Isolation and characterization of broad spectrum coaggregating bacteria from different water systems for potential use in bioaugmentation

Abstract

The bridging bacteria with broad-spectrum coaggregation ability play an important role during multispecies-biofilm development. In this study, through a visual and semi-quantitative assay, twenty-two bacterial strains with aggregation ability were obtained from 8 different water environments, and these strains were assigned to 7 genera according to their 16S rDNA and they were Aeromonas, Bacillus, Comamonas, Exiguobacterium, Pseudomonas, Shewanella and Comamonas. Furthermore, all possible 231 pairwise combinations among these 22 strains were explored for coaggregation ability by spectrophotometric assay. Among all these strains, it was found that Bacillus cereus G5 and Bacillus megaterium T1 coaggregated with themajority of assayed other strains, 90.5% (19 of 21 strains) and 76.2% respectively (17 of 21 strains) at a higher coaggregation rates (A.I. greater than 50%), indicating they have a broad-spectrum coaggregation property. The images of coaggregates also confirmed the coexistence of G5 and T1 with their partner strains. Biofilm biomass development of G5 cocultured with each of its partner strains were further evaluateded. The results showed that 15 of 21 strains, when paired with G5, developed greater biofilm biomass than the monocultures. Furthermore, the images from both fluorescence microscopy and scanning electron microscopy (SEM) demonstrated that G5 and A3-GFP (a 3,5-dinitrobenzoic acid-degrading strain, staining with gfp),could develop a typical spatial structure of dual-species biofilm when cocultured. These results suggested that bridging-bacteria with a broad spectrum coaggregating ability, such as G5,could mediate the integration of exogenous degrading bacteria into biofilms and contribute to the bioaugmentation treatment.

Figure 1. Numbers of strains pairings and their percentages for each of the 22 strains at 2 h (a) and 20 h (b) in different A.I. ranges (A.I.: coaggregation index).

The bacteria were incubated at 20°C for 2 h or 20 h.

Figure 2. Scanning electron micrograph of the coaggregates.

a: G5 with A3; b: G5 with M22; c: T1 with A3; d: T1 with M22; Larger G5 and T1 cells were surrounded by many smaller bacterial cells (A3 or M22), showing stable coaggregate mixtures.

Figure 3. Biofilm biomass compare of G5 with different strains.

The bacteria were incubated at 30°C at 150 rpm for 20 h in 5% LB. The error bars represent ± SD of the assay performed in triplicate.

Figure 4. Fluorescence microimages (a) and Scanning electron micrograph (b) of G5-A3/GFP biofilms.

Biofilms were developed on slide surfaces and were visualized with FM and SEM using G5 and A3/GFP (expressing GFP). Two different sizes bacteria were observed in the structural biofilms.