Metagenomics, metatranscriptomics, and proteomics reveal the metabolic mechanism of biofilm sequencing batch reactor with higher phosphate enrichment capacity under low phosphorus load.

The biofilm sequencing batch reactor (BSBR) process has higher phosphate recovery efficiency and enrichment multiple when the phosphorus load is lower, but the mechanism of phosphate enrichment at low phosphorus load remains unclear. In this study, we operated two BSBR operating under low and high phosphorus load (0.012 and 0.032 kg/(m3·d)) respectively, and used metagenomic, metatranscriptomic, and proteomics methods to analyze the community structure of the phosphorus accumulating organisms (PAOs) in the biofilm, the transcription and protein expression of key functional genes and enzymes, and the metabolism of intracellular polymers. Compared with at high phosphorus load, the BSBR at low phosphorus load have different PAOs and fewer types of PAOs, but in both cases the PAOs must have the PHA, PPX, Pst, and acs genes to become dominant. Some key differences in the metabolism of PAOs from the BSBR with different phosphorus load can be identified as follows. When the phosphorus load is low, the adenosine triphosphoric acid (ATP) and NAD(P)H in the anaerobic stage come from the TCA cycle and the second half of the EMP pathway. The key genes that are upregulated include GAPDH, PGK, ENO, ppdk in the EMP pathway, actP in acetate metabolism, phnB in polyhydroxybutyrate (PHB) synthesis, and aceA, mdh, sdhA, and IDH1 in the TCA cycle. In the meantime, the ccr gene in the PHV pathway is inhibited. As a result, the metabolism of the PAOs features low glycogen with high PHB, Pupt, Prel, and low PHV. That is, more ATP and NAD(P)H flow to phosphorus enrichment metabolism, thus allowing the highly efficient enrichment of phosphorus from low concentration phosphate thanks to the higher abundance of PAOs. The current results provide theoretical support and a new technical option for the enrichment and recovery of low concentrations of phosphate from wastewater by the BSBR process.

A new insight in enhancing phosphate enrichment in biofilm process: Comparison of the key metabolic pathways in highly-efficient and dominant PAOs based on metagenomics.

The formation of dominant phosphate-accumulating organisms (PAOs) is essential for the high enrichment of phosphate in biofilm sequencing batch reactors (BSBR) for phosphorus recovery. The dominant PAOs in the biofilm process have not been isolated and purified, and the key metabolic pathways that promote the formation of dominant PAOs are still unclear. In this study, four strains of highly-efficient PAOs were obtained by an innovative isolation procedure. The relationship between the abundance of highly-efficient and dominant PAOs and the phosphate removal ability was compared. We found that the abundance of PAOs was positively correlated with the phosphate removal efficiency in vitro pure culture and complex biofilm process. Metagenomics analysis revealed that compared with highly-efficient PAOs cultured in vitro, dominant PAOs in biofilms had unique key metabolic pathways, F-ATPases and N-Acyl homoserine lactones (AHLs). F-ATPases are important for maintaining the proton motive force (PMF) required for the uptake of carbon sources by PAOs, and AHLs are participating in phosphate metabolism through quorum sensing (QS) mediated secretion of extracellular polymeric substance (EPS). The formation of dominant PAOs was promoted by optimizing carbon source uptake and phosphate metabolism. This study revealed that the difficult isolation of dominant PAOs was due to the AHLs-mediated QS, and we identified the key pathways regulating the formation of dominant PAOs in biofilms through genomics analysis. Our findings provide insights in enhancing phosphate enrichment in BSBR by modulating the components of microbial community under the low concentration of carbon source consumption.

Metagenomics reveals the metabolism of polyphosphate-accumulating organisms in biofilm sequencing batch reactor: A new model.

This study assessed the impact of the operating conditions of the biofilm sequencing batch reactor (BSBR) on the community structure and the growth/metabolic pathways of its polyphosphate-accumulating organisms (PAOs). There are significant difference with reference to the enhanced biological phosphorus removal (EBPR) process. The leading PAOs in BSBR generally are capable of high affinity acetate metabolism, gluconeogenesis, and low affinity phosphate transport, and have various carbon source supplementation pathways to ensure the efficient circulation of energy and reducing power. A new model of the metabolic mechanism of PAOs in the BSBR was formulated, which features low glycogen metabolism with simultaneous gluconeogenesis and glycogenolysis and differs significantly from the classic mechanism based on Candidatus_Accumulibacter and Tetrasphaera. The findings will assist the efficient recovery of low concentration phosphate in municipal wastewater.

Study on community structure and metabolic mechanism of dominant polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) in suspended biofilm based on phosphate recovery.

Biofilm sequencing batch reactor (BSBR) can achieve efficient phosphate (P) removal and enrichment, but its process performance and metabolic mechanisms for P removal and enrichment of municipal wastewater remain largely unclear. In the present study, we assessed the P removal and enrichment of municipal wastewater at influent P concentrations of 2.5 mg/L and 10 mg/L. The efficiency of P removal and enzyme activity in polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) were compared, and the growth and metabolic characteristics of dominant PAOs and GAOs at different influent P concentrations were studied with the macro-sequencing technology. The results showed that the P recovery efficiencies were 70.03% and 76.19% when the influent P concentration was 2.5 mg/L and 10 mg/L in BSBR, respectively, and the maximum P concentration of recovery liquid was 81.29 mg/L and 173.12 mg/L, respectively. There were no phosphate kinase (PPK) and phosphate hydrolase (PPX) in extracellular polymeric substances (EPS). The dominant PAOs were Candidatus_Contendobacter, Dechloromonas, and Flavobacterium, and the dominant GAO was Candidatus_Competibacter. The abundance of Candidatus_Contendobacter was the highest with the most potential contribution to P removal. PAOs had competitive advantages in carbon (C) source uptake, glycogen metabolism, P metabolism, and adenosine triphosphate (ATP) metabolism. HMP was unique to PAOs, EMP had the highest abundance in glycogen metabolism, and ED was contained in PAOs of BSBR. These results indicated that BSBR provided sufficient reducing power and ATP for PAOs through different glycogen decomposition pathways to promote P uptake and obtained competitive advantages in P metabolism, C source uptake, and ATP utilization to achieve efficient P removal and enrichment. Collectively, our current findings provided valuable insights into the P removal and enrichment mechanism of BSBR in municipal sewage.

Comparative transcriptomic analysis of silkworm Bmovo-1 and wild type silkworm ovary.

The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm(+Bmovo-1)) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm(+Bmovo-1) ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm(+Bmovo-1) and 98.87% for WT silkworm ovaries. Comparative transcriptome analysis showed 123 upregulated and 111 downregulated genes in silkworm(+Bmovo-1) ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis.

Bmovo-1 regulates ovary size in the silkworm, Bombyx mori.

The regulation of antagonistic OVO isoforms is critical for germline formation and differentiation in Drosophila. However, little is known about genes related to ovary development. In this study, we cloned the Bombyx mori ovo gene and investigated its four alternatively spliced isoforms. BmOVO-1, BmOVO-2 and BmOVO-3 all had four C2H2 type zinc fingers, but differed at the N-terminal ends, while BmOVO-4 had a single zinc finger. Bmovo-1, Bmovo-2 and Bmovo-4 showed the highest levels of mRNA in ovaries, while Bmovo-3 was primarily expressed in testes. The mRNA expression pattern suggested that Bmovo expression was related to ovary development. RNAi and transgenic techniques were used to analyze the biological function of Bmovo. The results showed that when the Bmovo gene was downregulated, oviposition number decreased. Upregulation of Bmovo-1 in the gonads of transgenic silkworms increased oviposition number and elevated the trehalose contents of hemolymph and ovaries. We concluded that Bmovo-1 was involved in protein synthesis, contributing to the development of ovaries and oviposition number in silkworms.

Reducing blood glucose levels in TIDM mice with an orally administered extract of sericin from hIGF-I-transgenic silkworm cocoons.

In previous studies, we reported that the blood glucose levels of mice with type I diabetes mellitus (TIDM) was reduced with orally administered silk gland powder from silkworms transgenic for human insulin-like growth factor-I (hIGF-I). However, potential safety hazards could not be eliminated because the transgenic silk gland powder contained heterologous DNA, including the green fluorescent protein (gfp) and neomycin resistance (neo) genes. These shortcomings might be overcome if the recombinant hIGF-I were secreted into the sericin layer of the cocoon. In this study, silkworm eggs were transfected with a novel piggyBac transposon vector, pigA3GFP-serHS-hIGF-I-neo, containing the neo, gfp, and hIGF-I genes controlled by the sericin-1 (ser-1) promoter with the signal peptide DNA sequence of the fibrin heavy chain (Fib-H) and a helper plasmid containing the piggyBac transposase sequence under the control of the Bombyx mori actin 3 (A3) promoter, using sperm-mediated gene transfer to generate the transformed silkworms. The hIGF-I content estimated by enzyme-linked immunosorbent assay was approximately 162.7 ng/g. To estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered sericin from the transgenic silkworm. The blood glucose levels of the mice were significantly reduced, suggesting that the extract from the transgenic hIGF-I silkworm cocoons can be used as an orally administered drug.