Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater.

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

Microbial community diversity of organically rich cassava sago factory waste waters and their ability to use nitrate and N2O added as external N-sources for enhancing biomethanation and the purification efficiency.

Water shortage necessitated South Indian sago factory owners, extracting starch out of cassava tubers, to install biogas plants where a starch utilizing microbial community multiplies and reduces the biological oxygen demand (BOD) of the waste waters by presently about 30%. The purification efficiency of sago factory waste waters, rich in solid particles and having wide C/N ratios, around 250, through unstirred biogas plants needs to be improved. Our approach was to apply instead of animal slurry nitrate (NO3(-)) and nitrous oxide (N2O) as external N-sources anticipating a better N-distribution in the unstirred biogas plants. Estimated cell numbers, bacterial community changes, on the basis of 16S rRNA gene clone libraries and changing CO2-, CH4-, N2O releases due to the presence of nitrate or N2O suggest that acid tolerant Lactobacillus spp. dominate the biogas plant inflows (pH 3.5). They were very less or not found in the outflows (pH 7.3). Assumingly, the phyla Bacteroidetes (Prevotella spp.), Proteobacteria (Rhizobium spp., Defluvibacter sp.), Firmicutes (Megasphaera spp., Dialister spp., Clostridium spp.) and Synergistetes (Thermanaerovibrio spp.), not-detectable in the biogas plant inflows, replaced them. Anaerobes, about 400cellsml(-1) in the inflows, increased to about 10(6)cellsml(-1) in the outflows. The methane formation, as confirmed by the incubation experiments, suggests that methanogens must have been present among the anaerobes. In the biogas plant in- and outflows also about 300cellsml(-1) denitrifying bacteria and up to 10(4)cfu fungi were found. Despite the low number of denitrifying bacteria nitrate added to the biogas plant in- and outflows was widely consumed and added N2O decreased considerably. Thus, wide C/N ratios substrates like sago factory waste waters keep the N2O emissions low by using N2O either as electron acceptor or by incorporating it into the growing biomass what needs to be confirmed. The biogas plant inflow samples have emitted tentatively more CO2 and the outflow samples released more CH4.

Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection.

BACKGROUND: The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1. RESULTS: Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites. CONCLUSION: The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.

Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization.

DNA vaccines have been successful in eliciting potent immune responses in mice. Their efficiency, however, is restricted in larger animals. One reason for the limited performance of the DNA vaccines is the lack of molecular strategies to enhance immune responses. Additionally, genes directly cloned from pathogenic organisms may not be efficiently translated in a heterologous host expression system as a consequence of codon bias. To evaluate the influence of codon optimization on the immune response, we elected to use the Tat antigens of human immunodeficiency virus type 1 (HIV-1) (subtype C) and HIV-2, as these viral antigens are poorly immunogenic in natural infection and in experimental immunization and they are functionally important in viral infectivity and pathogenesis. Substituting codons that are optimally used in the mammalian system, we synthetically assembled Tat genes and compared them with the wild-type counterparts in two different mouse strains. Codon-optimized Tat genes induced qualitatively and quantitatively superior immune responses as measured in a T-cell proliferation assay, enzyme-linked immunospot assay, and chromium release assay. Importantly, while the wild-type genes promoted a mixed Th1-Th2-type cytokine profile, the codon-optimized genes induced a predominantly Th1 profile. Using a pepscan strategy, we mapped an immunodominant T-helper epitope to the core and basic domains of HIV-1 Tat. We also identified cross-clade immune responses between HIV-1 subtype B and C Tat proteins mapped to this T-helper epitope. Developing molecular strategies to optimize the immunogenicity of DNA vaccines is critical for inducing strong immune responses, especially to antigens like Tat. Our identification of a highly conserved T-helper epitope in the first exon of HIV-1 Tat of subtype C and the demonstration of a cross-clade immune response between subtypes B and C are important for a more rational design of an HIV vaccine.

Codon optimization and ubiquitin conjugation of human immunodeficiency virus-1 Tat lead to enhanced cell-mediated immune responses.

The transactivator protein, Tat, is a potential candidate for developing a vaccine against human immunodeficiency virus (HIV-1). Since Tat is not immunodominant, especially when delivered as a genetic vaccine, we expressed codon-optimized subtype-C Tat as a molecular conjugate of ubiquitin, to elicit antigen-specific cell-mediated immune responses. Immunization of mice with different ubiquitin-Tat constructs elicited a strong cellular, but not a humoral, immune response. The combination of codon-optimization and ubiquitin-mediated processing of Tat induced a Th-1 type cellular immune response that was detectable without in vitro stimulation, suggesting its potential utility for destruction of virus-infected cells via CTL-mediated lysis. Preliminary attempts at characterizing the immunodominant regions identified a novel T-helper epitope within the core domain of Tat.