Digestat fra biogassproduksjon som substrat og vektor for introduksjon av N2O-respirerende bakterier til landbuksjord

Anthropogenic nitrous oxide (N2O) emissions are largely driven by the input of N-based fertilizers in agriculture. N2O emissions from agricultural soils in Europe are estimated to 0.51 Tg annually (Fig. I), which sums to 48 % of total European N2O emissions and 35 % of the climate forcing from European agriculture. Yet, N2O emission mitigation from agriculture is still hampered by a lack of implemented abatement options. Whilst several biogeochemical reactions may release N2O (Fig. I) the enzyme nitrous oxide reductase (Nos) is the only known enzyme to reduce nitrous oxide. Nos is expressed in denitrifying and non-denitrifying prokaryotes and catalyzes the reduction of N2O to N2. The complete denitrification pathway is the stepwise reduction NO3- → NO2- → NO → N2O → N2, catalyzed by the enzymes Nar/Nap, Nir, Nor, and Nos that are encoded by the genes nar/nap, nirK/nirS, nor, and nosZ, respectively (Fig. I). A significant proportion of the denitrifying community in soils have truncated denitrification pathways, i.e. lacking one to three of the genes encoding the enzymes in the stepwise reduction of NO3- to N2. The consequence of such modularity is that organisms lacking nosZ are net N2O emitters, while organisms with nosZ only are net sinks for N2O. However, organisms equipped with a complete denitrification pathway can also be strong sinks or sources of N2O depending on their regulatory biology. N2O emissions from soils make up a substantial fraction of the climate forcing from food production and mitigation beyond that achieved by “good management practices” are needed if we are to limit global warming by 2 °C, as set in the Paris Agreement. One approach for reducing N2O emissions is to modify the soil microbiome, increasing the proportion of N2O-respiring bacteria (NRB) resulting in reduced N2O emissions. This would, however, be costly and impractical as a standalone operation. As an element towards a low-carbon circular economy, the volume of organic wastes channeled through AD is expected to increase in the coming decades. This presents a unique possibility for mitigation of N2O emissions as the residues of biogas production, digestates, destined as bio-fertilizers in agriculture, could be enriched with N2O-respiring bacteria before soil fertilization. Thus, providing a cost-efficient N2O mitigation measure (Fig. I). Here we demonstrate the use of biogas digestates from anaerobic digestion (AD) as a widely available, low-cost vector for NRB to agricultural soils. A primary task was to search for suitable organisms that 1) could grow to high cell densities in digestate and 2) would act as net N2O sinks in soil. To achieve this, enrichment culturing under anaerobic conditions with N2O as the sole electron acceptor was used. The enrichment cultures were monitored both by measuring the gas kinetics and by inspecting the composition of the microbiota by genomics and proteomics. Based on genomic information and targeted isolation, we obtained axenic cultures of the organisms that became dominant in the enrichment cultures. As a first approach, we enriched indigenous N2O-respiring bacteria in anaerobically digested sewage sludge (digestate) by anoxic incubation with N2O. The gas kinetics predicted that N2O-respiring organisms grew to high cell densities, which was confirmed by metagenomic and metaproteomic (omics-) analyses of the enriched digestate. The omics demonstrated dominance of organisms equipped with the nosZ clade II (coding for N2O-reductase), but also with the genes for the preceding steps of the denitrification pathway. Three digestate-derived N2O-reducing bacteria were isolated, of which one (Azonexus sp.) matched the recovered Metagenome-Assembled Genome (MAG) of the dominant N2O reducer with an average nucleotide identity (ANI) of 98.2%. This MAG also demonstrated a high complement of Nos in the enrichment as quantified by metaproteomics. Gas kinetics and meta-omics indicated that the anaerobic consortium of the digestate remained active during anaerobic incubation with N2O and that N2O-respiring bacteria grew by harvesting fermentation intermediates. The latter was supported by screening carbon catabolism profiles of the isolated organisms. The isolated Azonexus sp. demonstrated regulatory traits that would predict the organism to be a strong N2O sink, and it reduced immediate N2O emissions from digestate-amended soils. However, the Azonexus sp. was probably not an ideal N2O-respiring inoculant in soil because it was equipped with a full-fledged denitrification pathway and because its capacity to utilize soil carbon was limited. The importance of an active methanogenic community throughout the enrichments, providing fermentation intermediates as a carbon source for the N2O-respiring organisms, would predict a selective advantage for organisms with a streamlined (narrow) catabolic capacity, which was the case for the Azonexus sp.. It was evident that we needed to refine our search, to find organisms with a broader catabolic repertoire. A new procedure to obtain more ideal isolates was designed, involving a deliberate enrichment of N2O-respiring organisms with the characteristics of strong growth both in digestate and soil. We thought this could be achieved by “dual enrichment culturing”, i.e. a sequence of enrichment cultures where a fraction of a batch enrichment was passaged to the next batch, alternating between sterile soil and sterile digestate as substrate. Our point of departure was to model this approach, using a simple logistic model for the competition for a common substrate, between three distinctive groups; 1: Organisms with a competitive advantage in digestate (digestate specialists), 2: Organisms with a competitive advantage in soil (soil specialists), and 3: organisms capable of sustaining growth in both environments (generalists). The modelling revealed that generalists could indeed become dominant within a limited number of batch cultures, depending on their competitive edge vis a vis the specialists. Based on this we realized a dual enrichment experiment, using the microbiota of wastewater digestate and soil as initial inocula, sterile digestate and sterile soil as substrate, and monitored the gas kinetics and the community composition (by 16S rDNA amplicon sequencing) throughout seven consecutive enrichment cultures. The gas kinetics corroborated the model’s prediction of a gradual enrichment of organisms that grew both in soil and digestate, and the generalists that became dominant were identified as a limited number of Operational Taxonomic Units (OTUs, based on 16S rDNA sequencing). OTUs that became dominant circumscribed isolates obtained from the enrichment cultures. These OTUs also portrayed the targeted generalist as predicted by the modelling. Most isolates obtained had traits of strong N2O sinks, of which a dominating Cloacibacterium sp., carrying Nos (Clade II) as the sole N-reductase, significantly reduced N2O emissions in digestate amended soils of both neutral and acidic pH. A full-fledged denitrifying Pseudomonas sp. was able to persist in the soil for at least one month whereby significant N2O emissions reduction was obtained upon a fertilization event. Genome analysis of the isolated organisms shed some light as to why these organisms had a competitive advantage in both soil and digestate. Although the ideal isolate is yet to be found, we’ve opened an avenue to a concept that, within the expected expansion of AD, could be scaled to secure a substantial reduction in N2O emissions.Menneskeskapte utslipp av drivhusgassen lystgass (N2O) skyldes i stor grad tilførsel av nitrogenholdig gjødsel til landbruksjord. N2O-utslipp fra landbruksjord i Europa er estimert til 0,51 Tg årlig (Fig. I), som utgjør om lag 48% av de totale utslippene av N2O, som igjen representerer 35 % av det totale klimagassfotavtrykket fra europeisk landbruk. Begrensning av disse utslippene har vært utfordrende grunnet mangel på implementerte metoder og teknologier som effektivt reduserer lystgassutslippet fra landbruksjord. Flere biogeokjemiske reaksjoner kan frigjøre N2O (Fig. I), men enzymet lystgassreduktase (Nos) er det eneste kjente enzymet som reduserer N2O til N2. Nos uttrykkes av denitrifiserende prokaryoter og katalyserer reduksjonen av N2O til N2. Denitrifiserende prokaryoter katalyserer den trinnvise reduksjon av NO3- → NO2- → NO → N2O → N2, som katalyseres av enzymene Nar/Nap, Nir, Nor og Nos som er kodet av genene nar/nap, nir, nor og nosZ (Fig. I). Men, en betydelig andel av det denitrifiserende mikrobesamfunnet i jord er trunkert, dvs. en andel av denitrifikantene mangler ett til tre av genene som koder enzymene involvert i reduksjonen av NO3- til N2. En organisme som kun mangler nosZ vil produsere N2O. I motsatt tilfelle vil en organisme som kun er utstyrt med nosZ bare evne å redusere N2O. Organismer utstyrt med et komplett sett av gener for en fullstendig denitrifikasjon kan være både sterke og svake N2O-reduktanter. Dette bestemmes av deres regulatoriske biologi. N2O-utslipp fra jord utgjør en betydelig mengde av det totale klimafotavtrykket fra matproduksjon og en reduksjon av dette utslippet er nødvendig om vi skal nå de målene som er satt i Parisavtalen og begrense global oppvarming til 2 °C. En mulighet for å redusere N2O-utslipp er å modifisere jordmikrobiomet ved å øke andelen N2O-respirerende bakterier (NRB) – noe som vil redusere utslippene av N2O. Men, som ett frittstående tiltak vil en storskala modifisering av mikrobiologien i jordsmonnet være svært ressurskrevende. Som et ledd i overgangen til en lav-karbon sirkulærøkonomi forventes anaerob utråtning (AD) å øke i omfang og rekkevidde de neste årene. Denne utviklingen skaper en unik mulighet for å redusere N2O-utslipp dersom digestater, restproduktet fra AD, som brukes som organisk gjødsel i landbruket, kan anrikes med N2O-reduserende bakterier før disse digestatene benyttes som gjødsel (Fig. I). Her demonstrerer vi at lett tilgjengelige digestater kan benyttes som vekstsubstrat og en vektor for å overføre NRB til jord. En slik modifikasjon være et svært kostnadseffektivt N2O-reduserende tiltak. Det primære målet i denne avhandlingen var å lete etter egnede organismer som 1) kan gro til høy celletetthet i digestater, og 2) redusere N2O-utslipp fra jord. For å oppnå dette ble anrikninger av slike organismer ved bruk av N2O som eneste elektronakseptor gjennomført. Anrikningskulturene ble monitorert ved å måle gasskinetikk og ved overvåking av samfunnsprofiler og bakteriell populasjonsdynamikk ved bruk av DNA- og proteomanalyser. Med basis i den genetiske informasjonen var målet å isolere dominerende organismer fra anrikningskulturene. Som en første tilnærming anriket vi N2O-reduserende bakterier som er naturlig tilstedeværende i digestat i anoksiske inkubasjoner hvor N2O ble tilsatt som eneste elektronakseptor. Gasskinetikk predikerte at NRB vokste til høye celletettheter under inkubasjonen, som ble bekreftet av metagenom- og metaproteomanalyser av det anrikede digestatet. Meta-omikk analysene viste at organismer utstyrt med nosZ Type II (genet for N2O-reduktase), men også med de øvrige genene for et komplett denitrifiseringsspor, dominerte anrikningen. Tre N2O-reduserende bakterier ble isolert hvorav det ene isolatet, en Azonexus sp., samsvarte med et gjenvunnet Dechloromonas-beslektet metagenom som dominerte anrikningen med en aminosyreidentitet på 98,2% delt med det dominerende metagenomet. Metaproteomikk viste at dette metagenomet utrykte brorparten av Nos under anrikningen. Gasskinetikk og meta-omikk avslørte videre at det metanogene konsortiet i digestatet forblir aktivt også under den anaerobe inkubasjonen med N2O, og at dominerende bakterier med en anaerob respiratorisk metabolisme sannsynligvis vokste ved å høste fermenteringsmellomprodukter fra det metanogene samfunnet. Det sistnevnte ble støttet ved karbonkatabolismeprofiler for de isolerte organismene. Den isolerte Azonexus sp. demonstrerte regulatoriske egenskaper som ville forutsi at organismen var en sterk N2O-reduktant, og den reduserte N2O-utslipp fra jord gjødslet med Azonexus anriket digestat. Likevel så var anrikningsvinneren sannsynligvis ikke en ideell N2O-reduserende inokulant i jord fordi dens evne til å overleve i jord-miljøet sannsynligvis var begrenset. Betydningen av et aktivt metanogent bakteriesamfunn, som produsenter av karbonkilder for NRB igjennom anrikningene, gav sannsynligvis en selektiv fordel for organismer med en strømlinjeformet (smal) katabolsk kapasitet, som var tilfelle for Azonexus sp.. Det var tydelig at vi trengte å videreforedle anrikningsprosedyrene våre for å anrike kompetente organismer en bredere metabolsk fleksibilitet. En ny tilnærming for å oppnå mer ideelle isolater som evner å vokse i både jord og i digestat ble designet med utgangspunkt i å selektivt anrike organismer med disse egenskapene. Vi antok at slike organismer kunne anrikes ved en «dobbelt-anrikning»-prosedyre der miljøet ble vekslet mellom jord og digestat. Mao: En sekvens av batch-anrikningskulturer hvor en overfører en fraksjon av anrikningen til en ny batch og vekslet mellom jord og digestat som vekstsubstrat. Med dette utgangspunktet ble logistisk vekst, kun med konkurranse om tilgjengelig karbon, modellert for tre ulike bakteriegrupper; 1) Organismer med konkurransefortrinn i digestat (digestat-spesialister), 2) Organismer med konkurransefortrinn i jord (jordspesialister), og 3) organismer som er i stand til å opprettholde vekst/aktivitet i begge miljøer (generalister). Modelleringen avslørte at generalister teoretisk sett kunne anrikes ved å passere fraksjoner av disse anrikningene mellom digestat og jord, avhengig av generalistenes konkurransefortrinn relativt til spesialistene. Basert på denne modelleringen realiserte vi et nytt anrikningseksperiment med bruk av digestat og jord som initielt inokulum og sterilt digestat og jord som vekstsubstrat og lot populasjonene konkurrere om tilgjengelig karbon med tilsats av N2O. Monitorering av gasskinetikk og populasjonsdynamikk (ved 16S amplikonsekvensering) igjennom syv sammenhengende anrikninger viste en populasjonsutvikling slik predikert fra modelleringen: Gasskinetikken støttet modellprediksjonen om en gradvis ankrikning av organismer som vokste i jord og digestat, og 16S-analysen vist at et fåtall operasjonelle taksonomiske enheter (OTUer) dominerte anrikningen. Isolatene fra disse anrikningskulturene var omsluttet av en dominerende gruppe OTUer som portretterte vekstegenskaper igjennom hele anrikningsserien som representerte de ønskede generalistvinnerne. Ett av isolatene, en Cloacibacterium sp., hvis genom kun kodet for genet for Nos, dominerte anrikningene, og denne reduserte også N2O-utslipp i jord med lav pH. Et annet isolat, en Pseudomonas sp., demonstrert en mer langvarig N2O reduserende aktivitet i jord da aktiviteten var fremtredende selv 30 dager etter gjødsling. Genomanalyse av isolerte organismer kastet noe lys kring hvorfor disse organismer kunne ha et konkurransefortrinn i anrikningene. Selv om det ideelle isolatet ennå ikke er funnet, har vi åpnet en vei for et konsept som, i kontekst av den forventede utviklingen av AD, kan skaleres for å sikre betydelig reduksjon i N2O-utslipp.Vestfjorden Avløpsselskap (VEAS

Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

publishedVersio

Effect of salinity shifts on microbial community composition in different nitrifying biofilms in continuous moving bed biofilm reactors.

Salinity is considered a common stress factor when nitrification is applied in waste water treatment. Observations often show a sub-optimal nitrification performance as a response to variations in in-fluent salinity. Recently, researchers have coupled microbial community dynamics and changes in the community structure to process stability, and there is a need for interdisciplinary research at the borderline between microbial ecology and process engineering to understand these links.The aim of this study was to investigate and compare the community changes in two different nitrifying cultures adapted to different salinities as a response to a change in salinity. Two continuous moving bed bio-film reactors were first operated at salinities corresponding to the community origin. These were a 0 ppt salinity adapted culture, originating from low salinity municipal waste water, and a 33 ppt salinity adapted culture originating from a recirculated aquaculture filter. After a period of continuous operation at those salinities the salinity were switched: operating the 0 ppt salinity adapted culture with a 33 ppt salinity based cultivation medium, and the 33 ppt adapted culture with a 0 ppt salinity based cultivation medium. Changes in community structure and community dynamics were monitored over time with denaturing gradient gel electrophoresis (DGGE).Average Bray-Curtis similarities within each community showed that a static nitrifying community was not essential for complete nitrification, but rather an advantageous community trait that gave a higher resilience towards fluctuations in environmental factors such as pH, temperature and nitrogen loading.The results showed that the nitrifying culture adapted to 33 ppt salinity was more robust towards a change in salinity, and that the culture was halotolerant. Full nitrification was achieved from day 38 after the salinity change. The microbial adaptation strategy was not determined by either acclimation or by population shift, but rather a combination of the two determined by the community's inherent prerequisites. Further, it was demonstrated that during low salt adaptation a stable nitrification performance was not necessarily coupled to a stable community structure.Population shift was probably the main adaptation strategy for the 0 ppt adapted culture when adapting to 33 ppt salinity. Observations during continuous operation at this salinity showed only partial nitrification from day 54 after the salinity change with low ammonium oxidation rates. Up towards this point in time, and towards the end of the experiment, the community structure was constantly changing

Understanding structure/function relationships in nitrifying microbial communities after cross-transfer between freshwater and seawater

In this study, nitrification before and after abrupt cross-transfer in salinity was investigated in two moving bed biofilm reactors inoculated with nitrifying cultures that had adaptation to freshwater (FR) and seawater salinities (SR). FR and SR MBRRs were exposed to short and long term cross-transfer in salinity, and the functional capacity of nitrifying microbial communities was quantified by the estimation of ammonia and nitrite oxidation rates. Salinity induced successions were evaluated before and after salinity change by deep sequencing of 16S rRNA gene amplicons and statistical analysis. The bacterial community structure was characterized and Venn diagrams were included. The results indicated that after salinity cross-transfer, the FR was not significantly recovered at seawater salinity whereas SR showed high resistance to stress caused by low-salt. Succession and physiological plasticity were the main mechanisms of the long-term adaption of the nitrifying communities exposed to abrupt salinity changes. Independently of salinity, some nitrifiers presented high physiological plasticity towards salinity and were very successful at both zero and full seawater salinity. SR culture is robust and suitable inoculum for ammonium removal from recirculating aquaculture systems and industrial wastewaters with variable and fast salinity changes. Our findings contradict the current perspective of the significance of salinity on the structure of nitrifying communities

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates

publishedVersio

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates

Organic waste fractions such as sewage sludge, food waste and manure can be stabilized by anaerobic digestion (AD) to produce renewable energy in the form of biogas. Following AD, the digested solid fraction (digestate) is usually dewatered to reduce the volume before transportation. Post-AD treatments such as the Post-AD thermal hydrolysis process (Post-AD THP) have been developed to improve the dewatering, but the mode of action is not well understood. In this study, samples from 32 commercial full-scale plants were used to assess the impact of Post-AD THP on a broad range of raw materials. Maximum dewatered cake solids after Post-AD THP was predicted by thermogravimetric analysis (TGA). Post-AD THP changed the moisture distribution of the samples by increasing the free water fraction. A consistent improvement in predicted dewatered cake solids was achieved across the 32 samples tested, on average increasing the dry solids concentration by 87%. A full-scale trial showed that dewatering Post-AD THP digestate at 80 °C improved dewatered cake solids above the predictions by TGA at 35 °C. In conclusion, dewatered cake solids were significantly improved by Post-AD THP, reducing the volume of dewatered cake for disposal.publishedVersio

Protecting Thermally Hydrolyzed Biosolids from Pathogenic Bacterial Growth by Addition of Compost

Sludge biosolids for agricultural application represent a valuable fertilizer but also a health risk unless pathogens are effectively reduced, and recontamination controlled. The Post Anaerobic Digestion Thermal Hydrolysis Process (Post-AD THP) is gaining interest due to improved dewaterability, reducing the volume and thus transportation costs of biosolids. However, Post-AD THP results in sterile biosolids easily exposed to recontamination by pathogens due to the lack of microbial competitors. In theory, this could be suppressed by establishing a competing community of harmless bacteria. The theory was tested by monitoring the abundance of Escherichia coli (viable counts) and gene abundance (ddPCR) in wastewater recontaminated Post-AD THP biosolids, with and without addition of compost. Respiration, total bacterial population and bacterial diversity (16S rRNA amplicon sequencing) were used to monitor the microbial community. Biosolids from the regulatory approved methods thermophilic AD (TAD) and Pre-AD THP were tested in parallel for comparison. The results demonstrated that regulatory requirements can be reached by storing the TAD and Pre-AD THP biosolids for 3 days after recontamination and the Post-AD THP biosolids for more than 13 days. However, addition of compost suppressed growth of E. coli in Post-AD THP biosolids, reducing the time to comply with regulative requirements. In conclusion, pathogen growth in Post-AD THP biosolids can be controlled by inoculation with compost

Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

publishedVersio

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates

Organic waste fractions such as sewage sludge, food waste and manure can be stabilized by anaerobic digestion (AD) to produce renewable energy in the form of biogas. Following AD, the digested solid fraction (digestate) is usually dewatered to reduce the volume before transportation. Post-AD treatments such as the Post-AD thermal hydrolysis process (Post-AD THP) have been developed to improve the dewatering, but the mode of action is not well understood. In this study, samples from 32 commercial full-scale plants were used to assess the impact of Post-AD THP on a broad range of raw materials. Maximum dewatered cake solids after Post-AD THP was predicted by thermogravimetric analysis (TGA). Post-AD THP changed the moisture distribution of the samples by increasing the free water fraction. A consistent improvement in predicted dewatered cake solids was achieved across the 32 samples tested, on average increasing the dry solids concentration by 87%. A full-scale trial showed that dewatering Post-AD THP digestate at 80 °C improved dewatered cake solids above the predictions by TGA at 35 °C. In conclusion, dewatered cake solids were significantly improved by Post-AD THP, reducing the volume of dewatered cake for disposal

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates

Organic waste fractions such as sewage sludge, food waste and manure can be stabilized by anaerobic digestion (AD) to produce renewable energy in the form of biogas. Following AD, the digested solid fraction (digestate) is usually dewatered to reduce the volume before transportation. Post-AD treatments such as the Post-AD thermal hydrolysis process (Post-AD THP) have been developed to improve the dewatering, but the mode of action is not well understood. In this study, samples from 32 commercial full-scale plants were used to assess the impact of Post-AD THP on a broad range of raw materials. Maximum dewatered cake solids after Post-AD THP was predicted by thermogravimetric analysis (TGA). Post-AD THP changed the moisture distribution of the samples by increasing the free water fraction. A consistent improvement in predicted dewatered cake solids was achieved across the 32 samples tested, on average increasing the dry solids concentration by 87%. A full-scale trial showed that dewatering Post-AD THP digestate at 80 °C improved dewatered cake solids above the predictions by TGA at 35 °C. In conclusion, dewatered cake solids were significantly improved by Post-AD THP, reducing the volume of dewatered cake for disposal