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

Cognitive Control Functions in Unipolar Major Depression with and without Co-Morbid Anxiety Disorder

Background: Impaired cognitive control functions have been demonstrated in both major depression (MDD) and anxiety disorder (A), but few studies have systematically examined the impact of MDD with co-morbid A (MDDA), which is the main aim of this study. Method: We compared patients with MDD with (MDDA; n = 24) and without co-morbid A (n = 37) to a group of healthy controls (HC; n = 92) on three subtests from the Cambridge Neuropsychological Test Automated Battery; intra–extra dimensional, stop signal task, and spatial working memory. These tasks correspond to a theoretical model consisting of three separable but interrelated executive control functions: Shifting, Inhibition, and Updating. A simple psychomotor speed measure was also included. Results: After controlling for age, gender, and education level, the results showed that the MDDA group displayed significantly impaired performance on the functions Shifting and Updating compared to HC. There emerged no significant differences between any of the patient groups and HC regarding Inhibition. The pure MDD group did not display dysfunctions relative to the HC group on the main executive control variables, but displayed slowed psychomotor speed. Contrary to expectation there were no significant differences between the MDDA and the MDD groups. Conclusion: Co-morbid anxiety should be taken into account when studying cognitive control functions in major depression

Serotonin Transporter Polymorphism Modulates N-Back Task Performance and fMRI BOLD Signal Intensity in Healthy Women

CONTEXT: Exploring intermediate phenotypes within the human brain's functional and structural circuitry is a promising approach to explain the relative contributions of genetics, complex behaviors and neural mechanisms in the development of major depressive disorder (MDD). The polymorphic region 5-HTTLPR in the serotonin transporter gene (SLC6A4) has been shown to modulate MDD risk, but the neural underpinnings are incompletely understood. OBJECTIVE: 37 right handed healthy women between 21 and 61 years of age were invited to participate in an fMRI modified n-back study. The functional polymorphism 5-HTTLPR located in the promoter region of the SLC6A4 gene was genotyped using polymerase chain reaction (PCR). RESULTS: Short 5-HTTLPR allele carriers showed more blood-oxygen-level-dependent (BOLD) bilateral prefrontal cortex activation in the right [F(2, 30) = 4.8, η(2) = .25, p = .026] and left [F(2, 30) = 4.1, η(2) = .22, p = .015] inferior frontal gyrus pars triangularis with increasing n-back task difficulty relative to long 5-HTTLPR allele carriers. Short 5-HTTLPR allele carriers had inferior task performance on the most difficult n-back condition [F(2, 30) = 4.9, η(2) = .26, p = .014]. CONCLUSIONS: This activation pattern found in healthy at risk individuals resembles an activation pattern that is typically found in patients suffering from acute MDD. Altered function in these areas may reflect intermediate phenotypes and may help explain the increased risk of depression in short 5-HTTLPR allele carriers

Within-Network Connectivity in the Salience Network After Attention Bias Modification Training in Residual Depression: Report From a Preregistered Clinical Trial

Alterations in resting state networks (RSNs) are associated with emotional- and attentional control difficulties in depressed individuals. Attentional bias modification (ABM) training may lead to more adaptive emotional processing in depression, but little is known about the neural underpinnings associated with ABM. In the current study a sample of 134 previously depressed individuals were randomized into 14 days of computerized ABM- or a closely matched placebo training regime followed by a resting state magnetic resonance imaging (MRI) scan. Using independent component analysis (ICA) we examined within-network connectivity in three major RSN’s, the default mode network (DMN), the salience network (SN) and the central executive network (CEN) after 2 weeks of ABM training. We found a significant difference between the training groups within the SN, but no difference within the DMN or CEN. Moreover, a significant symptom improvement was observed in the ABM group after training.Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT02931487

Repeated bronchoscopy in health and obstructive lung disease: is the airway microbiome stable?

Objective Little is known concerning the stability of the lower airway microbiome. We have compared the microbiota identified by repeated bronchoscopy in healthy subjects and patients with ostructive lung diseaseases (OLD). Methods 21 healthy controls and 41 patients with OLD completed two bronchoscopies. In addition to negative controls (NCS) and oral wash (OW) samples, we gathered protected bronchoalveolar lavage in two fractions (PBAL1 and PBAL2) and protected specimen brushes (PSB). After DNA extraction, we amplified the V3V4 region of the 16S rRNA gene, and performed paired-end sequencing (Illumina MiSeq). Initial bioinformatic processing was carried out in the QIIME-2 pipeline, identifying amplicon sequence variants (ASVs) with the DADA2 algorithm. Potentially contaminating ASVs were identified and removed using the decontam package in R and the sequenced NCS. Results A final table of 551 ASVs consisted of 19 × 106 sequences. Alpha diversity was lower in the second exam for OW samples, and borderline lower for PBAL1, with larger differences in subjects not having received intercurrent antibiotics. Permutational tests of beta diversity indicated that within-individual changes were significantly lower than between-individual changes. A non-parametric trend test showed that differences in composition between the two exams (beta diversity) were largest in the PSBs, and that these differences followed a pattern of PSB > PBAL2 > PBAL1 > OW. Time between procedures was not associated with increased diversity. Conclusion The airways microbiota varied between examinations. However, there is compositional microbiota stability within a person, beyond that of chance, supporting the notion of a transient airways microbiota with a possibly more stable individual core microbiome.publishedVersio

Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

publishedVersio

Protected sampling is preferable in bronchoscopic studies of the airway microbiome

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem.publishedVersio

The effect of 5-HTTLPR variability on cognitive control functioning : implications of emotional processing and sex

Background: Although it is widely accepted that serotonin plays a pivotal role in emotional perception and processing, the role of serotonin in cognition is less clear. The present study investigated the implications of introducing emotional faces in a measure of cognitive control functioning. The measure was explored in association with the serotonin transporter polymorphism (5-HTTLPR), linked to serotonin transmission in the brain. An integrative model of emotional processing was used to illustrate cognitive control functioning in emotional processing and potential 5-HTTLPR-dependent diatheses for depressive symptomatology. As previous studies have shown the effect of sex on the relationship between 5-HTTLPR subtypes and cognitive measures, we also included sex as a variable in our analyses. Methods: Sixty healthy participants were recruited in an experimental design. The participants underwent an extensive screening procedure and gave blood samples for 5-HTTLPR analysis. A new computerized test, labeled the Emo n-back, was constructed to explore the implications of presenting emotional categories of human faces in a paradigm measuring aspects of cognitive control functioning. Emotional categories within the Emo n-back were analyzed to spot 5-HTTLPR-dependent variation in cognitive and emotional perception and processing in males and females. Results: Exposure to different emotional categories had specific effects on cognitive control functions as measured by the Emo n-back. Participants showed significantly decreased accuracy on the n-back task when presented with successive images from the negative emotional categories, and this effect was most pronounced for sad compared with neutral faces. A three-way interaction effect was found between sex, 5-HTTLPR polymorphism and emotional categories within the Emo n-back. Examination of this interaction revealed a distinct pattern for female short 5-HTTLPR carriers indicating an increased sensitivity to the sad emotional category in this subgroup of participants. Conclusion: Sex and 5-HTTLPR polymorphism had a significant impact on accuracy in a measure of cognitive control function. The effect is most pronounced when presented with sad human faces, indicating altered sex- 5-HTTLPR-dependent processing linked to specific emotional content. The Emo n-back indicated mechanisms that might represent diatheses for depressive symptomatology by demonstrating the different genotypes to reveal distinct patterns in the way they process adverse environmental stimuli