Inhibition by ammonium at concentrations above 1000 mgN/L is known to harm the methanogenesis phase of anaerobic digestion. We anaerobically digested swine waste and achieved steady state COD-removal efficiency of around 52% with no fatty-acid or H[subscript 2] accumulation. As the anaerobic microbial community adapted to the gradual increase of total ammonia-N (NH[subscript 3]-N) from 890 ± 295 to 2040 ± 30 mg/L, the Bacterial and Archaeal communities became less diverse. Phylotypes most closely related to hydrogenotrophic Methanoculleus (36.4%) and Methanobrevibacter (11.6%), along with acetoclastic Methanosaeta (29.3%), became the most abundant Archaeal sequences during acclimation. This was accompanied by a sharp increase in the relative abundances of phylotypes most closely related to acetogens and fatty-acid producers (Clostridium, Coprococcus, and Sphaerochaeta) and syntrophic fatty-acid Bacteria (Syntrophomonas, Clostridium, Clostridiaceae species, and Cloacamonaceae species) that have metabolic capabilities for butyrate and propionate fermentation, as well as for reverse acetogenesis. Our results provide evidence countering a prevailing theory that acetoclastic methanogens are selectively inhibited when the total ammonia-N concentration is greater than ~1000 mgN/L. Instead, acetoclastic and hydrogenotrophic methanogens coexisted in the presence of total ammonia-N of ~2000 mgN/L by establishing syntrophic relationships with fatty-acid fermenters, as well as homoacetogens able to carry out forward and reverse acetogenesis.

The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).

Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.