好氧反硝化生物脱氮技术的研究进展

好氧反硝化生物脱氮技术自提出以来,凭借能实现同步硝化反硝化、节省基建投资及运行费用等诸多优点,受到国内外环境领域学者的广泛关注。本文首先总结了近年来好氧反硝化菌种的筛选分离情况,以及环境因子对好氧反硝化菌脱氮效能的影响,包括溶解氧(dissolved oxygen,DO)、碳氮比(C/N)、温度等。然后深入探讨了好氧反硝化生物脱氮技术的原理,好氧反硝化过程中的关键功能基因及酶,同时介绍了分子生物技术在好氧反硝化研究过程中的应用,以及好氧反硝化生物脱氮技术在实际应用方面的研究现状。最后,基于目前的研究瓶颈问题,对未来好氧反硝化生物脱氮技术的研究方向提出了科学展望。     

Influence of nutrient inputs, hexadecane, and temporal variations on denitrification and community composition of river biofilms

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P < or = 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.     

Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms.

The microbial community of a denitrifying sand filter in a municipal wastewater treatment plant was examined by conventional and molecular techniques to identify the bacteria actively involved in the removal of nitrate. In this system, denitrification is carried out as the last step of water treatment by biofilms growing on quartz grains with methanol as a supplemented carbon source. The biofilms are quite irregular, having a median thickness of 13 to 20 microns. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp. in the sand filter. 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization. Stringency requirements for the probes were adjusted by use of a formamide concentration gradient to achieve complete discrimination of even highly similar target sequences. Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven of the 56 isolates hybridized with the genus-specific probes. In situ hybridization identified dense aggregates of paracocci in detached biofilms. Probes complementary to the type strains of P. denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting the presence of a new Paracoccus species in the sand filter. Analysis using confocal laser scanning microscopy detected spherical aggregates of morphologically identical cells exhibiting a uniform fluorescence. Cell quantification was performed after thorough disruption of the biofilms and filtration onto polycarbonate filters. An average of 3.5% of total cell counts corresponded to a Paracoccus sp., whereas in a parallel sand filter with no supplemented methanol, and no measurable denitrification, only very few paracocci (0.07% of cells stained with 4',6-diamidino-2-phenylindole) could be detected. Hyphomicrobium spp. constituted approximately 2% of all cells in the denitrifying unit and could not be detected in the regular sand filter. This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process. Possible selective advantages favoring the paracocci in this habitat are discussed.     

The effects of organic carbon, ammoniacal-nitrogen, and oxygen partial pressure on the stratification of membrane-aerated biofilms

The purpose of this study was to examine the effects of different nutrient (carbon, nitrogen, oxygen) concentrations on the microbial activity and community structure in membrane-aerated biofilms (MABs). MABs were grown under well-defined conditions of fluid flow, substrate concentration, and membrane oxygen partial pressure. Biofilms were then removed and thin-sliced using a cryostat/microtome parallel to the membrane. Individual slices were analyzed for changes with depth in biomass density, respiratory activity, and the population densities of ammonia-oxidizing and denitrifying bacteria populations. Oxygen-sensing microelectrodes were used to determine the depth of oxygen penetration into each biofilm. Our results demonstrated that ammonia-oxidizing bacteria grow near the membrane, while denitrifying bacteria grow a substantial distance from the membrane. However, nitrifying and denitrifying bacteria did not grow simultaneously when organic concentrations became too high or ammonia concentrations became too low. In conclusion, membrane-aerated biofilms exhibit substantial stratification with respect to community structure and activity. A fundamental understanding of the factors that control this stratification will help optimize the performance of full-scale membrane-aerated biofilm reactors for wastewater treatment.     

Influence of microbial interactions and EPS/polysaccharide composition on nutrient removal activity in biofilms formed by strains found in wastewater treatment systems

The study of biofilm function, structure and microbial interactions might help to improve our understanding of biofilm wastewater treatment processes. However, few reports specifically address the influence of interactions within multispecies biofilms on microbial activity and biofilm composition. Thus, the relationship between biofilm formation, denitrification activity, phosphorus removal and the composition of extracellular polymeric substances (EPS), exopolysaccharides and the bacterial community was investigated using biofilms of denitrifying and phosphorus removing strains Comamonas denitrificans 110, Brachymonas denitrificans B79, Aeromonas hydrophila L6 and Acinetobacter calcoaceticus ATCC23055. Denitrification activity within the biofilms generally increased with the amount of biofilm while phosphorus removal depended on bacterial growth rate. Synergistic effects of co-growth on denitrification (B. denitrificans B79 and A. hydrophila L6) and phosphorus removal (C. denitrificans 110 with either A. calcoaceticus or A. hydrophila L6) were observed. B. denitrificans B79 was highly affected by interspecies interactions with respect to biofilm formation, denitrification activity and EPS composition, while C. denitrificans 110 remained largely unaffected. In some of the dual and quadruple strain biofilms new exopolysaccharide monomers were detected which were not present in the pure strain samples.     

Metagenomic analysis reveals distinct patterns of denitrification gene abundance across soil moisture,nitrate gradients

This study coupled a landscape-scale metagenomic survey of denitrification gene abundance in soils with in situ denitrification measurements to show how environmental factors shape distinct denitrification communities that exhibit varying denitrification activity. Across a hydrologic gradient, the distribution of total denitrification genes (nap/nar + nirK/nirS + cNor/qNor + nosZ) inferred from metagenomic read abundance exhibited no consistent patterns. However, when genes were considered independently, nirS, cNor and nosZ read abundance was positively associated with areas of higher soil moisture, higher nitrate and higher annual denitrification rates, whereas nirK and qNor read abundance was negatively associated with these factors. These results suggest that environmental conditions, in particular soil moisture and nitrate, select for distinct denitrification communities that are characterized by differential abundance of genes encoding apparently functionally redundant proteins. In contrast, taxonomic analysis did not identify notable variability in denitrifying community composition across sites. While the capacity to denitrify was ubiquitous across sites, denitrification genes with higher energetic costs, such as nirS and cNor, appear to confer a selective advantage in microbial communities experiencing more frequent soil saturation and greater nitrate inputs. This study suggests metagenomics can help identify denitrification hotspots that could be protected or enhanced to treat non-point source nitrogen pollution.     

A novel double-membrane system for simultaneous nitrification and denitrification in a single tank

A novel biological treatment system, which contains two types of membrane modules in a single tank, was developed for simultaneous nitrification and denitrification. Both of the modules were fed with the substrates on the tube side of the silicone tubes by diffusing them to the biofilms which form on the surface of the tubes. One module was fed with methanol for denitrification and the other one was fed with pure oxygen for nitrification. As a result, the interference of organic carbon on nitrification, and that of oxygen on denitrification, were both hindered by the diffusion barriers (biofilms), thereby allowing two different niches for nitrifiers and denitrifiers to coexist in a single tank. Besides saving space and the amount of alkalinity required for nitrification, this system also produced low residual chemical oxygen demand (COD) and high nitrogen removal rates (2.9-3.4 gN m-2 d-1 of surface area of membrane).     

Characterization of mono- and mixed-culture Campylobacter jejuni biofilms

Campylobacter jejuni, one of the most common causes of human gastroenteritis, is a thermophilic and microaerophilic bacterium. These characteristics make it a fastidious organism, which limits its ability to survive outside animal hosts. Nevertheless, C. jejuni can be transmitted to both humans and animals via environmental pathways, especially through contaminated water. Biofilms may play a crucial role in the survival of the bacterium under unfavorable environmental conditions. The goal of this study was to investigate survival strategies of C. jejuni in mono- and mixed-culture biofilms. We grew monoculture biofilms of C. jejuni and mixed-culture biofilms of C. jejuni with Pseudomonas aeruginosa. We found that mono- and mixed-culture biofilms had significantly different structures and activities. Monoculture C. jejuni biofilms did not consume a measurable quantity of oxygen. Using a confocal laser scanning microscope (CLSM), we found that cells from monoculture biofilms were alive according to live/dead staining but that these cells were not culturable. In contrast, in mixed-culture biofilms, C. jejuni remained in a culturable physiological state. Monoculture C. jejuni biofilms could persist under lower flow rates (0.75 ml/min) but were unable to persist at higher flow rates (1 to 2.5 ml/min). In sharp contrast, mixed-culture biofilms were more robust and were unaffected by higher flow rates (2.5 ml/min). Our results indicate that biofilms provide an environmental refuge that is conducive to the survival of C. jejuni.     

Mapping field-scale spatial patterns of size and activity of the denitrifier community

There is ample evidence that microbial processes can exhibit large variations in activity on a field scale. However, very little is known about the spatial distribution of the microbial communities mediating these processes. Here we used geostatistical modelling to explore spatial patterns of size and activity of the denitrifying community, a functional guild involved in N-cycling, in a grassland field subjected to different cattle grazing regimes. We observed a non-random distribution pattern of the size of the denitrifier community estimated by quantification of the denitrification genes copy numbers with a macro-scale spatial dependence (6–16 m) and mapped the distribution of this functional guild in the field. The spatial patterns of soil properties, which were strongly affected by presence of cattle, imposed significant control on potential denitrification activity, potential N₂O production and relative abundance of some denitrification genes but not on the size of the denitrifier community. Absolute abundance of most denitrification genes was not correlated with the distribution patterns of potential denitrification activity or potential N₂O production. However, the relative abundance of bacteria possessing the nosZ gene encoding the N₂O reductase in the total bacterial community was a strong predictor of the N₂O/(N₂ + N₂O) ratio, which provides evidence for a relationship between bacterial community composition based on the relative abundance of denitrifiers in the total bacterial community and ecosystem processes. More generally, the presented geostatistical approach allows integrated mapping of microbial communities, and hence can facilitate our understanding of relationships between the ecology of microbial communities and microbial processes along environmental gradients.     

Nitrogen-Cycling Genes in Epilithic Biofilms of Oligotrophic High-Altitude Lakes (Central Pyrenees,Spain)

Microbial biofilms in oligotrophic environments are the most reactive component of the ecosystem. In high-altitude lakes, exposed bedrock, boulders, gravel, and sand in contact with highly oxygenated water and where a very thin epilithic biofilm develops usually dominate the littoral zone. Traditionally, these surfaces have been considered unsuitable for denitrification, but recent investigations have shown higher biological diversity than expected, including diverse anaerobic microorganisms. In this study, we explored the presence of microbial N-cycling nirS and nirK (denitrification through the conversion of NO₂ ^? to NO), nifH (N₂ fixation), anammox (anaerobic ammonium oxidation), and amoA (aerobic ammonia oxidation, both bacterial and archaeal) genes in epilithic biofilms of a set of high-altitude oligotrophic lakes in the Pyrenees. The concentrations of denitrifying genes determined by quantitative PCR were two orders of magnitude higher than those of ammonia-oxidizing genes. Both types of genes were significantly correlated, suggesting a potential tight coupling nitrification-denitrification in these biofilms that deserves further confirmation. The nifH gene was detected after nested PCR, and no signal was detected for the anammox-specific genes used. The taxonomic composition of denitrifying and nitrogen-fixing genes was further explored by cloning and sequencing. Interestingly, both microbial functional groups were richer and more genetically diverse than expected. The nirK gene, mostly related to Alphaproteobacteria (Bradyrhizobiaceae), dominated the denitrifying gene pool as expected for oxygen-exposed habitats, whereas Deltaproteobacteria (Geobacter like) and Cyanobacteria were the most abundant among nitrogen fixers. Overall, these results suggest an epilithic community more metabolically diverse than previously thought and with the potential to carry out an active role in the biogeochemical nitrogen cycling of high-altitude ecosystems. Measurements of activity rates should be however carried out to substantiate and further explore these findings.     

Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2(-) and O2 concentrations.

The oxygen control of denitrification and its emission of NO/N2O/N2 was investigated by incubation of Nycodenz-extracted soil bacteria in an incubation robot which monitors O2, NO, N2O and N2 concentrations (in He+O2 atmosphere). Two consecutive incubations were undertaken to determine (1) the regulation of denitrification by O2 and NO2(-) during respiratory O2 depletion and (2) the effects of re-exposure to O2 of cultures with fully expressed denitrification proteome. Early denitrification was only detected (as NO and N2O) at 相似文献   

Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms

The bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial wastewater were investigated by combination of the 16S rRNA gene clone analysis, fluorescence in situ hybridization (FISH) and microelectrodes. These results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (IWTP). Both nitrification and denitrification occurred in the primary denitrification (PD) tank and denitrification occurred in the secondary denitrification (SD) tank. In contrast, nitrification and denitrification rates were very low in the nitrification (N) tank. 16S rRNA gene clone sequence analysis revealed that the bacteria affiliated with Alphaproteobacteria, followed by Betaproteobacteria, were numerically important microbial groups in three tanks. The many clones affiliated with Alphaproteobacteria were closely related to the denitrifying bacteria (e.g., Hyphomicrobium spp., Rhodopseudomonas palustris, and Rhodobacter spp.). In addition, Methylophilus leisingeri affiliated with Betaproteobacteria, which favorably utilized methanol, was detected only in the SD-tank to which methanol was added. Nitrosomonas europaea and Nitrosomonas marina were detected as the ammonia-oxidizing bacteria affiliated with Betaproteobacteria throughout this plant, although the dominant species of them was different among three tanks. Nitrifying bacteria were mainly detected in the upper parts of the PD-biofilm whereas their populations were low in the upper parts of the N-biofilm. The presence of denitrifying bacteria affiliated with Hyphomicrobium spp. in SD- and N-biofilms was verified by FISH analysis. Microelectrode measurements showed that the nitrifying bacteria present in the N- and PD-biofilms were active and the bacteria present in the SD-biofilm could denitrify.     

Effects of photoperiod on growth of and denitrification by Pseudomonas chlororaphis in the root zone of Glyceria maxima,studied in a gnotobiotic microcosm

The emergent macrophyte Glyceria maxima was subjected to different photoperiods and grown with ammonium or nitrate as nitrogen source in presterilized microcosms with spatially separated root and non-root compartments. The microcosms were inoculated with the denitrifying bacterium Pseudomonas chlororaphis. The effect of the plant and the photoperiod on growth and denitrification by P. chlororaphis was assessed. The plant had a strong positive effect on the growth of the bacteria. The bacterial numbers in the root compartment of the planted microcosms were 19-32 times higher than found in the non-root sediment of the unplanted systems. Lengthening the photoperiod resulted in elevated bacterial numbers due to the higher carbon exudation of the plant. This effect was greater still with the nitrate-fed plants, where additional P. chlororaphis growth could proceed via denitrification, indicating oxygen-limiting conditions in the microcosms. Higher porewater N₂O concentrations in the root compartments as compared to the non-root compartments, which were highest for the long photoperiod, were also indicative of a plant-induced stimulation of denitrification. An effect of a diurnal oxygen release pattern of G. maxima on denitrification could not be detected. The gnotobiotic microcosm used in this study represents a potential system for the study of the behaviour and interactions of important bacterial groups, such as nitrifying and denitrifying bacteria where plant roots drive bacterial activity.     

Impact of anodophilic biofilm bioelectroactivity on the denitrification behavior of air-cathode microbial fuel cells

Generally, high bioelectroactivity of anodophilic biofilm favors high power generation of microbial fuel cell (MFC); however, it is not clear whether it can promote denitrification of MFC synchronously. In this study, we studied the impact of anodophilic biofilm bioelectroactivity on the denitrification behavior of air-cathode MFC (AC-MFC) in steady state and found that high bioelectroactivity of anodophilic biofilm not only favored high power generation of the AC-MFC, but also promoted the growth of denitrifers at the anodes and strengthened denitrification. Anodophilic biofilms of AC-MFC with various bioelectroactivity were acclimated at conditions of open circuit (OC), R_ext of 1000 Ω and 20 Ω (denoted as AC-MFC-OC, AC-MFC-1000Ω, and AC-MFC-20Ω, respectively) and performed for over 100 days. Electrochemical tests and microbial analysis results showed that the anode of the AC-MFC-20Ω delivered higher current response of both oxidation and denitrification and had higher abundance of electroactive bacteria than the AC-MFC-OC, AC-MFC-1000Ω, demonstrating a higher bioelectroactivity of the anodophilic biofilms. Moreover, these electroactive bacteria favored the accumulation of denitrifers, like Thauera and Alicycliphilus, probably by consuming trace oxygen through catalyzing oxygen reduction. The AC-MFC-20Ω not only delivered a 61.7% higher power than the AC-MFC-1000Ω, but also achieved a stable and high denitrification rate constant (k_DN) of 1.9 h^?1, which was 50% and 40% higher than that of the AC-MFC-OC and AC-MFC-1000Ω, respectively. It could be concluded that the high bioelectroactivity of the anodophilic biofilms not only favored high power generation of the AC-MFC, but also promoted the enrichment of denitrifers at the anodes and strengthened denitrification. This study provided an effective method for enhancing power generation and denitrification performance of the AC-MFC synchronously.     

Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH₄Cl), phosphorus (KH₂PO₄), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N₂, suggesting that emission of N₂O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P ≤ 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.     

Denitrification under various aeration conditions in Comamonas sp., strain SGLY2

Abstract A number of bacteria were isolated from different anoxic reactors. Those having denitrifying potential were tested for their ability to denitrify under aerobic conditions. The activity of their denitrifying enzymes varied from partial inactivation by oxygen (strains NO2B9 and TCET1) to oxygen-independent activity in a strain named SGLY2 which was tentatively identified as Comamonas sp. The effect of different aeration conditions on growth and on denitrification of SGLY2 was studied more extensively. This strain was able to consume oxygen and nitrate simultaneously with the production of nitrogen and without build-up of nitrite. The dissimilatory nitrate-reductase of nitrate-adapted cells was found to be more active in the presence of oxygen than in micro-aerobic or strictly anaerobic conditions.     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。     

Influence of the Pseudomonas quinolone signal on denitrification in Pseudomonas aeruginosa

Denitrification is a well-studied respiratory system that is also important in the biogeochemical nitrogen cycle. Environmental signals such as oxygen and N-oxides have been demonstrated to regulate denitrification, though how denitrification is regulated in a bacterial community remains obscure. Pseudomonas aeruginosa is a ubiquitous bacterium that controls numerous genes through cell-to-cell signals. The bacterium possesses at least two N-acyl-L-homoserine lactone (AHL) signals. In our previous study, these quorum-sensing signals controlled denitrification in P. aeruginosa. In addition to the AHL signals, a third cell-to-cell communication signal, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been characterized. In this study, we examined the effect of PQS on denitrification to obtain more insight into the respiratory regulation in a bacterial community. Denitrification in P. aeruginosa was repressed by PQS, which was partially mediated by PqsR and PqsE. Measuring the denitrifying enzyme activities indicated that nitrite reductase activity was increased by PQS, whereas PQS inhibited nitric oxide reductase and the nitrate-respiratory chain activities. This is the first report to demonstrate that PQS influences enzyme activities, suggesting this effect is not specific to P. aeruginosa. Furthermore, when iron was supplied to the PQS-added medium, denitrifying activity was almost restored, indicating that the iron chelating property of PQS affected denitrification. Thus, our data indicate that PQS regulates denitrification primarily through iron chelation. The PQS effect on denitrification was relevant in a condition where oxygen was limited and denitrification was induced, suggesting its role in controlling denitrification where oxygen is present.     

Titration biosensors for the estimation of the biochemical nitrate demand of municipal and industrial wastes

An anoxic titrimetric test was investigated for measuring denitrification potential of different wastewaters, both municipal and industrial, and to quantify the denitrifying activity in an activated sludge system. The method measures the amount of acid that is required to maintain the pH set-point value in a batch denitrification experiment, and it was performed using a DENICON (denitrification controller) biosensor. The amount of acid is proportional to the nitrate used to oxidise the biodegradable chemical oxygen demand present in the wastewater, while the acid consumption rate is used to derive the denitrifying activity. The wastewaters tested were a municipal wastewater (MW), an industrial-municipal wastewater (MIW; 70% and 30%, respectively), and four industrial wastewaters drawn from an ice-cream factory (IW1), a beet-sugar factory (IW2), a brewery (IW3), and a tuna cannery industry (IW4). Good correlation between titration data and analyses was found.