Isolation And Characterization Of Co-aggregation Bacterium Strain And Its Co-aggregate Mechanism

Coaggregation is a process by which genetically distinct bacteria become attached to one another via specific molecules. Cumulative evidence suggests that such adhesion influences the development of complex multispecies biofilms and the diversity of biofilm communities. Survey demonstrated that coaggregation was a common phenomenon between a broad range of genera, but Bridging bacterium which acts as a core or bridging organism in the development of complex multi-species biofilms and can be coaggregated with multiple species of bacteria strongly is found a few only.In order to acquire the Bridging bacterium having a wide range of coaggregation, 23 Strains were isolated from biofilms from 7 ecosystems including municipal sewage and industrial waste water treatment systems et al in this article. Then coaggregation percertage of a total of 263 matching combinations were determined, the results show that:There are 3 strains G5,G8 and T1 hold coaggregation percertage more than 50% respectively with 20 (91% of the total measured strains),20 (91% of the total measured strains) and 17 (77% of the total measured strains), showing extensive coaggregation ability. And in the study of biofilm forming, this 3 strains were able to increase the biofilm biomass of 18/19,16/16 and 20/20 pairing bacterial in 10%LB/5%LB medium, respectively, showing theis ability of promoting biofilm forming. Analysis by the morphological,physiological,biochemical and 16S rDNA sequence, T1,G5 and G8 were identified as Bacillus megaterium,Bacillus cereus and Bacillus marisflavi, respectively.To investigate the influence of physical and chemical environment on the coaggregation ability and the stability of aggregates, using T1,G5,G8 and Shewanella sp. H3 isolated from the biofilm of sewage treatment system as materials, the impact of Growth phase,Temperature,pH,Hydraulic shear stress,Surfactant(SDS, Tween 80),Chelating agent(EDTA-Ca2+/Mg2+) and other chemical agents (H2O2, Succinate) on Tl'ability of co-aggregate with three other bacteria was studied. The results showed that: Tl'coaggregation percertage with G5 was little changed in the range of pH 5-9, but with H3 or G8,it has a greater degree of reducement, it proved that:acidic conditions has a certain extent influence on the coaggregation ability; The coaggregation percertage of T1 paired with three strains holding a high level in 10℃-40℃, but lower at 4℃and 50℃; overall it did not show the characteristics of extreme sensitivity to temperature. The coaggregation percertage of T1 paired with three strains holding a high level under static condition, but 0-300 r/min of rotation speed did not produce significant effects. This rule also applies to T1'Growth phase. Ca2+,Mg2+ increased coaggregation percertage of three paired strains significantly, and the surfactant (SDS, Tween 80) or oxidant (H2O2) play the opposite role, indicating that the Tl'ability of co-aggregate with three other bacteria is not sensitive to general environmental changes, but to surfactant and oxidant is yes. This work provides a frame of reference to us in the application of coaggregate bacterium to sewage treatment.To explore the molecular mechanism of coaggregation, using trypsin digestion (to remove cell surface protein non-specificity),80℃heat shock (to detect heat sensitive protein,sodium oxide (to remove glycoprotein chain of cell surface non-specificity),amber anhydride (acylation modification of lectin non-specificity) and sugar inhibition test (determine the type lectin), to probe how does T1 coaggregate with paired strains G5, G8, H3, and I1(Bacillus cereus). The results that after treatment by trypsin and heat shock of 80℃, T1'aggregation ability had fell (p<0.05), showing that thermal sensitivity surface proteins of T1 involved in this processs. After treatment by sodium periodate oxidation, coaggregation percertage of T1 paired with I1 or H3 decreased significantly (p<0.05), paired with G5 or G8 were not changes significantly. And the influence of Succinic anhydride on T1 was on the contrary. Both results show that:lectin receptor in T1'surface involved with its coaggregate with I1 and H3, lectin in Tl'surface involved with its coaggregate with G5 and G8. Consider of the coaggregation of T1 and I1,H3, G5, G8 can be inhibited by Mannose and Lactose(50 mM. p<0.05),N-Acetyl-D-Glucosamine(50 mM. p<0.05),D(+)-Galactose(50 mM. p<0.05),D-Lactose(50 mM, p<0.05), We can preliminary conclude that:In the surface of T1, lectin receptors sensitived to Mannose and Lactose were involved in its coaggregation with I1; Sensitived to N-Acetyl-D-Glucosamine, with H3; Lectin sensitived to D(+)-Galactose and D-Lactose play the key role in the process to G5 and G8. Correspondingly, I1'cell surface has lectin sensitived to Mannose and Lactose be involved in this course; H3, lectin sensitived to N-Acetyl-D-Glucosamine; G5 and G8, lectin receptors sensitived to D(+)-Galactose and D-Lactose. This work about coaggregational mechanism supply a frame of reference to the further study.