Polyhydroxybutyrate-producing cyanobacteria from lampenflora: The case study of the "Stiffe" caves in Italy

Abstract

This study aimed to estimate the green formation lampenflora of "Stiffe" caves in order to evaluate their suitability as an isolation source of cyanobacteria useful for the production of polyhydroxyalkanoates (PHAs). The cave system was chosen as the sampling site due to its touristic use and the presence of high-impact illuminations. The biofilms and the mats of the illuminated walls were sampled. Samples were investigated by 16S rRNA gene analysis and culturable cyanobacteria isolation. The isolated strains were then screened for the production of PHAs under typical culturing and nutritional starvation. Cultures were checked for PHA accumulation, poly-β-hydroxybutyrate (PHB) presence (infrared spectroscopy), and pigment production. The 16S rRNA gene metabarcoding. Highlighted a considerable extent of the pressure exerted by anthropogenic activities. However, the isolation yielded eleven cyanobacteria isolates with good PHA (mainly PHB)-producing abilities and interesting pigment production rates (chlorophyll a and carotenoids). Under normal conditions (BG11₀), the accumulation abilities ranged from 266 to 1,152 ng mg dry biomass^-1. The optimization of bioprocesses through nutritional starvation resulted in a 2.5-fold increase. Fourier transform infrared (FTIR) studies established the occurrence of PHB within PHAs extracted by cyanobacteria isolates. The comparison of results with standard strains underlined good production rates. For C2 and C8 strains, PHA accumulation rates under starvation were higher than Azospirillum brasilense and similar to Synechocystis cf. salina 192. This study broadened the knowledge of the microbial communities of mats and biofilms on the lightened walls of the caves. These findings suggested that these structures, which are common in tourist caves, could be used to isolate valuable strains before remediation measures are adopted.

Keywords: 16S rRNA gene metabarcoding; biopolymers; deep biosphere; microbial communities of caves; polyhydroxyalkanoates.

FIGURE 1

Cave survey of the “Stiffe” caves. Sampling areas are highlighted in the plan view and pictures on the bottom show the thermic camera and normal photos for each site.

FIGURE 2

Taxonomy barplots of the main ASVs (abundances > 0.5%) at phylum (A) and genus level (B).

FIGURE 3

Polyhydroxyalkanoates contents recorded for “Stiffe” caves’ cyanobacteria and Synechocystis cf. salina, grown in BG11₀, and for A. brasilense and H. eurihalina, cultured on NFCC and MH, respectively. Uppercase letters refer to the comparison among BG11₀ growths and A. brasilense and H. eurihalina. Lowercase letters refer to the comparison among BG11 starving growths and A. brasilense and H. eurihalina. For both conditions, results followed by the same case letters are not significantly different according to Fisher’s LSD post hoc test (p > 0.05) (LSD value, 40.3).

FIGURE 4

Polyhydroxyalkanoates production curves recorded for “Stiffe” caves’ cyanobacteria and Synechocystis cf. salina, grown in BG11 modified for starvation. (A) C1-C6 curves; (B) C7-C11 and CCALA192 curves.

FIGURE 5

Polyhydroxyalkanoates contents recorded for “Stiffe” caves’ cyanobacteria and Synechocystis cf. salina, grown in BG11 modified for starvation, and for A. brasilense and H. eurihalina, cultured on NFCC and MH, respectively. Uppercase letters refer to the comparison among BG11₀ growths and A. brasilense and H. eurihalina. Lowercase letters refer to the comparison among BG11 starving growths and A. brasilense and H. eurihalina. For both conditions, results followed by the same case letters are not significantly different according to Fisher’s LSD post hoc test (p > 0.05) (LSD value, 192.1).

FIGURE 6

ATR-FTIR spectra obtained from C1–C11 isolates (A) and comparison between the average spectrum obtained for C1–C11 isolates with that obtained for Synechocystis cf. salina (C192) (B).

FIGURE 7

ATR-FTIR spectra comparison between the average spectrum obtained for C1–C11 isolates with that obtained for PHB pure standard.