Ammonia based sanitation technology

Water-borne sanitation of toilet waste is not a viable option for the estimated 2.6 billion people that lack improved sanitation throughout the world. In Environmental Systems Analysis, source separating sewage systems have proven to be of interest, since both energy and nutrients are saved compared with conventional systems. As the urine and faecal matter contribute with the majority of nutrients to wastewater but constitute a small part of the volume, these fractions are suitable for nutrient recycling to agriculture. The potential content of pathogenic (disease causing) microorganisms makes it a necessity to sanitise the material before use as a fertiliser, especially as many pathogens are zoonotic, infecting both man and animal. The main objective of this study was to evaluate ammonia based sanitation technology for source separated urine and faeces aiming for production of safe fertilisers. To achieve this objective, the inactivation kinetics of several groups of organisms was investigated in relation to concentration of free ammonia, NH3, temperature and dry matter content. Inactivation of Ascaris suum eggs, Salmonella spp. Enterococcus spp., S. Typhimurium phage 28B, an f-specific RNA phage MS2 and a coliphage ΦX 174 was monitored in spiked human urine and faeces. Storage of urine diluted 1:0, 1:1 and 1:3 with water was studied at 4, 14, 24, and 34°C. Faecal material, source separated dry, was treated with urea at concentrations ranging from 0.5% to 2% at 14, 24, and 34°C. Faecal material with ash amendments was studied at 24 and 34°C, separately and with supplementary addition of 1% urea. Temperature was found to be a key factor for the efficiency of the ammonia based sanitation, both through synergy and by affecting transformation of ammonia into NH3. At 34°C the NH3 concentrations in urine and faecal material resulted in short decimal reduction (D) values for microorganism concentrations, except for the bacteriophage 28B, which showed little inactivation in stored faecal material. At 24°C, treatments of both urine and faeces with NH3 concentrations of 50 mM and above gave significant reductions whereas at lower concentrations (urine 1:3 and storage of faecal matter) little inactivation of bacteriophage 28B and ascaris eggs was observed. This means that urine must be collected as concentrated as possible in order to contain sufficient ammonia to reduce pathogens by storage. Treatment with urea, a 2% addition resulted in stable pH and NH3 concentrations that resulted in fast Salmonella spp. inactivation even at 4°C and 14°C, and inactivation of ascaris and the bacteriophage at temperature 24°C and above. Coverage with ash and lime during collection can give an enhanced pathogen inactivation when later treated in closed containers. Accompanying urea treatment of faeces collected with ash is possible but with a high pH (>10) in the material urea will not be degraded and thus not contribute to inactivation

Supporting students in higher education in transforming their thesis into a scientific article

Detta verk är licensierat under en Creative Commons Erkännande-IckeKommersiell 4.0 Internationell-licens.According to our experience from education in health and welfare, there are increased expectations in academia for students to publish their work as scientific international articles. Reasons and motivation for transforming the thesis to a scientific article vary; however, it is essential to understand that the work includes substantial revision to meet the requirements of a scientific article. We have long experience of teaching in higher education and have supported students from various educational programs in health and welfare to publish their theses as articles in scientific journals. This demands time, engagement and competence from both supervisors and students, demands that to our knowledge rarely are acknowledged in higher education organisations. A coherent guide on how to support students aiming for publication would have been helpful, and this article is anchored in our experiences and reflections on this matter. This article highlights eight preparatory reflective questions and presents a guide for supervisors in supporting actions for the transformation of a thesis to an article.publishedVersio

Nitrogen based sanitation of source separated faecal matter

To fulfil the UN millennium goals for sustainable development, there is an urgent need for alternatives to conventional water based sanitation. Faeces and urine contain valuable plant nutrients and should be considered as resources rather than wastes. Collection with efficient water usage enables faeces and urine to be reused and environmental pollution may be better prevented. When using human excreta as plant fertilisers, it is important to prevent disease transmission by reducing the content of gastrointestinal pathogens. Nitrogen based treatment of faeces and manure with ammonium hydroxide and urea has been shown to be an efficient method for inactivating bacterial pathogens, nematode eggs and protozoan cysts. The substance responsible for microbial inactivation is uncharged ammonia, NH3 (aq). As ammonia is a weak base, the ammonia equilibrium can be controlled by additions of alkaline agents such as lime. A pH above 8 is needed to produce substantial amounts of ammonia in the form of NH3. The objective of this study was to examine the potential of nitrogen based treatment for reduction of human bacterial and parasitic pathogens in faeces collected separately from a dry sanitation system. The faeces samples were inoculated with Enterococcus faecalis, Salmonella Typhimurium, Escherichia coli O157:H7 and Ascaris suum eggs prior to treatment. Treatments were performed at 14°C and consisted of ammonia (1% w/w), urea (0.5, 1 and 2% w/w) and/or slaked lime and storage. Inactivation of bacteria was monitored by plate count methods and viability of ascaris eggs by microscopy. Ammonia content was determined by distillation and titration and NH3 concentration calculations based on pH. Addition of ammonia, urea and/or lime resulted in an increase in pH from the initial 7.14, whereas the pH in the storage treatment decreased. The 1% ammonia treatment with equimolar addition of total ammonia as 2% urea resulted in a higher pH (10.2) than 2% urea (9.2). Addition of urea gave a more stable pH over time compared to addition of lime, although pH declined with time in all treatments, thus affecting NH3 concentration. On day 21, 1% ammonia and 2% urea were the only treatments with sufficiently high pH to produce a substantial amount of NH3, although reductions in bacteria fitted well to an exponential function even after day 21. No viable A. suum eggs were observed after 41 days in the 0.5% urea, 2% urea and storage treatments. On day 12, no significant differences in viability could be observed between the different treatments. However a tendency for reduced A. suum viability according to the urea gradient could be observed. E. faecalis was less sensitive to the treatments than any of the pathogenic bacteria studied and E. coli was more sensitive than S. Typhimurium, although the differences were small. The 1% ammonia and 2% urea treatments were the most efficient at reducing bacteria, resulting in a decimal reduction time of 0.13-5 days for 1% ammonia treatment and 0.2-41 days for 2% urea treatment. Regression analysis of the coefficients k for the bacterial reduction function and the concentrations of NH3 in the treatments revealed a significant linear correlation for all bacteria studied. However, pH was only significantly correlated to k for E. faecalis, though weaker than to NH3. The relationship between NH3 concentration and reduction coefficient gave a change in k of 0.022 units per mM NH3 for E. coli and 0.014 units for S. Typhimurium. The pathogenic bacteria were more sensitive to NH3 concentration than E. faecalis, with a change in k of 0.00054 units per mM NH3. As E. faecalis seemed to have a higher threshold concentration for inactivation by ammonia based treatments and its reduction time exceeded that of the pathogenic bacteria studied, E. faecalis might not be a suitable indicator organism for this method. In conclusion, treatment with 1% ammonia, 2% urea or 1% urea with lime addition was sufficient to give a 6 log10 reduction of the pathogenic bacteria studied, within 3 weeks of treatment. A. suum had a rapid inactivation but seemed to be affected by other environmental parameters in addition to ammonia concentration.För att uppnå de av FN uppsatta milleniummålen, att halvera det antal människor som saknar fullgod sanitet, måste alternativ till vattenburen sanitet utvecklas och implementeras. Fekalier och urin måste därvid ses som en resurs och en väg att sluta näringskretsloppet och därmed minimera miljöpåverkan. För att säkerställa mänsklig hälsa när fekalier och urin används för jordbruksproduktion krävs behandling som förhindrar spridning av sjukdomsalstrande mikroorganismer. Kvävebaserade behandlingar med ammoniumhydroxid och urea och har visat sig effektiva att reducera bakteriella patogener, nematodägg och protozoer i fekalier och gödsel. Det är oladdad ammoniak, NH3, som påverkar mikrobiell inaktivering. Då ammoniak är en svag bas påverkas jämvikten mellan oladdad och laddad ammoniak av pH. Därmed kan andelen ammoniak som förekommer som NH3 styras med tillsatser av alkaliska substanser, såsom kalk. Ett pH över 8 är nödvändigt för att uppnå betydande koncentration av NH3. Syftet med denna studie var att undersöka potentialen hos kvävebaserade behandlingar för reduktion av bakteriella och parasitiska patogener i källsorterade fekalier. Fekalier från ett torrsorterande system behandlades vid 14°C med ammoniak (1%), urea (0,5, 1 och 2%) och/eller kalk och lagring. Enterococcus faecalis, Salmonella Typhimurium, Escherichia coli O157:H7 and Ascaris suum ägg tillsattes före behandling. Den bakteriella reduktionen studerades med plattmetoder och överlevnaden hos ascaris ägg observerades i mikroskop. Innehållet av ammoniak destillerades och titrerades för koncentrationsbestämmning och andelen NH3 beräknades utifrån pH. Alla behandlingar förutom lagring resulterade i en ökning av pH från det initiala 7,14. Behandling med 1% ammoniak med samma molara tillsats av total ammoniak som behandling med 2% urea gav ett högre pH jämfört med 2% urea, 10,2 respektive 9,2. Behandling med urea gav ett pH som var stabilare över tid jämfört med pH från behandlingar med kalk. I alla behandlingarna minskade pH över tid vilket således påverkade koncentrationen av NH3. Dag 21 var det bara behandlingarna med 1% ammoniak och 2% urea som höll ett tillräckligt högt pH för att ge någon betydande andel NH3. Dock följde den bakteriella reduktionen en exponentiell avdödning även i de behandlingar som efter dag 21 höll låg NH3(aq) koncentration. Inga levande A. suum ägg kunde observeras efter 41 dagar i de studerade behandlingarna: urea 0,5% och 2% och lagring. Dag 12 kunde man se en trend med lägre överlevnad med ökad ureatillsats, dock inte statistiskt signifikant. Av de studerade bakterierna visade sig E. faecalis minst känslig för de olika behandlingarna. E. coli var generellt känsligare för behandlingarna än S. Typhimurium, dock var resultaten för dessa två patogena bakterier mer lika vad gäller känslighet och reduktionstid jämfört med E. faecalis. Behandling med 1% ammoniak eller 2% urea visade sig vara mest effektiv för bakteriell avdödning med decimalreduktionstid 0,13-5 dagar för 1% ammoniak och 0,2-41 dagar för 2% urea. Regressionsanalys av reduktionskoefficienter k mot koncentrationen NH3 visade ett linjärt samband för alla studerade bakterier. Bara E. faecalis gav ett signifikant samband mellan k och pH, dock svagare än för NH3. Det linjära sambandet gav en förändring av k med 0,022 enheter per mM NH3 för E. coli och 0,014 for S. Typhimurium. E. faecalis som var mindre känslig för behandlingarna hade en förändring av koefficienten med 0,00054 enheter per mM NH3. E. faecalis visade sig mycket mer tålig än de patogena bakterierna som studerades och en högre tröskelkoncentration av oladdad ammoniak verkar krävas för effektiv reduktion. Eftersom reduktionstiden för E. faecalis i vissa fall överskred reduktionstiden för de patogena bakterierna mångfalt verkar inte E. faecalis vara ett passande val av indikatororganism för reduktion av bakteriella patogener med denna metod. Slutsatsen är att behandling med 1% ammoniak, 2% urea eller 1% urea med kalktillsats verkar vara goda behandlingsalternativ för källsorterade fekalier då dessa behandlingar gav en 6 log10 reduktion av de patogena bakterierna inom tre veckor. A.suum verkar även ha påverkats av andra faktorer än NH3 koncentration då även lagring gav en snabb reduktion av överlevnaden

Nitrogen based sanitation of source separated faecal matter

To fulfil the UN millennium goals for sustainable development, there is an urgent need for alternatives to conventional water based sanitation. Faeces and urine contain valuable plant nutrients and should be considered as resources rather than wastes. Collection with efficient water usage enables faeces and urine to be reused and environmental pollution may be better prevented. When using human excreta as plant fertilisers, it is important to prevent disease transmission by reducing the content of gastrointestinal pathogens. Nitrogen based treatment of faeces and manure with ammonium hydroxide and urea has been shown to be an efficient method for inactivating bacterial pathogens, nematode eggs and protozoan cysts. The substance responsible for microbial inactivation is uncharged ammonia, NH3 (aq). As ammonia is a weak base, the ammonia equilibrium can be controlled by additions of alkaline agents such as lime. A pH above 8 is needed to produce substantial amounts of ammonia in the form of NH3. The objective of this study was to examine the potential of nitrogen based treatment for reduction of human bacterial and parasitic pathogens in faeces collected separately from a dry sanitation system. The faeces samples were inoculated with Enterococcus faecalis, Salmonella Typhimurium, Escherichia coli O157:H7 and Ascaris suum eggs prior to treatment. Treatments were performed at 14°C and consisted of ammonia (1% w/w), urea (0.5, 1 and 2% w/w) and/or slaked lime and storage. Inactivation of bacteria was monitored by plate count methods and viability of ascaris eggs by microscopy. Ammonia content was determined by distillation and titration and NH3 concentration calculations based on pH. Addition of ammonia, urea and/or lime resulted in an increase in pH from the initial 7.14, whereas the pH in the storage treatment decreased. The 1% ammonia treatment with equimolar addition of total ammonia as 2% urea resulted in a higher pH (10.2) than 2% urea (9.2). Addition of urea gave a more stable pH over time compared to addition of lime, although pH declined with time in all treatments, thus affecting NH3 concentration. On day 21, 1% ammonia and 2% urea were the only treatments with sufficiently high pH to produce a substantial amount of NH3, although reductions in bacteria fitted well to an exponential function even after day 21. No viable A. suum eggs were observed after 41 days in the 0.5% urea, 2% urea and storage treatments. On day 12, no significant differences in viability could be observed between the different treatments. However a tendency for reduced A. suum viability according to the urea gradient could be observed. E. faecalis was less sensitive to the treatments than any of the pathogenic bacteria studied and E. coli was more sensitive than S. Typhimurium, although the differences were small. The 1% ammonia and 2% urea treatments were the most efficient at reducing bacteria, resulting in a decimal reduction time of 0.13-5 days for 1% ammonia treatment and 0.2-41 days for 2% urea treatment. Regression analysis of the coefficients k for the bacterial reduction function and the concentrations of NH3 in the treatments revealed a significant linear correlation for all bacteria studied. However, pH was only significantly correlated to k for E. faecalis, though weaker than to NH3. The relationship between NH3 concentration and reduction coefficient gave a change in k of 0.022 units per mM NH3 for E. coli and 0.014 units for S. Typhimurium. The pathogenic bacteria were more sensitive to NH3 concentration than E. faecalis, with a change in k of 0.00054 units per mM NH3. As E. faecalis seemed to have a higher threshold concentration for inactivation by ammonia based treatments and its reduction time exceeded that of the pathogenic bacteria studied, E. faecalis might not be a suitable indicator organism for this method. In conclusion, treatment with 1% ammonia, 2% urea or 1% urea with lime addition was sufficient to give a 6 log10 reduction of the pathogenic bacteria studied, within 3 weeks of treatment. A. suum had a rapid inactivation but seemed to be affected by other environmental parameters in addition to ammonia concentration.För att uppnå de av FN uppsatta milleniummålen, att halvera det antal människor som saknar fullgod sanitet, måste alternativ till vattenburen sanitet utvecklas och implementeras. Fekalier och urin måste därvid ses som en resurs och en väg att sluta näringskretsloppet och därmed minimera miljöpåverkan. För att säkerställa mänsklig hälsa när fekalier och urin används för jordbruksproduktion krävs behandling som förhindrar spridning av sjukdomsalstrande mikroorganismer. Kvävebaserade behandlingar med ammoniumhydroxid och urea och har visat sig effektiva att reducera bakteriella patogener, nematodägg och protozoer i fekalier och gödsel. Det är oladdad ammoniak, NH3, som påverkar mikrobiell inaktivering. Då ammoniak är en svag bas påverkas jämvikten mellan oladdad och laddad ammoniak av pH. Därmed kan andelen ammoniak som förekommer som NH3 styras med tillsatser av alkaliska substanser, såsom kalk. Ett pH över 8 är nödvändigt för att uppnå betydande koncentration av NH3. Syftet med denna studie var att undersöka potentialen hos kvävebaserade behandlingar för reduktion av bakteriella och parasitiska patogener i källsorterade fekalier. Fekalier från ett torrsorterande system behandlades vid 14°C med ammoniak (1%), urea (0,5, 1 och 2%) och/eller kalk och lagring. Enterococcus faecalis, Salmonella Typhimurium, Escherichia coli O157:H7 and Ascaris suum ägg tillsattes före behandling. Den bakteriella reduktionen studerades med plattmetoder och överlevnaden hos ascaris ägg observerades i mikroskop. Innehållet av ammoniak destillerades och titrerades för koncentrationsbestämmning och andelen NH3 beräknades utifrån pH. Alla behandlingar förutom lagring resulterade i en ökning av pH från det initiala 7,14. Behandling med 1% ammoniak med samma molara tillsats av total ammoniak som behandling med 2% urea gav ett högre pH jämfört med 2% urea, 10,2 respektive 9,2. Behandling med urea gav ett pH som var stabilare över tid jämfört med pH från behandlingar med kalk. I alla behandlingarna minskade pH över tid vilket således påverkade koncentrationen av NH3. Dag 21 var det bara behandlingarna med 1% ammoniak och 2% urea som höll ett tillräckligt högt pH för att ge någon betydande andel NH3. Dock följde den bakteriella reduktionen en exponentiell avdödning även i de behandlingar som efter dag 21 höll låg NH3(aq) koncentration. Inga levande A. suum ägg kunde observeras efter 41 dagar i de studerade behandlingarna: urea 0,5% och 2% och lagring. Dag 12 kunde man se en trend med lägre överlevnad med ökad ureatillsats, dock inte statistiskt signifikant. Av de studerade bakterierna visade sig E. faecalis minst känslig för de olika behandlingarna. E. coli var generellt känsligare för behandlingarna än S. Typhimurium, dock var resultaten för dessa två patogena bakterier mer lika vad gäller känslighet och reduktionstid jämfört med E. faecalis. Behandling med 1% ammoniak eller 2% urea visade sig vara mest effektiv för bakteriell avdödning med decimalreduktionstid 0,13-5 dagar för 1% ammoniak och 0,2-41 dagar för 2% urea. Regressionsanalys av reduktionskoefficienter k mot koncentrationen NH3 visade ett linjärt samband för alla studerade bakterier. Bara E. faecalis gav ett signifikant samband mellan k och pH, dock svagare än för NH3. Det linjära sambandet gav en förändring av k med 0,022 enheter per mM NH3 för E. coli och 0,014 for S. Typhimurium. E. faecalis som var mindre känslig för behandlingarna hade en förändring av koefficienten med 0,00054 enheter per mM NH3. E. faecalis visade sig mycket mer tålig än de patogena bakterierna som studerades och en högre tröskelkoncentration av oladdad ammoniak verkar krävas för effektiv reduktion. Eftersom reduktionstiden för E. faecalis i vissa fall överskred reduktionstiden för de patogena bakterierna mångfalt verkar inte E. faecalis vara ett passande val av indikatororganism för reduktion av bakteriella patogener med denna metod. Slutsatsen är att behandling med 1% ammoniak, 2% urea eller 1% urea med kalktillsats verkar vara goda behandlingsalternativ för källsorterade fekalier då dessa behandlingar gav en 6 log10 reduktion av de patogena bakterierna inom tre veckor. A.suum verkar även ha påverkats av andra faktorer än NH3 koncentration då även lagring gav en snabb reduktion av överlevnaden

Contrasting Effects of Long-Term Nitrogen Deposition on Plant Phosphorus in a Northern Boreal Forest

Ecosystem responses of carbon and nitrogen (N) biogeochemistry to N deposition have a high variation across sites. Phosphorus (P), which can interact strongly with N, can be the cause of some of this variation. We quantified plant N and P concentrations and estimated P stocks in aboveground foliage, and soil O-horizon P concentrations and stocks after 18 years in a long-term stand-scale (0.1 ha) N addition experiment [12.5 kg (N1) and 50 kg (N2) N ha−1 year−1] in a c. 100-years-old boreal spruce [Picea abies (L.) Karst] forest. Basal area growth had increased by 65% in the N2 treatment compared to control, along with a higher leaf area index, and lower litter decomposition rates. The higher tree growth occurred during the initial c. 10-years period thereafter resuming to control rates. We hypothesized that increased plant demand for P together with decreased recycling of organic matter in this initially N limited system may have decreased plant-available P, with possible consequences for longer-term biogeochemistry and ecosystem production. However, resin-extractable P did not differ between the three treatments (0.32 kg P ha−1), and plant NP ratios and P concentrations and O-horizon P characteristics were similar in the N1 and control treatments. The N2 treatment doubled total P in the O-horizon (100 vs. 54 kg P ha−1), explained by an increase in organic P. The N concentration, NP ratio, and spruce needle biomass were higher in N2, while the P stock in current year needles was similar as in the control due to a lower P concentration. In addition to P dilution, increased light competition and/or premature aging may have caused the reduction of N-stimulated growth of the trees. For the dominant understory shrub [Vaccinium myrtillus (L.)] no changes in growth was apparent in N2 despite a significantly higher NP ratio compared to control (15 vs. 9, respectively). We therefore conclude that increased NP ratio of vegetation cannot be used as a sole indicator of P limitation. The vegetation and O-horizon changes in N2 were still large enough to merit further studies addressing whether such high N loads may alter ecosystem biogeochemistry toward P limitation. For the lower N addition rate, relevant from an anthropogenic N deposition perspective, we suggest it had no such effect

Fate of Ascaris at various pH, temperature and moisture levels

Soil-transmitted helminths (STH) are intestinal worms that infect 24% of the world’s population. Stopping the spread of STH is difficult, as the eggs are resilient (can withstand high pH) and persistent (can remain viable in soils for several years). To ensure that new sanitation systems can inactivate STH, a better understanding of their resilience is required. This study assessed the inactivation of Ascaris eggs under various conditions, in terms of moisture content (MC) (90%), temperature (20–50 C) and pH (7–12.5). The results highlight that the exposure of Ascaris eggs to elevated pH (10.5–12.5) at temperatures 27.5 C for >70 days had no effect on egg viability. Compounding effects of alkaline pH (10.5) or decreasing MC (<20%) was observed at 35 C, with pH having more of an effect than decreasing MC. To accelerate the inactivation of STH, an increase in the treatment temperature is more effective than pH increase. Alkaline pH alone did not inactivate the eggs but can enhance the effect of ammonia, which is likely to be present in organic wastes

Long‐term nitrogen enrichment does not increase microbial phosphorus mobilization in a northern coniferous forest

* Nitrogen (N) deposition can enhance carbon (C) capture and storage in northern coniferous forests but it may also enhance the demand for phosphorus (P). While it is well established that long‐term N enrichment can decrease decomposition and enhance the accumulation of C in soils, it remains uncertain if a higher demand and acquisition of P influence soil C. * We studied microbial phosphorus mobilization and growth within a long‐term N enrichment experiment in a Norway spruce forest, where N deposition was simulated by adding 0, 12.5 or 50 kg N ha−1 year−1 for 21 years (n = 12), by incubating microbial ingrowth cores with needles and humus with low and high P content, and with sand with and without mineral apatite P. * Long‐term N enrichment had no effect on microbial P mobilization in needles and humus and did not enhance the positive effect that apatite had on fungal growth. However, it consistently strengthened the retention of C in the soil by decreasing decomposition of needle and humus, both with low and high P content, and by increasing fungal growth in sand‐filled ingrowth cores. Furthermore, we did not find any evidence that higher microbial P mobilization in response to N enrichment affected soil C storage. * These results show that long‐term N enrichment in relatively young soils dominated by coniferous trees and ectomycorrhizal fungi can have relatively small impact on microbial P mobilization from organic sources and on the potential to mobilize P from minerals, and subsequently that elevated P demand due to N enrichment is unlikely to lead to a reduction in the soil C accumulation rate

Presence and activity of nitrogen-fixing bacteria in Scots pine needles in a boreal forest: a nitrogen-addition experiment

Endophytic nitrogen-fixing bacteria have been detected and isolated from the needles of conifer trees growing in North American boreal forests. Because boreal forests are nutrient-limited, these bacteria could provide an important source of nitrogen for tree species. This study aimed to determine their presence and activity in a Scandinavian boreal forest, using immunodetection of nitrogenase enzyme subunits and acetylene-reduction assays of native Scots pine (Pinus sylvestris L.) needles. The presence and rate of nitrogen fixation by endophytic bacteria were compared between control plots and fertilized plots in a nitrogen-addition experiment. In contrast to the expectation that nitrogen-fixation rates would decline in fertilized plots, as seen, for instance, with nitrogen-fixing bacteria associated with bryophytes, there was no difference in the presence or activity of nitrogen-fixing bacteria between the two treatments. The extrapolated calculated rate of nitrogen fixation relevant for the forest stand was 20 g N ha(-1) year(-1), which is rather low compared with Scots pine annual nitrogen use but could be important for the nitrogen-poor forest in the long term. In addition, of 13 colonies of potential nitrogen-fixing bacteria isolated from the needles on nitrogen-free media, 10 showed in vitro nitrogen fixation. In summary, 16S rRNA sequencing identified the species as belonging to the genera Bacillus, Variovorax, Novosphingobium, Sphingomonas, Microbacterium and Priestia, which was confirmed by Illumina whole-genome sequencing. Our results confirm the presence of endophytic nitrogen-fixing bacteria in Scots pine needles and suggest that they could be important for the long-term nitrogen budget of the Scandinavian boreal forest

Early and repeated nutrient additions support far greater stemwood production in Norway spruce than traditional late-rotation fertilisation

Silvicultural techniques aimed at promoting forest biomass production can help meet the growing demand for renewable materials and mitigate climate change. One-time nitrogen (N) addition late in the rotation is a wellestablished method to stimulate growth in coniferous forests in northern Europe, but the potential gains from earlier and repeated fertiliser application remain uncertain. Here, we tested the impact of repeated fertilisation in juvenile Norway spruce stands across 9 sites covering a wide range of growing conditions over a 700 km stretch from central to southern Sweden. We tested the fertilisation effects using two separate studies: i) an interval trial with a fertilisation frequency of one (F1), two (F2), or three years (F3) performed at plot-level across five sites (2002-2014), and ii) a practice-oriented trial with a two-year fertilisation interval (F2) applied at stand-level and replicated at four sites (2003-2013). The composition of the nutrient mix in each plot was optimised based on foliar nutrient analyses. In the interval trial, all three fertilisation schedules strongly increased periodic annual increment (PAI) (F1: 105 %, F2: 93 %, F3: 79 %) relative to the unfertilised control, resulting in more than a doubling of stem volume yield in the F1 and F2 treatments (110 % and 120 %, respectively) and a significantly smaller but still sizeable yield stimulation of 82 % in the F3 treatment. Nitrogen use efficiency (NUE, stemwood volume increase per unit mass of N added) was similar among fertilisation intervals (on average 130 m3 ha-1 1000 kg N-1), indicating that the extra N provided through yearly fertilisation (F1) is redundant given the similar stemwood yields in the F2 treatment. In the practice-oriented trial, the sole F2 treatment increased PAI by 95 % over the control, translating into a yield stimulation of 114 % and an almost identical NUE to that of the interval trial. NUE greatly exceeded the figures typically observed with traditional late-rotation fertilisation and correlated inversely with baseline site productivity (using site index as a proxy) in the F1 and F2 treatments (the latter pooled across the two trials). Our results clearly indicate that nutrient limitation restricts growth and carbon (C) capture in young Norway spruce plantations in northern Europe to less than half of their potential, highlighting repeated fertilisation at nutrient-poor sites as an effective management tool to support a growing bioeconomy and enhance C sequestration