Avaliação do potencial biotecnológico de microorganismos associados ao inseto-praga diabrotica speciosa na produção de polímeros biobaseados e biodegradáveis
Resumen
Technological development and market pressure turned polymers into widely used
structural materials for several different applications, being manufactured by a wide
range of monomers. However, traditional polymers usually show some drawbacks
regarding environmental aspects, as most used polymers are produced with nonrenewable
feedstock and generate huge amounts of non-biodegradable residues.
Therefore it is imperative the sustainable development of new bio-based and
biodegradable polymeric materials. The use of microorganisms for obtaining
biopolymers is a very promising reality. However, in order to achieve viable
production in industrial scale it is necessary to overcome economic barriers, by using
microbes with good assimilation of low-cost substrates and high biopolymer yields.
As such, the objective of this work was the isolation and identification of bacteria
associated with the insect Diabrotica speciosa, as well as the evaluation microbial
capacity of biopolymer production. The insect presented great microbial diversity,
identified as an underexplored niche with tremendous biotechnological potential for
the investigation of novel species and/or strains. In an attempt to find bacterial
isolates effective on the production of two classes of biopolymers,
polyhydroxyalkanoates (PHA) and exopolysaccharides (EPS), it was obtained 73
strains of bacteria associated with Diabrotica speciosa. These bacteria were
identified at genus level by genetic techniques using 16S rDNA sequencing and by
proteomic techniques using MALDI-TOF MS. Both characterization methods yielded
100% convergence on results. It was found 17 different bacterial genera, which were
submitted to qualitative screening assays in order to identify strains producing PHA
using Nile Red dye method, as well as for EPS by using the bacterial spot test.
Promising strains on both assays were selected for further quantitative studies and
structural characterization of the obtained biopolymers. Quantitative analyses for
PHA production corroborated satisfactorily with qualitative results, especially to
bacteria from genera Aurantimonas and Delftia which demonstrated high PHA
production capacity with 50 and 90% polymer yield on dry mass, both strains being
strains able to use substrates such as glucose, acetate and glycerol. GC-MS
analyses indicated that Aurantimonas sp. produced mostly a homopolymer of
polyhydroxybutyrate (PHB), while Delftia sp. was able to produce a copolymer having butyrate and valerate (PHBV), with up to 10% (w/w) of valerate. Regarding EPS
production, the screening showed that the isolates were able to produce polymers in
variable amounts, with vast and complex structural variations. Strains from genera
Acidovorax, Aurantimonas and Luteibacter were further selected for quantitative
analysis of EPS production and analytical characterization of the obtained
biopolymer. After analyses using NMR, MALDI-TOF, SEC-UV-ELSD and GC-MS,
bacteria from genus Luteibacter produced a highly complex polymer rich in mannose,
glucose, fucose and xylose; genus Acidovorax produced a glucomannan-type EPS
with a high degree of branching; and genus Aurantimonas was able to produce up to
2 g.L-1 of a water insoluble EPS. In face of these results, it was possible to conclude
that D. speciosa microbiota showed to be extremely rich in bacterial species viable
for exploratory studies with biotechnological context of biopolymer production.
Investigated strains showed promising characteristics to be further evaluated in
larger scale (fermenters), especially the bacteria Aurantimonas sp., able to produce
PHBV and EPS.