Role of hemocytin from Litopenaeus vannamei in immune response against microsporidian, Enterocytozoon hepatopenaei.

Hemocytin, a multidomain hemostasis-related protein, is a homologous protein of hemolectin in Drosophila melanogaster and von Willebrand factor (vWF) in humans. The vWF type D (VWD) domain in hemocytin is thought to be a major mediator of hemocyte aggregation and the prophenoloxidase (proPO) activation system. Here, we report for the first time the role of hemocytin from Litopenaeus vannamei (LvHCT) against Enterocytozoon hepatopenaei (EHP), the pathogenic microsporidian causing hepatopancreatic microsporidiosis in Pacific white shrimp (L. vannamei). The LvHCT gene contains 58,366 base pairs consisting of 84 exons encoding for 4267 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that LvHCT was clustered with crustacean hemocytins. Gene expression analysis by quantitative real-time RT-PCR showed that LvHCT in hemocytes was significantly upregulated at 9 and 11 days post-EHP cohabitation, which was consistent with EHP copy numbers in the infected shrimp. To further investigate the biological function of LvHCT in EHP infection, a recombinant protein containing an LvHCT-specific VWD domain (rLvVWD) was expressed in Escherichia coli. In vitro agglutination assays showed that rLvVWD was functionally representative of LvHCT and induced aggregation of pathogens, including Gram-negative and -positive bacteria, fungi, and EHP spore. LvHCT suppression resulted in higher EHP copy numbers and proliferation due to the lack of hemocytin-mediated EHP spore aggregation in LvHCT-silenced shrimp. Moreover, immune-related genes in the proPO-activating cascade and Toll, IMD and JAK/STAT signaling pathways were upregulated to eliminate the over-controlled EHP in LvHCT-silenced shrimp. Furthermore, the impaired phenoloxidase activity due to LvLGBP suppression was recovered after rLvVWD injection, suggesting that LvHCT may be directly involved in phenoloxidase activation. In conclusion, a novel LvHCT is involved in shrimp immunity against EHP via EHP spore aggregation and possible activation of the proPO-activating cascade.

Interaction investigations of crustacean ß-GBP recognition toward pathogenic microbial cell membrane and stimulate upon prophenoloxidase activation.

In invertebrates, crustaceans' immune system consists of pattern recognition receptors (PRRs) instead of immunoglobulin's, which involves in the microbial recognition and initiates the protein-ligand interaction between hosts and pathogens. In the present study, PRRs namely ß-1,3 glucan binding protein (ß-GBP) from mangrove crab Episesarma tetragonum and its interactions with the pathogens such as bacterial and fungal outer membrane proteins (OMP) were investigated through microbial aggregation and computational interaction studies. Molecular recognition and microbial aggregation results of Episesarma tetragonum ß-GBP showed the specific binding affinity toward the fungal ß-1,3 glucan molecule when compared to other bacterial ligands. Because of this microbial recognition, prophenoloxidase activity was enhanced and triggers the innate immunity inside the host animal. Our findings disclose the role of ß-GBP in molecular recognition, host-pathogen interaction through microbial aggregation, and docking analysis. In vitro results were concurred with the in silico docking, and molecular dynamics simulation analysis. This study would be helpful to understand the molecular mechanism of ß-GBP and update the current knowledge on the PRRs of crustaceans.

Potential role of quorum quenching activity of silver nanoparticles in controlling non-filamentous bulking within activated sludge process.

The effective prevention and control of non-filamentous bulking is a significant challenge. In this study, the underlying effect of quorum sensing (QS) on inducing non-filamentous bulking and the maintenance effect of silver nanoparticles (AgNPs) on sludge floc stability, aggregation and settleability based on the quorum quenching (QQ) activity during non-filamentous bulking were investigated. The results showed that the concentration of N-acyl homoserine lactone (AHL) increased significantly in the activated sludge system at a high organic load rate (OLR), triggering the AHL-mediated QS. Additionally, the triggered QS promoted exopolysaccharide secretion, reducing the surface charge and hydrophobicity of the sludge aggregates, and further deteriorating the settleability of the sludge aggregates. AgNPs, a quorum sensing inhibitor (QSI), inhibited the AHL-QS based on QQ activity under high OLR, which maintained the physicochemical properties of extracellular polymeric substances (EPS). AgNPs-QQ maintained the surface energy barrier and electrostatic barrier of sludge aggregates and the gel properties of exopolysaccharides, which is favorable for microbial aggregation. The appropriate concentrations of AgNPs (≤10 mg/L) had no negative effect on biological nutrient removal in the sequencing batch reactors (SBRs) at the high organic loading. Therefore, AgNPs effectively prevent and control non-filamentous bulking by their QQ activity in the activated sludge process. Thus, the present study provided new insights into controlling non-filamentous bulking during the activated sludge process.

A Review of the Role of Extracellular Polymeric Substances (EPS) in Wastewater Treatment Systems.

A review of the characterization and functions of extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems is presented in this paper. EPS represent the complex high-molecular-weight mixture of polymers excreted by microorganisms generated from cell lysis as well as adsorbed inorganic and organic matter from wastewater. EPS exhibit a three-dimensional, gel-like, highly hydrated matrix that facilitates microbial attachment, embedding, and immobilization. EPS play multiple roles in containments removal, and the main components of EPS crucially influence the properties of microbial aggregates, such as adsorption ability, stability, and formation capacity. Moreover, EPS are important to sludge bioflocculation, settleability, and dewatering properties and could be used as carbon and energy sources in wastewater treatment. However, due to the complex structure of EPS, related knowledge is incomplete, and further research is necessary to understand fully the precise roles in biological treatment processes.

Molecular Mechanisms Underpinning Aggregation in Acidiphilium sp. C61 Isolated from Iron-Rich Pelagic Aggregates.

Iron-rich pelagic aggregates (iron snow) are hot spots for microbial interactions. Using iron snow isolates, we previously demonstrated that the iron-oxidizer Acidithrix sp. C25 triggers Acidiphilium sp. C61 aggregation by producing the infochemical 2-phenethylamine (PEA). Here, we showed slightly enhanced aggregate formation in the presence of PEA on different Acidiphilium spp. but not other iron-snow microorganisms, including Acidocella sp. C78 and Ferrovum sp. PN-J47. Next, we sequenced the Acidiphilium sp. C61 genome to reconstruct its metabolic potential. Pangenome analyses of Acidiphilium spp. genomes revealed the core genome contained 65 gene clusters associated with aggregation, including autoaggregation, motility, and biofilm formation. Screening the Acidiphilium sp. C61 genome revealed the presence of autotransporter, flagellar, and extracellular polymeric substances (EPS) production genes. RNA-seq analyses of Acidiphilium sp. C61 incubations (+/- 10 µM PEA) indicated genes involved in energy production, respiration, and genetic processing were the most upregulated differentially expressed genes in the presence of PEA. Additionally, genes involved in flagellar basal body synthesis were highly upregulated, whereas the expression pattern of biofilm formation-related genes was inconclusive. Our data shows aggregation is a common trait among Acidiphilium spp. and PEA stimulates the central cellular metabolism, potentially advantageous in aggregates rapidly falling through the water column.

Calcium effect on microbial activity and biomass aggregation during anaerobic digestion at high salinity.

The potential effect of different Ca2+ additions (150, 300, 450, 600 and 1000 mg/L) on microbial activity and aggregation, during anaerobic digestion at moderate (8 g/L Na+) and high salinity (20 g/L Na+) has been investigated. Batch tests were carried out in duplicate serum bottles and operated for 30 days at 37 °C. At 8 g/L Na+, methanogenic activity and protein degradation were comparable from 150 to 450 mg/L Ca2+, and a significant inhibition was only observed at a Ca2+concentration of 1000 mg/L. In contrast, at 20 g/L Na+, 150 to 300 mg/L were the only Ca2+ concentrations to maintain chemical oxygen demand (COD) removal, protein hydrolysis and methane production. Overall, increasing Ca2+ concentrations had a larger impact on acetotrophic methanogenesis at 20 g/L than at 8 g/L Na+. Increasing Ca2+ had a negative effect on the aggregation behaviour of the dominant methanogen Methanosaeta when working at 8 g/L Na+. At 20 g/L Na+ the aggregation of Methanosaeta was less affected by addition of Ca2+ than at 8 g/L Na+. The negative effect appeared to be connected with Ca2+ precipitation and its impact on cell-to cell communication. The results highlight the importance of ionic balance for microbial aggregation at high salinity, bringing to the forefront the effect on Methanosaeta cells, known to be important to obtain anaerobic granules.

Multi-omics analysis reveals niche and fitness differences in typical denitrification microbial aggregations.

Suspended floc and fixed biofilm are two commonly applied strategies for heterotrophic denitrification in wastewater treatment. These two strategies use different carbon sources and reside within different ecological niches for microbial aggregation, which were hypothesized to show distinct microbial structures and metabolic fitness. We surveyed three floc reactors and three biofilm reactors for denitrification and determined if there were distinct microbial aggregations. Multiple molecular omics approaches were used to determine the microbial community composition, co-occurrence network and metabolic pathways. Proteobacteria was the dominating and most active phylum among all samples. Carbon source played an important role in shaping the microbial community composition while the distribution of functional protein was largely influenced by salinity. We found that the topological network features had different ecological patterns and that the microorganisms in the biofilm reactors had more nodes but less interactions than those in floc reactors. The large niche differences in the biofilm reactors explained the observed high microbial diversity, functional redundancy and resulting high system stability. We also observed a lower proportion of denitrifiers and higher resistance to oxygen and salinity perturbation in the biofilm reactors than the floc reactors. Our findings support our hypothesis that niche differences caused a distinct microbial structure and increased microbial ecology distribution, which has the potential to improve system efficiency and stability.

Understanding possible underlying mechanism in declining germicidal efficiency of UV-LED reactor.

Since ultraviolet light emitting diodes (UV-LEDs) have emerged as an alternative light source for UV disinfection systems, enhancement of reactor performance is a demanding challenge to promote its practical application in water treatment process. This study explored the underlying mechanism of the inefficiency observed in flow-through mode UV disinfection tests to improve the light utilization of UV-LED applications. In particular, the disinfection performance of UV-LED reactors was evaluated using two different flow channel types, reservoir and pathway systems, in order to elucidate the impact of physical circumstances on germicidal efficiency as the light profile was adjusted. Overall, a significant reduction in germicidal efficiency was observed when exposure time was prolonged or a mixing chamber was integrated. Zeta analysis revealed that the repulsion rate between microorganisms decreased with UV fluence transfer, and that change might cause the shielding effect of UV delivery to target microorganisms. In line with the above findings, the reduction in efficiency intensified when opportunities for microbial collision increased. Thus, UV induced microbial aggregation was implicated as being a disinfection hindering factor, exerting its effect through uneven UV illumination. Ultimately, the results refuted the prevailing belief that UV has a cumulative effect. We found that the reservoir system achieved worse performance than the pathway system despite it providing 15 times higher UV fluence: the differences in germicidal efficiency were 1-log, 1.4-log and 1.7-log in the cases of P.aeruginosa, E.coli and S.aureus, respectively.