Research Article: Microbial origin of bioflocculation components within a promising natural bioflocculant resource of Ruditapes philippinarum conglutination mud from an aquaculture farm in Zhoushan, China

Date Published: June 19, 2019

Publisher: Public Library of Science

Author(s): Jun Mu, Xia Cui, Mingjiao Shao, Yuxia Wang, Qiao Yang, Guangfeng Yang, Liying Zheng, O. Roger Anderson.


Ruditapes philippinarum conglutination mud (RPM) is a byproduct from the aquiculture of an important commercially bivalve mollusk R. philippinarum and has been recently reported as a promising natural bioflocculant resource. However the origin of bioflocculation components within RPM is still a pending doubt and impedes its effective exploitation. This study investigated the probability that RPM bioflocculation components originate from its associated microbes. RPM samples from an aquaculture farm in Zhoushan of China were applied to characterize its microbial community structure, screen associated bioflocculant-producing strains, and explore the homology between extracellular polysaccharides (EPS) from bioflocculant-producing isolates and RPM flocculation components. Results showed that RPM exhibited high bacterial biodiversity, with Proteobacteria, Bacteroidetes and Actinobacteria as the most abundant phyla; hgcI_clade, CL500_29_marine_group, Fusibacter, MWH_UniP1_aquatic_group and Arcobacter as the dominant genera. Fourteen highly efficient bioflocculant-producing strains were screened and phylogenetically identified as Pseudoalteromonas sp. (5), Psychrobacter sp. (3), Halomonas sp. (2), Albirhodobacter sp. (1), Celeribacter sp. (1), Kocuria sp. (1) and Bacillus sp. (1), all of which except Bacillus sp. were reported for the first time for their excellent flocculation capability. Furthermore, EPS from the bioflocculant-producing strains exhibited highly similar monosaccharide composition to the reported flocculation-effective RPM polysaccharides. On the other hand, the existence of fungi in RPM was rare and showed no flocculation functionality. Findings from Zhoushan RPM strongly supported that RPM flocculation components were of bacterial origin and make RPM reproduction possible by fermentation approach.

Partial Text

Microbial bioflocculants (MBF) mainly refer to extracellular polymer substances secreted by microorganisms during their special growth period and characteristic of harmlessness and biodegradability. There has been a history of exploiting bioflocculants from microorganisms of myriad habitats[1–5]. Although MBF are superior to traditional chemical flocculants due to their nontoxicity and environmental friendliness, high production costs and complicated fermentation/recovery processes have become the bottlenecks restraining their widespread commercial application, since most strains yield non-precipitating macromolecule polymers like extracellular polysaccharides (EPS) into the fermentation broth, and high-speed centrifugation and a large quantity of organic solvents are required for reclamation [6–8]. Ruditapes philippinarum conglutination mud (RPM), which refers particularly to the settled sludge from R. philippinarum when freshly harvested clams are kept in sterilized seawater for mud spitting, has been recently reported as a promising natural bioflocculant resource[9]. RPM possesses good flocculation capability, decoloration and heavy metal removal activity, especially exhibits easy recovery and fast settlement before and after treating pollutants[9–11].

R. philippinarum is a filter feeding bivalve which takes in water that has plankton floating in it[28]. With two typical siphons (one in and one out), a large quantity of microorganisms is sucked in during the feeding process and simultaneously wastes are expelled out as RPM. RPM comprises not only cell debris, but also live microbiota which reflects the characteristic endogenous microcosm of R. philippinarum. In our previous studies, RPM was discovered to be a promising natural bioflocculant resource showing excellent flocculation and decoloration activities[9, 11]. Naturally collected RPM exhibits high flocculation activities to kaolin clay both in deionized water and sea water assay systems, and also could flocculate marine microalgae[9]. Further research triggered speculation that bioflocculant activities of RPM might be attributed to R. philippinarum-associated microbiota. An experiment was performed using antibacterial and antifungal antibiotics to selectively inhibit bacteria or fungi in the temporary cultivation system of R. philippinarum. The result showed that RPM from antibacterial temporary cultivation group exhibited a significant decrease in flocculation activity, while RPM from the antifungal selectively inhibited group still kept a high flocculation activity similar to a normal cultivation group, which indirectly suggested that bacteria may be the key factor in the flocculation activity development of RPM [29]. However, more comprehensive and direct proofs were still needed to support the hypothesis of RPM flocculation origin from microorganisms. This study combined non-culturable, culturable and microbial EPS analytical approaches to verify that the RPM associated microbes played a key role in RPM flocculation.