Biofilm stratification in counter-diffused membrane biofilm bioreactors (MBfRs) for aerobic methane oxidation coupled to aerobic/anoxic denitrification: Effect of oxygen pressure.

Aerobic methane oxidation coupled with denitrification (AME-D) executed in membrane biofilm bioreactors (MBfRs) provides a high promise for simultaneously mitigating methane (CH4) emissions and removing nitrate in wastewater. However, systematically experimental investigation on how oxygen partial pressure affects the development and characteristics of counter-diffusional biofilm, as well as its spatial stratification profiles, and the cooperative interaction of the biofilm microbes, is still absent. In this study, we combined Optical Coherence Tomography (OCT) with Confocal Laser Scanning Microscopy (CLSM) to in-situ characterize the development of counter-diffusion biofilm in the MBfR for the first time. It was revealed that oxygen partial pressure onto the MBfR was capable of manipulating biofilm thickness and spatial stratification, and then managing the distribution of functional microbes. With the optimized oxygen partial pressure of 5.5 psig (25% oxygen content), the manipulated counter-diffusional biofilm in the AME-D process obtained the highest denitrification efficiency, due mainly to that this biofilm had the proper dynamic balance between the aerobic-layer and anoxic-layer where suitable O2 gradient and sufficient aerobic methanotrophs were achieved in aerobic-layer to favor methane oxidation, and complete O2 depletion and accessible organic sources were kept to avoid constraining denitrification activity in anoxic-layer. By using metagenome analysis and Fluorescence in situ hybridization (FISH) staining, the spatial distribution of the functional microbes within counter-diffused biofilm was successfully evidenced, and Rhodocyclaceae, one typical aerobic denitrifier, was found to survive and gradually enriched in the aerobic layer and played a key role in denitrification aerobically. This in-situ biofilm visualization and characterization evidenced directly for the first time the cooperative path of denitrification for AME-D in the counter-diffused biofilm, which involved aerobic methanotrophs, heterotrophic aerobic denitrifiers, and heterotrophic anoxic denitrifiers.

[Establishment of the human papillomavirus type 31 positive cervical cancer cell line].

The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.

[Expression, purification and antigenicity of P35 gene of Toxoplasma gondii].

OBJECTIVE: P35 is an important surface protein for Toxoplasma gondii. To obtain the highly pure and specific antigenicity protein, the gene P35 was cloned and its product was expressed in E. coli Rosetta. The expressed protein was purified and its immunogenecity was studied. METHODS: A pair of primers was designed according to cDNA sequence of P35, and then the P35 gene amplified by PCR was cloned into the vector pGEM-T and proved by DNA sequencing. The P35 gene was subcloned into prokaryotic expression vector pET-KDO, its expression was induced by IPTG, and the target protein was obtained by affinity chromatography. RESULTS: The P35 gene was successfully amplified from genome DNA of Toxoplasma gondii RH strain and a fusion protein was expressed in E. coli. The molecular weight of the expressed protein was about Mr 42 000. Western blotting indicated that the antigenicity of the protein was specific. CONCLUSIONS: The plasmid pET-KDO-p35 is constructed and the high efficient expression of P35 fusion protein has been achieved in E. coli. The fusion protein shows a specific antigenicity, the P35 fusion protein has a potential value in the diagnosis of toxoplasmosis.

[Expression of porcine beta-defensin 1 gene in Pichia pastoris].

PBD-1 is an antibacterial peptide that plays an important role in defence system of porcine. To produce PBD-1 with bioactivity in Pichia pastoris, according to published amino acid sequence of porcine beta-defensin 1(PBD-1) and the partiality codon of yeast, the PBD-1 gene was synthesized by PCR and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-PBD-1, the obtained recombinant plasmid was linearized by Sal I, and then transformed into SMD1168 by electroporation. Under the control of the promoter AOX1, an approximately 4.5 kD PBD-1 peptide was expressed. Antibacterial activity assay shows that the PBD-1 has the antibacterial activity on Staphylococcus aureus. This is the first secreted expression of porcine beta-defensin 1 gene in Pichia pastoris.