Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m³/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m³/day at a nitrogen loading rate of 1.0 kg-N/m³/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m³/day at a nitrogen loading rate of 0.93 kg-N/m³/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5–6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.     

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell-cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.     

Identification of the actinorhodin monomer and its related compound from a deletion mutant of the actVA-ORF4 gene of Streptomyces coelicolor A3(2)

An oxygenated derivative of dihydrokalafungin (DHK) was isolated from a deletion mutant of the actVA-ORF4 gene involved in the biosynthesis of a dimeric benzoisochromanequinone (BIQ) antibiotic, actinorhodin (ACT), in Streptomyces coelicolor A3(2). Spectroscopic analysis elucidated its structure as 8-hydroxy-DHK, corresponding to the monomeric unit of ACT. Further metabolite analysis identified its related compound, clearly derived from the reduction of 8-hydroxy-DHK. The structures of these metabolites indicate the essential role of ActVA-ORF4 in ACT biosynthesis, specifically in dimerization of a BIQ intermediate via C-C bond formation.     

Precocene II,a Trichothecene Production Inhibitor,Binds to Voltage-Dependent Anion Channel and Increases the Superoxide Level in Mitochondria of Fusarium graminearum

Precocene II, a constituent of essential oils, shows antijuvenile hormone activity in insects and inhibits trichothecene production in fungi. We investigated the molecular mechanism by which precocene II inhibits trichothecene production in Fusarium graminearum, the main causal agent of Fusarium head blight and trichothecene contamination in grains. Voltage-dependent anion channel (VDAC), a mitochondrial outer membrane protein, was identified as the precocene II-binding protein by an affinity magnetic bead method. Precocene II increased the superoxide level in mitochondria as well as the amount of oxidized mitochondrial proteins. Ascorbic acid, glutathione, and α-tocopherol promoted trichothecene production by the fungus. These antioxidants compensated for the inhibitory activity of precocene II on trichothecene production. These results suggest that the binding of precocene II to VDAC may cause high superoxide levels in mitochondria, which leads to stopping of trichothecene production.     

Detection of bacteria associated with harmful algal blooms from coastal and microcosm environments using electronic microarrays

With the global expansion of harmful algal blooms (HABs), several measures, including molecular approaches, have been undertaken to monitor its occurrence. Many reports have indicated the significant roles of bacteria in controlling algal bloom dynamics. Attempts have been made to utilize the bacteria/harmful algae relationship in HAB monitoring. In this study, bacterial assemblages monitored during coastal HABs and bacterial communities in induced microcosm blooms were investigated. Samples were analysed using denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene. DGGE bands with peculiar patterns before, during, and after algal blooms were isolated and identified. Probes for six ribotypes representing organisms associated with Chatonella spp., Heterocapsa circularisquama, or Heterosigma akashiwo were used for analysis on NanoChip electronic microarray. In addition, a new approach using cultured bacteria species was developed to detect longer (533 bp) polymerase chain reaction-amplified products on the electronic microarray. The use of fluorescently labelled primers allowed the detection of individual species in single or mixed DNA conditions. The developed approach enabled the detection of the presence or absence and relative abundance of the HAB-related ribotypes in coastal and microcosm blooms. This study indicates the ability of electronic microarray platform to detect or monitor bacteria in natural and induced environments.     

Combined process of urea nitrogen removal in anaerobic Anammox co-culture reactor

In this study, the feasibility of biological urea nitrogen removal in anaerobic Anammox co-culture was investigated. After 100 days of operation, complete urea nitrogen removal of 0.35 g (NH(2))(2)CO-N L(-1) d(-1) was achieved. The pure Anammox bacteria were obtained by percoll density-gradient centrifugation and found to be of incapable to hydrolyze urea. The ureolytic bacteria were isolated from the Anammox co-culture by the spread plate and streak. Comparative analysis of partial 16S rDNA sequence presented it belongs to Bacillus sp., and so named as Bacillus sp. LST-1. Fluorescence in situ hybridization was applied to identify the ratio of Bacillus sp. and Anammox in the reactor and the value was approximately 1:4. Urea nitrogen removal was realized in this autotrophic, anoxic reactor via the combined process of urea hydrolysis by Bacillus sp. LST-1 and ammonium oxidizing by Anammox. The investigation of this combined process might have an actual significance in engineering application for its low operational cost.     

Characterization of functional microbial community in a membrane-aerated biofilm reactor operated for completely autotrophic nitrogen removal

The 16S rDNA-based molecular technique was applied to investigate the functional microbial community of a membrane-aerated biofilm (MAB) that was used for completely autotrophic nitrogen removal over nitrite (CANON). The relationships among two kinds of key bacteria responsible for CANON: aerobic ammonia-oxidizing bacteria (AOB) and Anammox bacteria, and their possible distributions in the MAB were discussed based on the microbial community analysis. FISH analysis showed the existence of two visible active layers in experimental MAB. One is the partial nitrifying layer located in the region of oxygen-rich membrane-biofilm interface, dominated by NSO190-positive AOB. The other is the Anammox active layer located in the region of anoxic liquid-biofilm interface, dominated by PLA46 and AMX820-positive Anammox microorganisms. As a result of this study, the AOB as well as Anammox bacteria were present and active in experimental MABR, and the cooperation between AOB and Anammox bacteria was considered to be responsible for CANON.     

Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells

Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1+-transfectant cells (GM1+ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1+ cells. Binding of 125I-NGF to PC12 cells was almost equivalent between GM1+ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1+ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1+ cells. p75NTR and Ras also moved from the raft to non-raft fraction in GM1+ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1+ cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.     

Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions

Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (Fc gammaRIIB and Fc gammaRIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other Fc gammaR (Fc gammaRI and Fc gammaRIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of Fc gammaR and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.