Abstract

Faecal contamination of water has both anthropogenic and zoogenic origins that can shade various point and nonpoint/diffuse sources of pollution. Due to the dual origin and number of sources of faecal contamination, there are immense challenges in the implementation of effective measures to protect water bodies from pollution that poses threats to human and environmental health. The main health threats refer to infections, illnesses and deaths caused by enteric pathogenicmicrobes, in particular those responsible for waterborne zoonoses. To detect and identify the origins and sources of faecal pollution simultaneously, various methods and indicators have been compiled into a comprehensivemeasuring toolbox. Molecular diagnostics using genetic markers derived from Bacteroidales 16S rRNA gene sequences are quite prevalent in the current methodological implementation for the identification of faecal contamination sources in water. For instance, a culture- and library-independent microbial source tracking toolbox combining micro- and molecular biology tests run as a three-step procedure has been implemented in Norway. Outcomes from the Norwegian studies have identified two general trends in dominance of contributors to faecal water contamination in urban environments. Firstly, there is a tendency of higher contributions from anthropogenic sources during the cold season. Secondly, the identification of the dominance of zoogenic sources in faecalwater contamination during warm periods of the year.

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Abstract

In anaerobic digestion, studies of feeding frequency have produced conflicting results. Hence, the effect of feeding frequency on process variables and microbial community structure was investigated by comparing a laboratory-scale digester fed steam exploded food waste 10 times daily vs. one fed an equivalent amount once daily. The Frequently Fed Digester (FFD) produced on average 20% more methane and had lower effluent concentrations of long-chain fatty acids. Greater daily fluctuations in acetate, pH and biogas production rate could explain the lower specific methane yield and β-oxidation. Feeding frequency also influenced the microbial community whereby Tenericutes (42%) dominated in FFD but Firmicutes (31%) was most abundant in the Daily Fed Digester (DFD). Feeding frequency effects are therefore postulated to occur more often in digesters fed labile feedstocks at high organic loading rates.

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Abstract

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S)envelope protein are currently used in universal vaccination programs and achieve protective immuneresponse in more than 90% of recipients. However, new vaccination strategies are necessary for successfulimmunization of the remaining non- or low-responders. We have previously characterized a novel HBVchimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L)envelope protein (genotype D). The S/preS121–47chimera produced in mammalian cells and Nicotianabenthamiana plants, induced a significantly stronger immune response in parenterally vaccinated micethan the S protein. Here we describe the transient expression of the S/preS121–47antigen in an edibleplant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral admin-istration of adjuvant-free Lactuca sativa expressing the S/preS121–47antigen, three times, at 1lg/dose,was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies wereable to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) moreefficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results sup-port the S/preS121–47antigen as a promising candidate for future development as an edible HBV vaccine.

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Abstract

Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most costeffective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.

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Abstract

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases ( e.g . cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca s ativa ) using Agrobacterium - mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2 Δ N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2 Δ N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.

Abstract

This study describes the first Norwegian microbial source tracking (MST) approach for water quality control and pollution removal from catchment run-off in a nature-based treatment system (NBTS) with a constructed wetland. The applied MST tools combined microbial analyses and molecular tests to detect and define the source(s) and dominant origin(s) of faecal water contamination. Faecal indicator bacteria Escherichia coli and host-specific Bacteroidales 16 s rRNA gene markers have been employed. The study revealed that the newly developed contribution profiling of faecal origin derived from the Bacteroidales DNA could quantitatively distinguish between human and non-human pollution origins. Further, the outcomes of the MST test have been compared with the results of both physicochemical analyses and tests of pharmaceutical and personal care products (PPCPs). A strong positive correlation was discovered between the human marker and PPCPs. Gabapentin was the most frequently detected compound and it showed the uppermost positive correlation with the human marker. The study demonstrated that the NBTS performs satisfactorily with the removal of E. coli but not PPCPs. Interestingly, the presence of PPCPs in the water samples was not correlated with high concentrations of E. coli. Neither has the latter an apparent correlation with the human marker.

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Abstract

Biochar and its properties can be significantly altered according to how it is produced, and this has ramifications towards how biochar behaves once added to soil. We produced biochars from corncob and miscanthus straw via different methods (slow pyrolysis, hydrothermal and flash carbonization) and temperatures to assess how carbon cycling and soil microbial communities were affected. Mineralization of biochar, its parent feedstock, and native soil organic matter were monitored using 13C natural abundance during a 1-year lab incubation. Bacterial and fungal community compositions were studied using T-RFLP and ARISA, respectively. We found that persistent biochar-C with a half-life 60 times higher than the parent feedstock can be achieved at pyrolysis temperatures of as low as 370 °C, with no further gains to be made at higher temperatures. Biochar re-applied to soil previously incubated with our highest temperature biochar mineralized faster than when applied to unamended soil. Positive priming of native SOC was observed for all amendments but subsided by the end of the incubation. Fungal and bacterial community composition of the soil-biochar mixture changed increasingly with the application of biochars produced at higher temperatures as compared to unamended soil. Those changes were significantly (P < 0.005) related to biochar properties (mainly pH and O/C) and thus were correlated to pyrolysis temperature. In conclusion, our results suggest that biochar produced at temperatures as low as 370 °C can be utilized to sequester C in soil for more than 100 years while having less impact on soil microbial activities than high-temperature biochars.

Abstract

Water quality problems in Norway are caused mainly by high phosphorus (P) inputs from catchment areas. Multiple pollution sources contributes to P inputs into watercourses, and the two main sources in rural areas are agricultural runoff and discharge from on-site wastewater treatment systems (OWTSs). To reduce these inputs, Constructed wetlands (CWs) treating catchment runoff have been implemented in Norway since early 1990s. These CWs have been proven effective as supplements to agricultural best management practices for water quality improvements and therefore there are more than 1000 CWs established in Norway at present. This study aims to present some overall data on the present status of CWs treating catchment runoff in Norway, and in particular recent results of source tracking and retention of sediments and total phosphorus (TP) in a model, full-scale, long-term operated CW, which in practice treats runoff from a typical rural catchment with pollution from both point and diffuse sources. Nutrient contributions from agricultural runoff and OWTSs have been quantified in eight catchments, while the source tracking and retention of sediments and P has been studied in the model CW. P runoff in the catchments was largely affected by precipitation and runoff situation, and varied both throughout the year (every single year) and from one year to another. Annual TP contribution that origins from OWTSs was in general limited, and only 1 % in the catchment of the model CW. Monthly contribution, however, was higher than 30 % during warm/dry season, and cold months with frost season. For the purpose of source tracking study, faecal indicator bacteria (reported in terms of Escherichia coli - E. coli) and host-specific 16S rRNA gene markers Bacteroidales have been applied. High E.coli concentrations were well associated with high TP inputs into waterbodies during dry or/and cold season with little or no agriculture runoff, and further microbial source tracking (MST) tests proved human contribution. There are considerable variations in retention of sediments and TP in the CW between the years, and the annual yearly retention was about 38 % and 16 %, respectively. During the study period, the average monthly retention of sediments and TP was 54 % and 32 %, respectively. E. coli concentrations were also reduced in water passing the CW. The study confirmed that runoff from agricultural areas is the main P source in watercourses, however, discharges from OWTS can also be of great importance for the water quality, especially during warm/dry- and cold/frosty periods. Small CWs treating catchment runoff contribute substantially to the reduction of sediments, TP and faecal indicator bacteria transport into water recipients.

Abstract

Elevated nutrient concentrations in streams in the Norwegian agricultural landscape may occur due to faecal contamination. Escherichia coli (E. coli) has been used conventionally as an indicator of this contamination; however, it does not indicate the source of faecal origin. This work describes a study undertaken for the first time in Norway on an application of specific host-associated markers for faecal source tracking of water contamination. Real-time quantitative polymerase chain reaction (qPCR) on Bacteroidales host-specific markers was employed for microbial source tracking (MST) to determine the origin(s) of faecal water contamination. Four genetic markers were used: the universal AllBac (Bacteroidales) and the individual specific markers BacH (humans), BacR (ruminants) and Hor-Bac (horses). In addition, a pathogenicity test was carried out to detect the top seven Shiga toxin-producing E. coli (STEC) serogroups. The ratio between each individual marker and the universal one was used to: (1) normalise the markers to the level of AllBac in faeces, (2) determine the relative abundance of each specific marker, (3) develop a contribution profile for faecal water contamination and (4) elucidate the sources of contamination by highlighting the dominant origin(s). The results of the qPCR MST analyses indicated the actual contributions of humans and animals to faecal water contamination. The pathogenicity test revealed that water samples were STEC positive at a low level, which was in proportion to the concentration of the ruminant marker. The outcomes were verified statistically by coupling the findings of major contamination sources with observations in the field regarding local land use (residential or agricultural). Furthermore, clear correlations between the human marker and E. coli counts as well as the ruminant marker and STEC quantity in faecally contaminated water were observed. The results of this study have the potential to help identify sources of pollution for targeted mitigation of nutrient losses.