Cumulative N2O Production Research Articles

Influence of Acetylene Concentration on N2O and N2 Emissions from an Intensive Vegetable Soil under Anoxic and Oxic Conditions

Acetylene (C2H2) is often employed to assess soil total denitrification (N2O + N2) due to its ease of implementation. However, this technique underestimates soil denitrification in soils with low nutrient contents, particularly those supporting grain yields. To our knowledge, there are limited studies that have specifically investigated the impact of C2H2 on nutrient-rich vegetable soils, especially concerning the emissions of N2 and N2O and the nitrogenous gas product ratio (i.e., N2O/(N2O + N2)). In this study, we conducted both anoxic and oxic incubations at various C2H2 concentrations (0%, 0.01%, and 10%, v/v) and utilized a robotized sampling and analysis system to quantify soil N2, N2O, and CO2 emissions. Our findings revealed that the cumulative N2O production in soil treated with 10%C2H2 was significantly lower than that in soil treated with 0.01%C2H2 and soil without C2H2. Contrarily, high concentrations of C2H2 (10%, v/v) led to increased N2 production. Similar trends were observed under oxic conditions, where 10%C2H2 concentration did not enhance N2O production but markedly increased N2 and CO2 emissions. Moreover, the N2O/(N2O + N2) product ratio was notably higher in soils treated with 0%C2H2 compared to the 10%C2H2 treatment under anoxic conditions. These findings indicate that high concentrations of acetylene could facilitate the reduction of N2O to N2 and lead to underestimated soil total denitrification in vegetable soil, regardless of anoxic or oxic conditions. This discovery underscores the drawbacks when employing high concentrations of acetylene to evaluate actual total denitrification in intensive greenhouse vegetable soils, highlighting the necessity for further investigation into alternative methodologies.

Efficacy of grape seed procyanidins for inhibiting denitrification varies by source, soil texture, and cropping history

Plant-derived denitrification inhibitors, such as procyanidins derived from grape seed extract (GSE), hold potential in mitigating nitrous oxide (N2O) emissions, a potent greenhouse gas and stratospheric ozone depleter. A deeper understanding of the effects of GSE – considering aspects like rate, source, application timing, storage, and interactions with soil properties and cropping histories – on denitrification inhibition remains elusive. We conducted a series of laboratory microcosm experiments to assess these effects using denitrification enzyme activity (DEA) and measurements of N2O and carbon dioxide (CO2) production rates under both anaerobic and aerobic conditions, soil inorganic nitrogen (N), and other major soil properties. Our results revealed that one commercial GSE brand (USP GSE) inhibited DEA, while another source was ineffective. Two non-procyanidin compounds in commercial GSE mixtures, tartaric acid and gallic acid, actually enhanced DEA. Inhibition efficiency of the USP GSE varied significantly across soils with differing crop histories and physical properties, exhibiting a positive dose-response in three out of the five soils examined. Greater efficiency was observed in sandy soils compared to silt loam soils and in soils with a history of continuous corn production as opposed to soybean. The GSE application timing relative to urea addition and a one-week room temperature storage had minor effects on denitrification inhibition. In corn-cultivated soil, co-application of GSE and urea reduced cumulative N2O production by 27 % compared to soils treated with urea alone. The CO2 production and nitrate levels remained largely unaffected by GSE application. These findings supported the potential of GSE as a soil amendment to reduce N2O emissions in corn-cultivated soils. However, challenges including purity of procyanidins, cost-effectiveness, and inconsistent performance under various systems warrant consideration for broader field applications.

DMPP mitigates N2O and NO productions by inhibiting ammonia-oxidizing bacteria in an intensified vegetable field under different temperature and moisture regimes

Vegetable soils with high nitrogen input are major sources of nitrous oxide (N2O) and nitric oxide (NO), and incorporation of the nitrification inhibitor 3, 4-dimethylpyrazole phosphate (DMPP) into soils has been documented to effectively reduce emissions. However, the efficiency of DMPP in terms of soil N2O and NO mitigations varies greatly depending on soil temperature and moisture levels. Thus, further evaluations of DMPP efficiency in diverse environments are required to encourage widespread application. A laboratory incubation study (28 d) was established to investigate the interactive effects of DMPP, temperature (15, 25, and 35 °C), and soil moisture (55% and 80% of water-holding capacity (WHC)) on net nitrification rate, N2O and NO productions, and gene abundances of nitrifiers and denitrifiers in an intensive vegetable soil. Results showed that incubating soil with 1% DMPP led to partial inhibition of the net nitrification rate and N2O and NO productions, and the reduction percentage of N2O production was higher than that of NO production (69.3% vs. 38.2%) regardless of temperature and soil moisture conditions. The increased temperatures promoted the net nitrification rate but decreased soil N2O and NO productions. Soil moisture influenced NO production more than N2O production, decreasing with the increased moisture level (80%). The inhibitory effect of DMPP on cumulative N2O and NO productions decreased with increased temperatures at 55% WHC. Conversely, the inhibitory effect of DMPP on cumulative N2O production increased with increased temperatures at 80% WHC. Based on the correlation analyses and automatic linear modeling, the mitigation of both N2O and NO productions from the soil induced by DMPP was attributed to the decreases in ammonia-oxidizing bacteria (AOB) amoA gene abundance and NO2--N concentration. Overall, our study indicated that DMPP reduced both N2O and NO productions by regulating the associated AOB amoA gene abundance and NO2--N concentration. These findings improve our insights regarding the implications of DMPP for N2O and NO mitigations in vegetable soils under various climate scenarios.

Effects of biodegradable plastics on soil properties and greenhouse gas production

ABSTRACT Microplastics cause environmental problems. Biodegradable plastics have become popular because they aim to avoid such problems. However, their decomposition in the soil may have an impact. This study aims to investigate the effects of biodegradable plastics on the physicochemical properties of soil, specifically the production of CO2 and N2O in the soil and plant growth. Three kinds of biodegradable plastics in the forms of 1) nonwoven fabric sheets made of poly-lactic acid (PLA) and polybutylene-succinate (referred to hereafter as fabric), 2) laminate sheets made of polybutylene adipate terephthalate (PBAT) and pulp (hereafter laminate), and 3) drinking cups made of PLA (hereafter cup), were cut into small pieces (<5 mm), added to soil, then water-holding capacity was determined and incubated aerobically for 4 weeks at 30°C in the dark. Soil and gas samples were collected weekly to measure soil pH, nitrate-nitrogen content, CO2, and N2O productions. These plastics were also tested in a pot experiment with Komatsuna (Brassica napa var. perviridis). We tested for seed germination, plant growth, leaf color, and fresh weight at harvest. Results showed that the water retention capacity was higher in the fabric plastics as compared to the cup plastics and the control. Soil pH with the fabric plastics dropped during the initial 2 weeks of incubation, then recovered to a similar pH to the control (without plastic). Nitrate contents in the soil with laminate plastics were lower than those in the control, while CO2 production in the soil with the laminate plastics was higher than that in the control and the other plastics during the incubation period, and even higher than the one of added plastic-C. N2O was produced rapidly within 1 week of incubation in the soil with the laminate plastics, and cumulative N2O production from incubation was more than that of the control. The seed germination and plant growth tended to be suppressed in the pot experiment with the fabric and laminate plastics. The results indicate that the influence of these biodegradable plastics on soil properties, greenhouse gas production, and plant growth on the kind of plastic and the timing.

Nitrous oxide production and nitrogen transformations in a soil amended with biosolids

The application of organic amendments to agricultural soils enables the recycling of nutrients, further reducing the inputs of synthetic fertilizers for crop production. However, the production of N2O emissions is a concern that arises from such a practice. A 35 d incubation experiment was conducted with soils receiving three contrasting types of biosolids — mesophilic anaerobic digested (BM), composted (BC), and alkaline-stabilized (BA) — at four water-filled pore spaces (WFPS): 28%, 40%, 52%, and 64%. A zero-N-addition control was also evaluated. Across all the three types of biosolids, N2O production increased with soil moisture content, with BM and BC producing the overall highest N2O fluxes. The most intense pulses of N2O production were exhibited by BC at the beginning of the incubation. The highest cumulative N2O production was found with 64% WFPS and from BC- (409 μg N2O–N·kg−1 soil) or BM-amended soils (390 μgN2O–N·kg−1 soil), which produced more than four and two times the emissions from the control and BA-amended soils at 64% WFPS, respectively. We also found the highest nitrification rates in the BM- and BC-amended soils. The total N2O production was exponentially associated with the NO3−–N concentration present at the end of the experiment (R2 = 0.83). Changes in the concentration of the soil available N indicated the occurrence of mineralization, nitrification, and denitrification over the incubation. These results provided insight into the interacting responses of N2O production to soil moisture contents, biosolids treatment stabilization and properties, and soil N availability.

Application methods influence biochar–fertilizer interactive effects on soil nitrogen dynamics

AbstractThe potential nitrogen (N) losses from soils with fertilizer addition can be reduced when biochar is co‐applied, but this effect is influenced by the methods of biochar and fertilizer application. In a 60‐d laboratory incubation experiment, we investigated how two fertilizer application methods (surface placement and soil incorporation) affected N transformation in soils under the following treatments: control (soil with no biochar and urea [C]), biochar (150 mg N g−1 soil [B]), urea (150 mg N g−1 soil [U]), and the combination of B + U (75 mg N g−1 soil each B and U). Our results showed that at Day 30, the concentrations of soil NH4+–N and NO3−–N remained significantly higher for U but were relatively similar to control for biochar‐included treatments, indicating that the presence of biochar slowed the mineralization of urea during that period. The concentration of soil NO3−–N and cumulative N2O production under B + U treatment at 60 d was around two times higher for incorporation treatment compared with the surface treatment, indicative of a longer‐term N regulatory effect of biochar with the surface application method. Additionally, we observed a higher number of amoA gene transcripts when B + U was incorporated in the soil compared with applied to the surface at the later stage of incubation, indicative of higher potential nitrification activity. These results suggest that the surface application of B + U can be used as a slow release N source that can provide long‐term N supply to the crops, while the soil incorporation method could be used for crops that need low N at the beginning of the growth but require a substantial amount of it later. Surface co‐application of B + U can also be a good strategy to reduce soil N losses by slowing down ammonification, nitrification, N2O emission, and ammonia oxidizing bacteria activity.

Variations in the denitrifying microbial community and functional genes during mesophilic and thermophilic anaerobic digestion of cattle manure

In this study, the anaerobic digestion (AD) of cattle manure was conducted at two temperatures (mesophilic: 35 °C; thermophilic: 55 °C) to analyze the dynamics of the denitrifying functional microbial community and functional genes. The cumulative N2O production under thermophilic conditions was 130.3% higher than that under mesophilic conditions. Thermophilic AD decreased the abundance of nosZ, which was more functional than other denitrifying genes. Firmicutes, Proteobacteria, and Bacteroidetes were the main phyla, and they were also related to denitrification during AD. Redundancy analysis indicated that pH, temperature, and NH4+-N mainly affected the functional bacterial community. Temperature altered the co-occurrence patterns of the bacterial community and the keystone genera in AD. Desulfovibrio in mesophilic AD and Thiobacillus in thermophilic AD were closely related to nitrogen transformation among the keystone genera. The variations in the abundances of members of the denitrifying microbial community and functional genes during AD suggest that thermophilic AD may have caused greater nitrogen losses.

The effect of lignite on nitrogen mobility in a low-fertility soil amended with biosolids and urea

Lignite has been proposed as a soil amendment that reduces nitrate (NO3−) leaching from soil. Our objective was to determine the effect of lignite on nitrogen (N) fluxes from soil amended with biosolids or urea. The effect of lignite on plant yield and elemental composition was also determined. Batch sorption and column leaching experiments were followed by a lysimeter trial where a low fertility soil was amended with biosolids (400kgN/ha equivalent) and urea (200kgN/ha equivalent). Treatments were replicated three times, with and without lignite addition (20t/ha equivalent). Lignite did not reduce NO3− leaching from soils amended with either biosolids or urea. While lignite decreased NO3− leaching from an unamended soil, the magnitude of this effect was not significant in an agricultural context. Furthermore, lignite increased cumulative N2O production from soils receiving urea by 90%. Lignite lessened the beneficial growth effects of adding biosolids or urea to soil. Further work could investigate whether coating urea granules with lignite may produce meaningful environmental benefits.

Effects of soil pH and salt on N2O production in adjacent forest and grassland soils in central Alberta, Canada

Purpose The effects of soil pH manipulation and KCl addition on N2O production in adjacent forest and grassland soils in central Alberta were studied in a 16-day laboratory incubation experiment. Materials and methods The soils were subjected to four pH and two salt treatments: CK (control)—no addition of acid or alkali solution (pH 4.50 and 4.48 for the forest and grassland soils, respectively; same below); HCl—addition of HCl solution to lower soil pH (3.95 and 3.75); L-KOH and H-KOH—addition of 6 mL of 0.2 (5.36 and 5.57) and 0.4 (6.41 and 6.72)molL �1 KOH solution, respectively, to increase soil pH to two different levels. In order to differentiate between the effect of a change in pH and of changed salt concentrations on N2O production, 6 mL of 0.2 (L-KCl) (4.56 and 4.41) or 0.4 molL �1 (H-KCl) (4.59 and 4.42) KCl solutions were also applied as treatments to create two levels of salt application rates. Results and discussion Increasing pH promoted gross nitrification and cumulative N2O production in both soils, particularly in the forest soil. However, cumulative N2O production decreased in the forest soil but increased in the grassland soil when pH decreased. Cumulative N2O production in the grassland soil was 36 times higher in the L-KCl treatment (1,442 μgNkg �1 ) than in the CK (40 μgNkg �1 ), whereas the H-KCl treatment reduced cumulative N2O production. In contrast, in the forest soil, both KCl treatments reduced cumulative N2O production. Conclusions (1) The most important factor to increase N2O production in this study was increasing soil pH, suggesting that careful soil pH management could be used as a tool to control soil N2O production; (2) salt effect was also involved in affecting N2O production.

Greenhouse gas emissions and nutrient supply rates in soil amended with biofuel production by-products

Ethanol production results in distiller grain, and biodiesel produces glycerol as by-product. However, there is limited information on effects of their addition on evolution of N2O and CO2 from soils, yet it is important to enable our understanding of impacts of biofuel production on greenhouse gas budgets. The objective of this study was to evaluate the direct effects of adding wet distillers grain (WDG), thin stillage (TS), and glycerol at three rates on greenhouse gas emissions (N2O and CO2) and nutrient supply rates in a cultivated soil from the Canadian prairies. The WDG and TS application rates were: 100, 200, or 400 kg N ha−1, whereas glycerol was applied at: 40, 400, or 4,000 kg C ha−1 applied alone (G − N) or in a combination with 300 kg N ha−1 (G + N). In addition, conventional amendments of urea (UR) and dehydrated alfalfa (DA) were added at the same rates of total N as the by-products for comparative purposes. The production of N2O and CO2 was measured over an incubation period of 10 days in incubation chambers and Plant Root Simulator™ resin membrane probes were used to measure nutrient (NH 4 + -N, NO 3 − -N, and PO 4 −3 -P) supply rates in the soil during incubation. Per unit of N added, urea tended to result in the greatest N2O production, followed by wet distillers grain and thin stillage, with glycerol and dehydrated alfalfa resulting in the lowest N2O production. Cumulative N2O production increased with increasing the rate of N-containing amendments and was the highest at the high rate of UR treatment. Addition of urea with glycerol contributed to a higher rate of N2O emission, especially at the low rate of glycerol. The DA and WDG resulted in the greatest evolution of CO2 from the soil, with the thin stillage resulting in less CO2 evolved per unit of N added. Addition of N fertilizer along with glycerol enhanced microbial activity and decomposition. The amendments had significant impacts on release of available nutrient, with the UR treatments providing the highest NO 3 − -N supply rate. The TS treatments supplied the highest rate of NH 4 + -N, followed by WDG compared to the other amendments. The WDG treatments were able to provide the greatest supply of PO 4 −3 -P supply in comparison to the other amendments. Microbial N immobilization was associated with glycerol treatments applied alone. This study showed that the investigated biofuel by-products can be suitable soil amendments as a result of their ability to supply nutrients and N2O emissions that did not exceed that of the conventional urea fertilizer.

The effect of biochar addition on N2O and CO2 emissions from a sandy loam soil – The role of soil aeration

Biochar application to soil has significant potential as a climate change mitigation strategy, due to its recalcitrant C content and observed effect to suppress soil greenhouse gas emissions such as nitrous oxide (N2O). Increased soil aeration following biochar amendment may contribute to this suppression.Soil cores from a Miscanthus X. giganteus plantation were amended with hardwood biochar at a rate of 2% dry soil weight (22 t ha−1). The cores were incubated at three different temperatures (4, 10 and 16 °C) for 126 days, maintained field moist and half subjected to periodic wetting events. Cumulative N2O production was consistently suppressed by at least 49% with biochar amendment within 48 h of wetting at 10 and 16 °C. We concluded that hardwood biochar suppressed soil N2O emissions following wetting at a range of field-relevant temperatures over four months. We hypothesised that this was due to biochar increasing soil aeration at relatively high moisture contents by increasing the water holding capacity (WHC) of the soil; however, this hypothesis was rejected.We found that 5% and 10% biochar amendment increased soil WHC. Also, 10% biochar amendment decreased bulk density of the soil. Sealed incubations were performed with biochar added at 0–10 % of dry soil weight and wetted to a uniform 87% WHC (78% WFPS). Cumulative N2O production within 60 h of wetting was 19, 19, 73 and 98% lower than the biochar-free control in the 1, 2, 5 and 10% biochar treatments respectively. We conclude that high levels of biochar amendment may change soil physical properties, but that the enhancement of soil aeration by biochar incorporation makes only a minimal contribution to the suppression of N2O emissions from a sandy loam soil. We suggest that microbial or physical immobilisation of NO3− in soil following biochar addition may significantly contribute to the suppression of soil N2O emissions.

Trait-based representation of biological nitrification: model development, testing, and predicted community composition.

Trait-based microbial models show clear promise as tools to represent the diversity and activity of microorganisms across ecosystem gradients. These models parameterize specific traits that determine the relative fitness of an “organism” in a given environment, and represent the complexity of biological systems across temporal and spatial scales. In this study we introduce a microbial community trait-based modeling framework (MicroTrait) focused on nitrification (MicroTrait-N) that represents the ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) using traits related to enzyme kinetics and physiological properties. We used this model to predict nitrifier diversity, ammonia (NH3) oxidation rates, and nitrous oxide (N2O) production across pH, temperature, and substrate gradients. Predicted nitrifier diversity was predominantly determined by temperature and substrate availability, the latter was strongly influenced by pH. The model predicted that transient N2O production rates are maximized by a decoupling of the AOB and NOB communities, resulting in an accumulation and detoxification of nitrite to N2O by AOB. However, cumulative N2O production (over 6 month simulations) is maximized in a system where the relationship between AOB and NOB is maintained. When the reactions uncouple, the AOB become unstable and biomass declines rapidly, resulting in decreased NH3 oxidation and N2O production. We evaluated this model against site level chemical datasets from the interior of Alaska and accurately simulated NH3 oxidation rates and the relative ratio of AOA:AOB biomass. The predicted community structure and activity indicate (a) parameterization of a small number of traits may be sufficient to broadly characterize nitrifying community structure and (b) changing decadal trends in climate and edaphic conditions could impact nitrification rates in ways that are not captured by extant biogeochemical models.

Nitrification Inhibitor Reduces Nitrous Oxide Production from Different Soil Profiles of an Andosol Soil

A laboratory incubation was conducted to evaluate nitrous oxide (N2O) production during nitrification and the effect of a nitrification inhibitor on N2O production from different profiles in a Japanese orchard Andosol. Soils were collected from five profiles: A1, A2, Bw1, Bw2, and BC. The soils were treated with ammonium sulfate at the rate of 200 mg N kg−1 with or without dicyandiamide (DCD) and incubated under aerobic conditions for 32 days. The net nitrification rate without DCD during the first 8 days was greater in the surface soils than in the subsurface soils. Accordingly, the surface soils showed a greater cumulative N2O production than the subsurface soils. Application of DCD significantly reduced the nitrification rate and thus N2O production from any depths of soils by 33.8 to 62.9%. Our study showed that substantial N2O was produced from the subsurface soil, although the amount was less than from the surface soils.

Dehydrogenase activity, redox potential, and emissions of carbon dioxide and nitrous oxide from Cambisols under flooding conditions

Samples from topsoils (0–10 cm) of 16 Polish arable Cambisols developed from different parent materials (sand, silt, sandy gravel, loess, loam and clay), were incubated under flooded conditions with NO3 –. Dehydrogenase activity, redox potential (Eh), and emissions of CO2 and N2O were measured. According to dehydrogenase activity, the soils were divided into two groups: those of low activity (I), where the final dehydrogenase activity was 0.03 nmol TPF g–1 min–1. Generation of CO2 and of N2O under flooded conditions was shown to be significantly related to dehydrogenase activity. Soil dehydrogenase activity increased curvilinearly with organic matter content, showed a maximum at pH 7.1, and decreased curvilinearly with Eh. The final cumulative CO2 production increased linearly with soil organic matter content and curvilinearly with dehydrogenase activity and decreased linearly with Eh. The most significant relationship was found with dehydrogenase activity (R 2=0.74, P<0.001). The final cumulative N2O production decreased linearly with Eh and increased curvilinearly with pH and dehydrogenase activity but linearly with organic matter content; the most significant relation being found with dehydrogenase activity (R 2=0.69, P<0.001). The CO2:N2O ratio in the gases evolved increased curvilinearly with Eh and decreased with dehydrogenase activity and N2O and CO2 production.

DENITRIFICATION FROM SANDY SOILS TREATED WITH LIQUID OR DRY GRANULAR NITROGEN FORM1

The forms of nitrogen (N) in fertilizers and soil properties influence gaseous loss of N by denitrification. A laboratory incubation study was conducted to evaluate the denitrification losses from liquid or dry granular forms of ammonium nitrate (at 200 mg N kg−1 soil) as nitrous oxide in acetylene-inhibited systems. Myakka fine sand (Spodosol), Parkwood sand (Alfisol), and Tavares fine sand (Entisol) sampled at 0 to 15 cm depths were used with or without the addition of a readily available carbon (C) source (200 mg C kg−1, as glucose). The soils were incubated anaerobically for 96 h at field capacity moisture with 10% (vol/vol) acetylene atmosphere. Nitrous oxide emission was not detectable in the Parkwood sand throughout the study, despite its high organic C content and microbial activity, possibly because of incomplete inhibition of N2O reduction by acetylene in Parkwood soil as a result of its high clay content, which could have hindered the free diffusion of acetylene. In the Myakka fine sand and the Tavares fine sand, cumulative N2O production for 96 h was greater when ammonium nitrate was applied in the liquid form rather than the dry granular form. The difference was more pronounced in the Myakka sand (2.3-fold) than in the Tavares sand (1.9-fold). Denitrification was enhanced by the addition of readily available carbon in the form of glucose. The soil microbial population and soil organic carbon content seemed to play a major role in determining denitrification losses. Cumulative N2O emission was correlated significantly (r2 = 0.92) with the microbial activity as measured by fluorescein diacetate hydrolytic activity.

Denitrification, N2-fixation and fermentation during anaerobic incubation of soils amended with glucose and nitrate

Nitrate and glucose additions were investigated for their role in the C and N dynamics during anaerobic incubation of soil. A gas-flow soil core method was used, in which the net production of N2, N2O, NO, CO2, and CH4 under a He atmosphere could be monitored both accurately and frequently. In all experiments clayey silt loam soil samples were incubated for 9 days at 25 °C. Addition of nitrate (50 mg KNO3-N kg-1 soil) had no effect on total denitrification and CO2 production rates, while the N2O/N2 ratio was affected considerably. The cumulative N2O production exceeded the cumulative N2 production for 6 days in the treatment with nitrate addition, compared to 1.2 days in the unamended treatment. Glucose addition stimulated the microbial activity considerably. The denitrification rates were limited by the growth rate of the denitrifying population. During denitrification no significant differences were observed between the treatments with 700 mg glucose-C kg-1 and 4200 mg glucose-C kg-1, both in combination with 50 mg KNO3-N kg-1. The N2 production rates were remarkably low, until NO inf3 sup- exhaustion caused rapid reduction of N2O to N2 at day 2. During the denitrification period 15–18 mg N kg-1 was immobilised in the growing biomass. After NO inf3 sup- shortage, a second microbial population, capable of N2-fixation, became increasingly important. This change was clearly reflected in the CO2 production rates. Net volatile fatty acid (VFA) production was monitored during the net N2-fixation period with acetate as the dominant product. N2-fixation faded out, probably due to N2 shortage, followed by increased VFA production. In the high C treatment butyrate became the most important VFA, while in the low C treatment acetate and butyrate were produced at equal rates. During denitrification no VFA accumulation occurred; this does not prove, however, that denitrification and fermentation appeared sequentially. The experiments illustrate clearly the interactions of C-availability, microbial population and nitrate availability as influencing factors on denitrification and fermentation.