We isolated a diverse group of endophytic microorganisms from samples collected in Bertioga, São Paulo State, Brazil, from locations with and without an oil spill, and from three different mangrove plant species. However, both locations, whether contaminated with oil or not, suffer from anthropogenic influences due to proximity to industrial and domestic sewers, and because of the petroleum spill and sewer runoff, there are heavy metals present in these environments.
The description of the bacterial diversity within the mangrove plant hosts contributes to our understanding of these microorganisms in this ecosystem. This highly adapted bacterial species is a potential source of biotechnological resources for future investigations, such as the search for bacteria that can promote bioremediation of a polluted environment. The endophytes colonize the inner tissues of their plant hosts without causing disease, and they can establish mutualistic associations with the hosts, promoting a better adaptation of the host plants to the environment through mechanisms including the immobilization of heavy metals that are toxic to the plants.
Multiple authors have demonstrated that Methylobacterium
spp. can be tolerant of to up to 4
mM Cd, 8
mM Pb, and 48
mM As [15
]. In this study, most of the isolates (70%) were tolerant to 1
mM Cd, As, and Pb, concentrations higher than would be found in the natural environment (soil and oceans or rivers). Moreover, two isolates tolerated Cd, Pb, and As tested at a concentration of 8
mM. This concentration of Cd tolerated by Methylobacterium
sp. was higher than that found in any previous studies, presumably due to this soil microorganism becoming an endophyte. Also, Cd is more labile in the soil, allowing the bacteria to become more tolerant to cadmium (plasmid associated) [27
]. On the other hand, Pb is not labile in the soil, thus the bacteria are less tolerant [26
]. Nevertheless, two isolates showed tolerance to heavy metals in concentrations higher than described previously in the literature (except for As), thus present a potential use in the bioremediation of locations contaminated with heavy metals, such as the mangrove forest with an oil spill or other locations that suffer anthropogenic action due to industrial and domestic sewers.
We detected endophytic methylotrophic bacteria in the three different species of mangroves. These bacteria displayed differing levels of resistance to heavy metals, from 0.1
mM to 8.0
mM of As, Cd, and Pb. Cadmium is the most selective heavy metal assessed in this work, followed by arsenic and lead. The methodologies used to identify and analyze the genetic diversity of pink-pigmented bacteria showed that the isolates from the forest that had undergone oil spillage were grouped, suggesting that oil can select microorganisms that tolerate or degrade oil compounds and can change the methylotrophic bacterial community accordingly.
The two isolates that are tolerant to cadmium, lead, and arsenic were selected by oil and present a potential for use in bioremediation of this environment by their action in immobilizing these metals. Further study is required to determine the mechanisms of tolerance. These tolerant bacteria are endophytic and present a direct association with plants, strongly suggesting that these bacteria immobilize the metal, promoting plant growth. Therefore, the isolation and characterization of the Methylobacterium
spp. is the first step to enable the future bioremediation of this area through removal of the heavy metals from of mangrove forest though a reactor or by phytoremediation with endophytic bacteria [28
To our knowledge, this is the first report of the genetic diversity of the Methylobacterium spp. community in a mangrove forest that showed tolerance to heavy metals in in vitro assays.