern coast of

Scotland

Schuett & Doepke, 2010

Marine Asparagopsis armata (Red algae) Epi Peniche, Portugal Horta et al., 2019

Cytobacillus Terrestrial Oryza sativa L. (Rice) End South Korea Dutta et al., 2022

Marine Hydroclathrus sp (Brown

Macroalgae)

Epi South Sulawesi,

Indonesia

Ethica et al., 2023

Paenibacillus Seashore Rhizocarpon geographicum L

(Lichen)

Epi La Pointe de Crozon,

France

Miral et al., 2022

Marine Enhalus acroides (Seagrass) Epi Papua New Guinea Hassenrück et al., 2014

Terrestrial Lonicera japonica (Medicinal plant) Endo Eastern China Zhao et al., 2015

Pantoea Terrestrial Brassica oleracea var. capitata

Spinacia oleracea

Epi Japan Oie et al., 2008

Terrestrial Pisumsativum

Gossypium hirsutum L.

End Netherlands

Alabama (U.S.A)

Elvira-Recuenco & van

Vuurde, 2000

McInroy & Kloepper, 1995

Psychrobacter Marine Anoxycalyx joubini (Sponge)

Lissodendoryx nobilis (Sponge)

Haliclonissa verrucosa (Sponge)

End Antartic Papaleo et al., 2012

Marine Sargassum polycystum

Padina antilarum

Dictyota sp.

Epi Anjuna, Kunkeshwar

and Malwan (India)

Kumar et al., 2022

Stenotrophomonas Terrestrial Cucumis sativus L.

(Cucumber plants)

End Alabama: U.S.A. Ryan et al., 2009

Marine Porites astreoides (Coral) End Belize Meyer et al., 2014

Marine Laminaria saccharin (Macroalga) Epi Baltic Sea: Germany Lage & Graça, 2016

(Epi = Epibiont), (Endo = Endobiont).

16 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

from U. lactuca in the Colombian Caribbean

are present in microbial communities associ-

ated with brown and red macroalgae, marine

invertebrates, and terrestrial plants from dif-

ferent regions of the world. However, to deter-

mine the unique functional and taxonomic

assemblages of microbial epibiont and endo-

biont communities in U. lactuca, the use of

metagenomics and the 16S rRNA metabarcod-

ing approach is necessary.

The advantage of using conventional cul-

ture methods to assess microbial community

composition is that the genetic material of the

isolated microorganisms can be sequenced,

thereby improving the ability to explore the bio-

technological potential of these isolates through

genomic bioprospecting. Genome mining, an

in silico bioprospecting method (Ziemert et al.,

2016), is ideal for bioprospecting the genomes

of the isolates. It will facilitate the exploration

of metabolic pathways, secondary metabolites,

and antimicrobials that contribute to differ-

ent aspects of holobiont interactions, such as

(i) the synthesis and production of enzymes

that degrade sugars produced by the macroal-

ga (Barbato et al., 2022), (ii) microorganisms

engaging in antagonistic interactions through

secondary metabolites, including antibiotics

(Goecke et al., 2010; Ortega-Morales et al.,

2008; Velupillaimani & Muthaiyan, 2019), (iii)

the production of phytohormones (Ulrich et

al., 2022), and motility/chemotactic substances

(Colin et al., 2021) that promote biofilm forma-

tion, and (iv) siderophores for iron scavenging

(Zoccarato et al., 2022). Today, this genomic

bioprospecting is possible because of access

to a global repository of genome and metage-

nome datasets through platforms such as the

Integrated Microbial Genomes and Microbi-

omes System (IMG/M) (Chen et al., 2020).

In addition, IMG/M provides the Genomes

OnLine Database (GOLD), a manually curated

database with a metadata reporting system that

allows users to tabulate and search the associ-

ated metadata associated with each submitted

genome (Mukherjee et al., 2021).

Laccase production: Laccases are found

in in plants, animals, insects, and both eukary-

otic and prokaryotic microorganisms (Janusz et

al., 2020). These polyphenol oxidases (PPOs)

perform essential oxidase functions in vari-

ous biological systems. It has been shown that

fungal laccases are involved in lignin deg-

radation, sporulation, pigment production,

morphogenesis, stress defense and plant patho-

genesis (Thurston, 1994). Plant laccases have

been implicated in the biosynthesis of lignin

polymers (Sterjiades et al., 1992; Tobimatsu &

Schuetz, 2019; Zhao et al., 2013), elongation

(Balasubramanian et al., 2016; Liang et al.,

2006), stress response (Cho et al., 2014), repair

of damaged plant tissues, iron accumulation

and the polymerization of phenolic compounds

(Gałązka et al., 2023). Insect laccases partici-

pate in cuticle sclerotization and pigmentation,

as well as other processes such as wound heal-

ing and the development and maintenance of

the immune system (Dittmer & Kanost, 2010).

Bacterial laccases, on the other hand, are

involved in pigmentation processes, endospore

coat protein synthesis, morphogenesis, toxin

oxidation and protection against oxidants and

UV light (Sharma et al., 2007). These enzymes

have been reported for many gram-negative

and gram-positive bacterial genera such as Azo-

spirillum, Streptomyces, Bacillus, Aeromonas,

Pseuomonas, Oscillatoria, Thermus, Escherichia

and Haloferax, and have been detected in soils,

rhizospheres, deep-sea sediments, and extreme

and marine environments (Janusz et al., 2020;

Singh et al., 2011). In addition, a previous study

identified genes encoding laccases by analyzing

the genome of the U. lactuca epibiont bacterium

Achromobacter denitrificans strain EPI24 (Niño

et al., 2023). Therefore, although there are no

reports of laccase activity for microorganisms

associated with U. lactuca, we were not sur-

prised to find that out of 16 cultivable genera

associated with U. lactuca, eight were positive

for laccase activity. Furthermore, Bacillus sp.,

the genus with the highest number of isolates,

also had the highest number of epibiont and

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endobiont isolates positive for this enzyme

(70 %). In this genus, laccases are part of the

endospore walls and are involved in the biosyn-

thesis of a brown spore pigment, a melanin-like

polymer responsible for protection against UV

radiation and heavy metals (Janusz et al., 2020).

Although Vibrio had a high number of

isolates, especially epibionts, only 16 % showed

laccase activity. These results are consistent

with the rare reports in the literature of posi-

tive laccase activity by Vibrio. Enterobacter,

Halomonas, Pseudomonas, Paenibacillus, Pries-

tia and Shewanella, were also found to be posi-

tive for laccase production. These genera have

been previously reported to be laccase produc-

ers by Edoamodu and Nwodo (2021), Kurian

and Kumar (2015), Mathews et al. (2016), and

Sinirlioglu et al. (2013). Conversely, to the best

of our knowledge, Priestia and Halomonas

have not been previously reported to produce

this enzyme.

In this study, endobionts exhibited a higher

number of isolates positive for laccase produc-

tion in comparison to epibionts. This imbalance

may indicate a significant biological function

for the laccase-producing endophytic bacteria

associated with Ulva lactuca. The microbial

laccase catalyzes the oxidation of small organic

compounds (including phenolic compounds,

diamines, and aromatic amines), producing

radicals that can be utilized by the host in ana-

bolic and catabolic pathways (Jeon & Chang,

2013) linked to processes that enhance the per-

formance of the algae, such as the neutralization

of toxic by-products released in biochemical

reactions, morphogenesis, toxin oxidation and

protection from UV light (Gałązka et al., 2023).

Evaluating the potential use in biotechnol-

ogy of laccases produced by the many isolates

detected in this study is worthwhile. Firstly,

bacterial laccases have advantages over those

produced by fungi. Comparative studies have

shown that the active sites of bacterial, fungal

and plant laccases are highly conserved, reflect-

ing a common reaction mechanism for copper

oxidation and O2 reduction in these enzymes

(Dwivedi et al., 2011). However, analysis of

laccase molecular surface areas, volumes and

cavity residues, revealed that bacterial laccase

has the largest putative substrate binding site

cavity, as compared to fungi and plants, which

could explain the different functions of lac-

cases (Dwivedi et al., 2011). Another impor-

tant difference of these polyphenol oxidases

(PPOs) between different biological groups is

their redox potential (E°) at the T1 site of the

enzyme (Gałązka et al., 2023; Morozova et al.,

2007; Singh et al., 2014). This is a fundamental

physicochemical property of laccases, as the

rate of a laccase oxidation reaction depends on

the difference in redox potential (Δ E°) between

the T1 site of the enzyme and the substrate (Xu

et al., 1996). Laccases of fungal origin tend to

have higher E° values (0.34 to 0.81 V vs. NHE,

Normal Hydrogen Electrode) (Munk et al.,

2015; Singh et al., 2014) compared to bacte-

rial laccases (0.4 to 0.5 V vs. NHE), which can

withstand more challenging conditions than

fungal laccases (Agarwal et al., 2022). Bacte-

rial polyphenol oxidases are active at high pH

values and much more stable at high tempera-

tures, in the presence of organic solvents and

over a range of salt concentrations (Janusz et

al., 2020). Although data for plants are scarce,

a few studies report that their laccases have

low E° values (Janusz et al., 2020; Munk et al.,

2015). Second, biotechnology now makes it

possible to modify biological properties. Even if

we find bacteria in the Caribbean with laccase

activity that has significant advantages over

fungal laccases, none of these bacterial enzymes

may be optimal for industry. However, genetic

engineering can help overcome these hurdles

by modifying some biological properties of

microorganisms so that their enzymes can be

used by industry. For example, most micro-

organisms have limited laccase yields, which

limits industrial production of the enzyme.

Many screens have been conducted to explore

laccase-producing microorganisms in different

environments to identify efficient hypersecre-

tory microorganisms for large-scale production

or laccase-producing microorganisms that can

be improved using recombinant DNA tools

(Yang et al., 2017).

18 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20252695, enero-diciembre 2025 (Publicado Oct. 28, 2025)

This study shows that a significant pro-

portion of microorganisms associated with U.

lactuca in the Colombian Caribbean can pro-

duce laccases. It is then worthwhile to search

for enzymes applicable in industrial settings

in the microbial community associated with

U. lactuca. It is worth further investigating

whether endobiont bacteria are more inclined

to synthesize laccases with biotechnological

potential and exploring the impact of their

interaction with the macroalgal host on

enzyme production.

The present study showed that among

the culturable microorganisms associated with

Ulva lactuca from the Colombian Caribbean, a

large proportion belongs to the genera Vibrio,

which grow mainly on the algal surface, and

Bacillus, which grows in the tissues of the mac-

roalga. These epibiont and endobiont bacteria,

especially Bacillus, can produce laccases.

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict 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 gratefully acknowledge the

support of the Center of Excellence in Marine

Sciences Corporation (CEMarin) Foundation

and The University of Bogotá Jorge Tadeo Loza-

no for funding this study. We thank the Ministry

of Environment and Sustainable Development

in Colombia for allowing us to conduct this

research. The project was carried out under

Contract for Access to Genetic Resources and

Their Derivative Products No. 320 of 2021, File

RGE 384, issued by the Directorate of Forests,

Biodiversity, and Ecosystem Services.

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