MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters.

Anaerobic digestion of organic waste into methane and carbon dioxide (biogas) is carried out by complex microbial communities. Here, we use full-length 16S rRNA gene sequencing of 285 full-scale anaerobic digesters (ADs) to expand our knowledge about diversity and function of the bacteria and archaea in ADs worldwide. The sequences are processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs) and are used to expand the MiDAS 4 database for bacteria and archaea in wastewater treatment systems, creating MiDAS 5. The expansion of the MiDAS database increases the coverage for bacteria and archaea in ADs worldwide, leading to improved genus- and species-level classification. Using MiDAS 5, we carry out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We reveal how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance, respectively, across samples depending on the amplicon primers used. Finally, we examine the global diversity of functional groups with known importance for the anaerobic digestion process.

Biological conversion of carbon monoxide and hydrogen by anaerobic culture: Prospect of anaerobic digestion and thermochemical processes combination.

Waste biomass is considered a promising renewable energy feedstock that can be converted by anaerobic digestion. However, anaerobic digestion application can be challenging due to the structural complexity of several waste biomass kinds. Therefore, coupling anaerobic digestion with thermochemical processes can offset the limitations and convert the hardly biodegradable waste biomass, including digestate residue, into value-added products: syngas and pyrogas (gaseous mixtures consisting mainly of H2, CO, CO2), bio-oil, and biochar for further valorisation. In this review, the utilisation boundaries and benefits of the aforementioned products by anaerobic culture are discussed. First, thermochemical process parameters for an enhanced yield of desired products are summarised. Particularly, the microbiology of CO and H2 mixture biomethanation and fermentation in anaerobic digestion is presented. Finally, the state-of-the-art biological conversion of syngas and pyrogas to CH4 mediated by anaerobic culture is adequately described. Extensive research shows the successful selective biological conversion of CO and H2 to CH4, acetic acid, and alcohols. The main bottleneck is the gas-liquid mass transfer which can be enhanced appropriately by bioreactors' configurations. A few research groups focus on bio-oil and biochar addition into anaerobic digesters. However, according to the literature review, there has been no research for utilising all value-added products at once in anaerobic digestion published so far. Although synergic effects of such can be expected. In summary, the combination of anaerobic digestion and thermochemical processes is a promising alternative for wide-scale waste biomass utilisation in practice.

A novel two-stage process for biological conversion of syngas to biomethane.

This study shows biomethane production in a novel two-stage syngas biomethanation consisting of the thermophilic anaerobic digestion of sewage sludge combined with an injection of syngas as the first stage. Since the syngas does not contain enough reducing equivalents, the produced biogas was connected to the second stage consisting of the trickle-bed reactor with the external H2 addition (ex-situ) to increase the CH4 content further. The aim was to evaluate the influence of different syngas compositions on the biomethane production in both stages. The results showed that H2 concentration in syngas is the main factor affecting the CH4 content in biogas. Moreover, the ex-situ reactor with H2 addition served to convert the residual CO and CO2, achieving the maximum CH4 content of 94.7% in the produced biomethane. In summary, the two-stage process enables biomethane production without any inhibitory effects on anaerobic sludge digestion.

Bioconversion of carbon dioxide to methane using hydrogen and hydrogenotrophic methanogens.

Biogas produced from organic wastes contains energetically usable methane and unavoidable amount of carbon dioxide. The exploitation of whole biogas energy is locally limited and utilization of the natural gas transport system requires CO2 removal or its conversion to methane. The biological conversion of CO2 and hydrogen to methane is well known reaction without the demand of high pressure and temperature and is carried out by hydrogenotrophic methanogens. Reducing equivalents to the biotransformation of carbon dioxide from biogas or other resources to biomethane can be supplied by external hydrogen. Discontinuous electricity production from wind and solar energy combined with fluctuating utilization cause serious storage problems that can be solved by power-to-gas strategy representing the production of storable hydrogen via the electrolysis of water. The possibility of subsequent repowering of the energy of hydrogen to the easily utilizable and transportable form is a biological conversion with CO2 to biomethane. Biomethanization of CO2 can take place directly in anaerobic digesters fed with organic substrates or in separate bioreactors. The major bottleneck in the process is gas-liquid mass transfer of H2 and the method of the effective input of hydrogen into the system. There are many studies with different bioreactors arrangements and a way of enrichment of hydrogenotrophic methanogens, but the system still has to be optimized for a higher efficiency. The aim of the paper is to gather and critically assess the state of a research and experience from laboratory, pilot and operational applications of carbon dioxide bioconversion and highlight further perspective fields of research.

Effect of S/N ratio on sulfide removal by autotrophic denitrification.

In this study a completely stirred tank reactor was used to study the effect of sulfide to nitrate (S/N) ratio on sulfide removal while nitrate was used as electron acceptor. Several S/N ratios were studied for this purpose ranging from 0.3 to 2.4 mol/mol. The complete sulfide removal was achieved when S/N ratio 0.85 mol/mol was used with the autotrophic denitrification efficiency up to 80 %. No nitrite accumulation was observed, and the main product of sulfide oxidation was sulfate. Dissimilatory nitrogen reduction to ammonia occurred and subsequently, elemental sulfur accumulated while S/N ratio was higher than 1.3 mol/mol. The specific autotrophic denitrification rates under S/N ratios 0.8 and 1.2 were 5 and 26 mg g(-1) h(-1) (N-NO3 (-), VSS), respectively. Thiobacillus denitrificans and Thiomicrospira denitrificans were detected in the reactor by fluorescent in situ hybridization, but their overall representation was not more than 5 % of the entire microbial populations.

Sulfur-oxidizing bacteria in environmental technology.

Hydrogen sulfide is widely known as the most undesirable component of biogas that caused not only serious sensoric and toxic problems, but also corrosion of concrete and steel structures. Many agricultural and industrial waste used in biogas production, may contain a large amount of substances that serve as direct precursors to the formation of sulfide sulfur-sources of hydrogen sulfide in the biogas. Biological desulfurization methods are currently promoted to abiotic methods because they are less expensive and do not produce undesirable materials which must be disposed of. The final products of oxidation of sulfides are no longer hazardous. Biological removal of sulfide from a liquid or gaseous phase is based on the activity of sulfur-oxidizing bacteria. They need an oxidizing agent such as an acceptor of electrons released during the oxidation of sulfides-atmospheric oxygen or oxidized forms of nitrogen. Different genera of sulfur-oxidizing bacteria and their technological application are discussed.

DNA vaccine against human papillomavirus type 16: modifications of the E6 oncogene.

Since its discovery, DNA vaccination has become an effective strategy for the development of vaccines against cancer including cervical carcinoma (CC). The formation of CC is associated with human papillomavirus (HPV) infection. Viral E6 and E7 oncoproteins are suitable targets for therapeutic vaccination. To adapt the HPV16 E6 oncogene for DNA immunisation, we performed several modifications. First we fused the E6 gene with the 5' or 3'-terminus of the Escherichia coli beta-glucuronidase (GUS) gene and showed enhanced immunogenicity of the 3' fusion (GUS.E6). Then, as the E6 oncogene contains two alternative introns that result in the production of truncated forms of the E6 protein, we abolished the 5' splice site in the E6 gene. This modification completely eliminated the expression of the truncated E6 transcripts and thus increased the production of the full-length E6 protein. At the same time, it moderately reduced the immunogenicity of the modified non-fused (E6cc) or fused (GUS.E6cc) genes, probably as a consequence of the substitution in the immunodominant E6 epitope following the abolishment of the splice site. Furthermore, we reduced the oncogenicity of the E6 protein by two point mutations (E6GT) that, together, prevented E6-mediated p53 degradation. Finally, we constructed the GUS.E6GT gene characterized by enhanced safety and immunogenicity when compared with the wild-type E6 gene.

Vaccination with human papillomavirus type 16-derived peptides using a tattoo device.

Tattooing has been shown to be very efficient at inducing immunity by vaccination with DNA vaccines. In this study, we examined the usability of tattooing for delivery of peptide vaccines. We compared tattooing with subcutaneous (s.c.) needle injection using peptides derived from human papillomavirus type 16 (HPV16) proteins. We observed that higher peptide-specific immune responses were elicited after vaccination with the simple peptides (E7(44-62) and E7(49-57)) and keyhole limpet hemocyanin-(KLH)-conjugated peptides (E7(49-57), L2(18-38) and L2(108-120)) with a tattoo device compared to s.c. inoculation. The administration of the synthetic oligonucleotide containing immunostimulatory CpG motifs (ODN1826) enhanced the immune responses developed after s.c. injection of some peptides (E7(44-62), KLH-conjugated L2(18-38) and L2(108-120)) to levels close to or even comparable to those after tattoo delivery of identical peptides with ODN1826. The highest efficacy of tattooing was observed in combination with ODN1826 for the vaccination with the less immunogenic E6(48-57) peptide and KLH-conjugated and non-conjugated E7(49-57) peptides which form the visible aggregates that could negatively influence the development of immune responses after s.c. injection but probably not after tattooing. In summary, we first evidenced that tattoo administration of peptide vaccines that might be useful in some cases efficiently induced both humoral and cell-mediated immune responses.

Enhancement of T cell-mediated and humoral immunity of beta-glucuronidase-based DNA vaccines against HPV16 E7 oncoprotein.

Therapeutic DNA vaccines against oncogenic infection with human papillomavirus type 16 (HPV16) are mostly targeted against viral oncoproteins E7 and E6. To adapt the E7 oncoprotein for DNA immunization, we have previously reduced its oncogenicity by modification of the Rb-binding site and enhanced immunogenicity of the modified E7GGG gene by the fusion with the 5'-terminus of the gene encoding E. coli beta-glucuronidase (GUS). In this study, we attempted to improve immunogenicity of the GUS-based anti-E7 vaccines by increasing the steady-state level of fusion proteins. We fused deletion mutants of E7GGG and codon-optimized E7GGG with the 5'-terminus of GUS and unaltered E7GGG with the 3'-terminus of GUS. Furthermore, we mutated the initiation codon of the GUS gene in the E7GGG.GUS construct, as GUS alone was produced from this fusion gene. We found that only the fusion of E7GGG with the 3'-terminus of GUS (GUS.E7GGG) and deletion mutants of E7GGG with the 5'-terminus of GUS increased the steady-state level of fusion proteins in transfected human 293T cells. Analysis of immune reactions induced in mice by vaccination via a gene gun showed that the increased steady-state level of fusion proteins resulted in augmented production of E7-specific antibodies, but did not enhance cell-mediated anti-tumor immunity. Finally, we joined the signal sequence of the adenoviral E3 protein with GUS.E7GGG. This modification led to the predominant localization of the fusion protein in the endoplasmic reticulum and enhancement of CD8+ T-cell response, while antibody production was reduced. In conclusion, we found modifications of the E7GGG.GUS fusion gene that augmented either humoral or cell-mediated immune responses.

DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants.

Tattooing is one of a number of DNA delivery methods which results in an efficient expression of an introduced gene in the epidermal and dermal layers of the skin. The tattoo procedure causes many minor mechanical injuries followed by hemorrhage, necrosis, inflammation and regeneration of the skin and thus non-specifically stimulates the immune system. DNA vaccines delivered by tattooing have been shown to induce higher specific humoral and cellular immune responses than intramuscularly injected DNA. In this study, we focused on the comparison of DNA immunization protocols using different routes of administrations of DNA (intradermal tattoo versus intramuscular injection) and molecular adjuvants (cardiotoxin pre-treatment or GM-CSF DNA co-delivery). For this comparison we used the major capsid protein L1 of human papillomavirus type 16 as a model antigen. L1-specific immune responses were detected after three and four immunizations with 50 microg plasmid DNA. Cardiotoxin pretreatment or GM-CSF DNA co-delivery substantially enhanced the efficacy of DNA vaccine delivered intramuscularly by needle injection but had virtually no effect on the intradermal tattoo vaccination. The promoting effect of both adjuvants was more pronounced after three rather than four immunizations. However, three DNA tattoo immunizations without any adjuvant induced significantly higher L1-specific humoral immune responses than three or even four intramuscular DNA injections supported by molecular adjuvants. Tattooing also elicited significantly higher L1-specific cellular immune responses than intramuscularly delivered DNA in combination with adjuvants. In addition, the lymphocytes of mice treated with the tattoo device proliferated more strongly after mitogen stimulation suggesting the presence of inflammatory responses after tattooing. The tattoo delivery of DNA is a cost-effective method that may be used in laboratory conditions when more rapid and more robust immune responses are required.

DNA vaccines based on chimeric potyvirus-like particles carrying HPV16 E7 peptide (aa 44-60).

Vaccine strategies for the treatment of human papillomavirus-induced cervical cancer are based mainly on the human papillomavirus 16 E7 (HPV16 E7) oncoprotein. The immunogenicity of the E7 gene has been enhanced by its fusion to many different genes. Here, we linked a short sequence coding for the E7 peptide (aa 44-60) containing immunodominant epitopes for B and T cells to the 3' end of the gene coding for the whole coat protein (CP) of the poty-virus, potato virus A (PVA), and its deleted form (CPdel) with a short C-terminal deletion of 5 amino acids (LGVKG). CP-E7 and CPdel-E7 fusion proteins, just like CP alone, spontaneously assembled into virus-like particles in both procaryotic and eucaryotic cells. The CP-E7 and CPdel-E7 fusion genes induced slightly stronger E7-specific cytotoxic T-lymphocyte responses than the whole E7 gene, although they were still lower than those elicited by the previously constructed fusion gene, Sig/E7GGG/LAMP-1. The E7- and CP-specific antibody responses were not detected in mice vaccinated with CP-E7 and CPdel-E7 fusion genes. The CP-E7 and CPdel-E7 fusion genes protected mice against the development of tumors induced by TC-1 cells producing the E7 antigen and were also effective in the therapeutic setting, i.e. when the vaccination was performed after tumor cell administration. Their antitumor effect was comparable to those of the whole E7 gene and Sig/E7GGG/LAMP-1 fusion gene. There was no relevant difference between immune responses elicited by CP-E7 and CPdel-E7 DNA vaccination.

Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect.

BACKGROUND: The E7 oncoprotein of human papillomavirus type 16 (HPV16) is frequently used as a model tumor-associated antigen. Its immunogenicity has been substantially enhanced by fusion with several proteins of various origins and functions. Different mechanisms have been responsible for increased vaccination efficacy of fusion proteins. METHODS AND RESULTS: We linked E7 and its mutated form (E7GGG) with the mouse heat-shock protein 70.1 (HSP70.1). Enhanced immunogenicity of both fusion genes administered via a gene gun was demonstrated by protection of C57BL/6 mice against oncogenic MHC class I positive TC-1 cells producing the HPV16 E7 oncoprotein but not against the MHC class I negative TC-1/A9 subline. To assess if the efficacy of E7-based DNA vaccines could be increased by combination of various fusion genes, we combined the HSP70.1 fusion genes (i.e. E7HSP or E7GGGHSP) with the fusion construct linking E7GGG with targeting signals of lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Treatment of mice 4 days after TC-1 cell inoculation showed moderately higher immunization potency of HSP70.1 fusion genes in comparison with the Sig/E7GGG/LAMP-1 gene. Any combination of two fusion genes given in the same gene gun shot neither was more effective compared with single genes nor protected mice against TC-1/A9 cells. As fusion of E7GGG with E. coli glucuronidase (E7GGG.GUS) had been previously proven to provide partial protection from TC-1/A9-induced tumors, we also combined E7GGGHSP with E7GGG.GUS. The genes were inoculated either in mix in two gene gun shots or separately each gene in one shot into opposite sides of the abdomen. Neither mode of combined immunization induced higher protection than E7GGG.GUS alone. However, doubling the DNA dose considerably enhanced the antitumor efficacy of E7GGG.GUS. CONCLUSIONS: We constructed highly immunogenic fusions of HPV16 E7 and E7GGG with mouse HSP70.1. Furthermore, we substantially enhanced protection against TC-1/A9 cells with downregulated MHC class I expression by doubling the pBSC/E7GGG.GUS dose, but we failed to demonstrate a beneficial effect of any combination of two fusion genes with different mechanisms causing enhancement of HPV16 E7 immunogenicity.

Combined immunization with DNA and transduced tumor cells expressing mouse GM-CSF or IL-2.

A combination of different types of vaccines usually induces enhanced immune responses in comparison to immunization with single vaccines. The highest efficacy of a heterologous prime-boost strategy is mostly achieved after priming with a DNA vaccine and boosting with a recombinant virus or a protein vaccine. The aim of this study was to determine whether the combination of a DNA and cellular vaccine elicits stronger antitumor immune responses than vaccines used alone and to find out whether the efficacy of this combined immunization depends on the sequence in which the vaccines were applied. We utilized experimental vaccines that proved to be partially effective in protection against mouse tumor cells representing models of human papillomavirus-induced malignancies. The fusion gene Sig/E7GGG/LAMP-1, inoculated via a gene gun, was used for DNA immunization. As cellular vaccines, HPV16 E6/E7 and H-ras transformed B9 or 181 cells transduced with the gene coding for GM-CSF or IL-2, respectively, were applied. In both preventive and therapeutic immunizations, inoculation first with the DNA vaccine followed by application of a cellular vaccine induced the best protection from tumor growth. These results were confirmed by detection of immune reactions with in vitro tests. We failed to enhance immune reactions by utilization of an equivalent mix of B9 and 181 cells, but the addition of the second DNA-vaccine dose applied simultaneously with a cellular-vaccine boost moderately increased antitumor response. Our findings suggest the benefit of the heterologous prime-boost strategy based on combination of a DNA vaccine with a cellular vaccine and importance of sequence in which the vaccines are administered.

Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli beta-glucuronidase.

BACKGROUND: Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb-binding site of the HPV16 E7 protein. METHODS AND RESULTS: To increase E7GGG immunogenicity we constructed fusion genes of E. coli beta-glucuronidase (GUS) with one or three copies of E7GGG. Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N-terminus). The expression of the fusion genes was examined in human 293T cells. Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady-state level in comparison with GUS or E7GGGpGUS. Still, the steady-state level of E7GGG.GUS was about 20-fold higher than that of the E7GGG protein. The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun. TC-1 cells and their clone TC-1/A9 with down-regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation. All mice were protected against challenge with TC-1 cells and most animals remained tumour-free in therapeutic-immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Significant protection was also recorded against TC-1/A9 cells. Both tetramer staining and ELISPOT assay showed substantially higher activation of E7-specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP-1. Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene. CONCLUSIONS: The findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP-1. The E7GGG.GUS-based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression.

Immunisation with modified HPV16 E7 genes against mouse oncogenic TC-1 cell sublines with downregulated expression of MHC class I molecules.

Human papillomavirus type 16 (HPV16)-transformed mouse TC-1 cells are extensively used in the evaluation of efficacy of experimental vaccines against tumours induced by HPVs. As these cells strongly express MHC class I molecules and downregulation of MHC class I surface expression is one of the important mechanisms that enable tumour escape from the host immune system, we undertook to derive TC-1 clones with reduced expression of MHC class I antigens. TC-1 cells were inoculated into mice preimmunised with an E7 gene-based DNA vaccine and from tumours developing in a portion of the animals, cell clones with downregulated MHC class I surface expression were isolated. Treatment with IFN-gamma resulted in an upregulation of MHC class I molecules in these cells, but after IFN-gamma removal, their expression gradually dropped again. When the expression of some components of the antigen-processing machinery (APM; LMP-2, TAP-1, and TAP-2) was tested, a reduced TAP-1 production was detected in cell lines with downregulated MHC class I expression. An enhanced immunoresistance of TC-1-derived clones with reduced MHC class I expression was observed in animals immunised with plasmids carrying modified E7 genes. Apart from the previously described fusion gene Sig/E7/LAMP-1, a new construct, Sig/E7GGG/LAMP-1, with a mutated Rb-binding site, was also used for immunisation. No significant change of immunogenicity was recorded for Sig/E7GGG/LAMP-1. Cell lines with downregulated MHC class I expression derived from TC-1 cells may represent a useful model for testing therapeutic anti-HPV vaccines in settings more relevant to clinical requirements.