High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH inbiofilm and suspended-biomass reactors by known chemolithotrophicbacteria. In the biofilm reactor, at low pH (4.3 ± 0.1)and low bulk ammonium concentrations (9.3 ± 3.3 mg ·liter^–1), a very high nitrification rate of 5.6 g of Noxidized · liter^–1 · day^–1 was achieved.The specific nitrification rate (0.55 g of N · g of biomass^–1· day^–1) was similar to values reported for nitrifyingreactors at optimal pH. In the suspended-biomass reactor, theaverage pH was significantly lower than that in the biofilmreactor (pH 3.8 ± 0.3), and values as low as pH 3.2 werefound. In addition, measurements in the suspended-biomass reactor,using isotope-labeled ammonium (¹⁵N), showed that in spite ofthe very low pH, biomass growth occurred with a yield of 0.1g of biomass · g of N oxidized^–1. Fluorescencein situ hybridization using existing rRNA-targeted oligonucleotideprobes showed that the nitrifying bacteria were from the monophyleticgenus Nitrosomonas, suggesting that autotrophic nitrificationat low pH is more widespread than previously thought. The resultspresented in this paper clearly show that autotrophic nitrifyingbacteria have the ability to nitrify at a high rate at low pHand in the presence of only a negligible free ammonia concentration,suggesting the presence of an efficient ammonium uptake systemand the means to cope with low pH.

INTRODUCTION

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Introduction

Nitrification is carried out mainly by aerobic chemolithotrophicbacteria and consists of oxidation of ammonia to nitrite byammonia-oxidizing bacteria (AOB), followed by oxidation of nitriteto nitrate by nitrite-oxidizing bacteria (NOB). Ammonium (thedominant ion under neutral and acidic conditions) and nitriteare oxidized according to the following stoichiometry (neglectingbiomass growth) (31):

Asshown in the above equation, the oxidation of every mole ofammonium produces 2 mol of acidity, which can result in a pHdecrease in poorly buffered environments. Autotrophic microbialnitrification is known to be highly sensitive to pH, and optimalconditions have been found to be within the narrow pH rangeof 7 to 8. Generally, below pH values of 6.5, growth of purecultures of autotrophic ammonia-oxidizing bacteria in liquidculture does not occur (5, 24). The high sensitivity of nitrificationto acidic conditions has been attributed mainly to the exponentialdecrease in free ammonia (NH₃) with decreasing pH (NH₃ + H⁺ ${leftrightarrow}$ NH₄⁺; pK_a = 9.25) (8). Free ammonia is considered to be thesubstrate for the primary enzyme ammonia monooxygenase (34),and the transport of free ammonia into the cells, unlike ammoniumions, is by passive diffusion (41).

Low rates of nitrification and the presence of autotrophic nitrifyingbacteria in acid soils have been reported by many researchers(5, 8, 13) with a pH as low as 3.3 but could not be replicatedin dedicated liquid batch culture systems. Possible mechanismsfor NH₃ supply to ammonia monooxygenase under negligible concentrationsin acid soils are currently ascribed to the availability ofother sources of nonionic ammonia-like urea (5, 8) or to thepresence of alkaline microsites where a favorable pH exists(8). There are also indications that acid-tolerant AOB of thegenus Nitrosospira isolated from acid soils can be active ata low pH without the need for special NH₃-generating mechanisms(8). Other studies have shown the necessity for high cell density,either biofilm or aggregates, for nitrification at a low pH,but only at a very low rate (1, 7).

Although the general protective nature of biofilms and aggregatesagainst environmental extremes is well documented (6), a specificprotective mechanism for autotrophic nitrifiers enabling localneutralization under bulk acidic conditions is debatable. Autotrophicnitrifying biofilms intrinsically produce acidity, and the diffusionalresistance to the transport of protons and inorganic carbonspecies in the biofilm will further decrease pH in the interiorof the biofilm (35). Moreover, evidence from biofilm and aggregateengineered systems for wastewater treatment has demonstratedrepeatedly that autotrophic nitrifying bacteria are inhibitedat low pH (24, 31, 40). In contrast, the present paper showsthat autotrophic nitrifying bacteria originating from a municipalwastewater treatment plant have the ability to nitrify at lowpH and at a high rate even with a negligible free ammonia concentrationwithout any special NH₃-generating mechanisms. The phenomenonof high-rate nitrification at low pH is demonstrated for bothbiofilm and suspended-biomass reactor systems.

MATERIALS AND METHODS

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Materials and Methods

Attached biomass (biofilm) reactor.
The attached-biomass reactor consisted of a double-column fluidizedbed made of transparent 40-mm-diameter polyvinyl chloride tubes.The main nitrifying column was 110 cm long (1 liter) and wasfilled with 350 g (700 ml) of 1- to 2-mm sintered glass particlesused as the biomass carrier (Schott AG, Mainz, Germany). Themain column was interconnected to a 50-cm-long aeration column.Pure oxygen was bubbled at a constant flow rate through theaeration column, and the oxygen-saturated solution was recirculatedvia a pump back to the main column. The oxygen flow was adjustedso that the dissolved-oxygen concentration at the outlet ofthe main nitrifying column was greater than 8 mg · liter^–1.The reactor operated at a high recirculation ratio to providecompletely mixed conditions. The upflow velocity in the columnwas between 50 and 60 m · h^–1, and the bed expansionwas 30%. All of the experiments were carried out at 30°C.The reactor was operated with continuous flow, using an Iwakidiaphragm pump. Depending on the nitrification rate, the influentflow was varied between 7 and 15 ml · min^–1. Theinfluent solution was made from tap water spiked only with variousconcentrations of ammonium chloride (200 to 1,000 mg ·liter^–1), monobasic potassium phosphate (2 to 5 mg ·liter^–1), and sodium bicarbonate buffer (600 to 3,000mg · liter^–1).

Reactor start-up was at a pH of >7 (excess buffer addition)with bacterial inoculum originating from the Haifa municipalwastewater treatment plant. The reactor working procedure atlow pH was based on three main operational conditions: (i) thereactor feeding solution was preloaded with a buffer contentslightly lower than the amount required to neutralize the protonsreleased from complete nitrification of the influent ammoniumconcentration, (ii) the reactor biomass concentration was highenough to change the influent water chemistry, and (iii) theloading rate was attuned to the activity of the bacteria sothat the influent alkalinity (the proton-accepting capacity)was completely destroyed.

Samples of biofilm-covered sintered glass particles were removedweekly from the reactor by creating a slight vacuum in a flexible10-mm-diameter tube inserted into the middle of the fluidizedbed (no stratification of bioparticles was observed). Duplicatesamples were analyzed for dry biomass content using the volatile-suspended-solids(VSS) method (2).

Suspended-biomass reactor.
The suspended-biomass reactor consisted of a 10-liter glassflask equipped with a magnetic stirrer and aerated by pure oxygen(dissolved oxygen, >8 mg · liter^–1). Biomasstaken from a nitrifying fixed-bed reactor already adapted towork at low pH (pH 5) was used as the inoculum. The same workingprocedure and inorganic influent solution used in the attached-biomassreactor were used for the suspended-biomass reactor. The concentrationsof ammonium, bicarbonate buffer, and phosphate were adjustedaccording to the nitrification rate. The reactor was operatedwith a continuous influent flow of 3.8 ml · min^–1,using a Cole Parmer peristaltic pump. Effluent was dischargedfrom the suspended-biomass reactor only once a day, resultingin a gradual accumulation of liquid in the reactor of from 1to 6.4 liters in a 24 h-period. After each 24-h period of operation,the reactor was allowed to stand for 15 min with no mixing,aeration, and influent flow to allow for biomass settling. A5.4-liter volume of supernatant was then drained from the reactor.Mixing, aeration, and influent flow were restarted immediatelyafter drainage. The supernatant was filtered using Whatman GFAfilter paper (1.6-µm pore size) in order to avoid bacterialloss and maintain high reactor biomass concentrations. The smallamount of filtered biomass (100 to 200 mg) collected on thefilter paper was gently rinsed and returned to the reactor.Daily cleaning of the reactor walls prevented any formationof biofilm. Due to the specific operating conditions, the overallreaction rate in the reactor (rate per unit volume x reactorvolume) was constant throughout the day. The reactor was monitoreda number of times throughout the 24-h period, and ammonium,nitrite, and nitrate concentrations were found to be constant.The pH was slightly lower (about 0.2 pH units) immediately followingsettling and slightly higher (about 0.3 pH units) during thefirst hour following the restart of influent flow. During therest of the day, the pH was constant. Nitrogen and pH resultsgiven in this paper are the measurements recorded at the endof each 24-h period, i.e., when the volume of the reactor was6.4 liters. Every week, a 25-ml sample for dry biomass and proteincontent analysis was removed from the suspended-biomass reactor.Protein was measured on triplicate 1.5-ml samples by using theBradford method (4), while VSS analysis (2) was performed onduplicate 10-ml samples.

Chemical analysis.
Duplicate samples from the reactors were analyzed for ammonium,nitrate, nitrite, and phosphate concentrations. Nitrate, nitrite,and phosphate concentrations were determined by using a Metrohm761 ion chromatograph equipped with a Metrosep Dual 1 anionseparating column and suppressor using a carbonate (1.8 mM)-bicarbonate(1.7 mM) eluent. Ammonium concentrations were determined usinga second Metrohm 761 ion chromatograph equipped with a MetrosepC2 cation separating column, using a tartaric acid (4 mM)-dipicolonicacid (1 mM) eluent. The error between the duplicate sampleswas never greater than 2%. Duplicate pH measurements were takenfrom the reactor systems at intervals of 10 min using a dailycalibrated portable Eutech pH meter equipped with a standardcombined electrode and temperature probe. The difference betweensuccessive measurements was never greater than 0.05 pH units.Alkalinity was measured using the Gran titration procedure (11).Oxygen was measured using a Eutech oxygen meter equipped witha galvanic electrode.

¹⁵N isotope measurements.
In addition to measuring protein and VSS content in the suspended-biomassreactor, biomass growth was determined using ¹⁵N isotope measurements.Five percent of the ammonium content in the feeding solutionwas replaced with the isotope ¹⁵NH₄⁺ for 26 days (from day 7to day 33). The percent of ¹⁵N that accumulated in the biomasswas measured with an elemental analyzer (ANCA-SL; PDZ Europa,Crewe, United Kingdom) connected to an isotope ratio mass spectrometer(20-20; PDZ Europa). Based on the measured ratio of ¹⁵N to ¹⁴Nand the amount of elemental nitrogen present in the biomasssample, the change in ¹⁵N content in the biomass over time wasdetermined. The yield coefficient was then calculated from theamount of ammonium oxidized together with the change in ¹⁵Nbiomass content.

Analysis of nitrifying population in the biofilm and suspended-biomass reactors.
After extended operation at about pH 4 (250 days), a samplefrom the biofilm reactor was fixed 1:1 with ethanol (96%) andanalyzed for bacterial populations by Vermicon AG (Munich, Germany).The biofilm sample was tested by using fluorescently labeledoligonucleotide probes for eubacteria, ß-proteobacterialammonia-oxidizing bacteria, ammonia-oxidizing bacteria fromthe Nitrosomonas europaea/eutropha, Nitrosococcus mobilis, Nitrosomonasoligotropha, and Nitrosospira groups and nitrite-oxidizing bacteriaof the genus Nitrospira (Table 1). The hybridization was preformedas described previously (21). Slides were dual stained with4',6'-diamidino-2-phenylindole (DAPI), and biomass cell viabilitywas indicated by the percentage of DAPI-stained cells hybridizedto the eubacteria probe. To determine the share of each bacterialgroup relative to eubacteria, 20 different microscopic fieldswere evaluated and the average fraction was calculated. Theerror was 5 to 10% of the values given in Tables 1 and 2.

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TABLE 1. Analysis of bacterial population from laboratory biofilm reactor^a

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TABLE 2. Analysis of bacterial population from suspended-biomass laboratory reactor at end of experimental period^a

In a similar manner, after 100 days of low-pH operation, a sampleof settled biomass from the suspended-biomass reactor was analyzedfor bacterial populations by Vermicon AG (Munich, Germany).A more extensive hierarchical set of fluorescently labeled oligonucleotideprobes targeting the ${alpha}$ -, ß-, and ${gamma}$ -proteobacterium groups,ammonia-oxidizing bacteria, and the major subgroups of ammonia-and nitrite-oxidizing bacteria was used (Table 2).

Tests were also carried out to determine if any significantheterotrophic bacterial or fungal nitrification occurred inthe biofilm and suspended-biomass reactor. Batch experimentswith allylthiourea (1 µg · ml^–1) and nitrapyrinat low concentrations (0.5 µg · ml^–1) showedsignificant inhibition, indicating autotrophic nitrification(26, 39). Batch tests with increasing concentrations of acetate(up to 20 mg · liter^–1) showed similar or evenlower nitrification rates, further indicating no significantheterotrophic nitrification (37). In addition, batch cultureswith streptomycin and tetracycline at bacteriocide concentrationsalmost completely inhibited nitrifying activity, ruling outthe presence of fungal nitrification (19). Furthermore, usingappropriate fluorescent oligonucleotide probes on a biomasssample from the suspended-biomass reactor, the important heterotrophicnitrifier groups Paracoccus denitrificans, Alcaligenes faecalis,and Pseudomonas putida were found to be absent (Table 2).

RESULTS AND DISCUSSION

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Results and Discussion

Attached biomass (biofilm) reactor.
An attached-biomass reactor with sintered glass as the biofilmcarrier was first operated at pH 7 (Fig. 1a) until a high nitrificationrate of 4.2 g of N oxidized · liter of reactor volume^–1· day^–1 and biomass content of 6.6 ± 0.2g of VSS · liter^–1 were achieved with a specificnitrification rate of 0.64 g of N oxidized · g of biomass^–1· d^–1. The transition from neutral to acidic conditionswas carried out by gradually reducing the ammonium load to thereactor over a period of 8 days, lowering the nitrificationrate from 4.6 to 1.1 g of N oxidized · liter of reactorvolume^–1 · day^–1, and by changing the feedingsolution to contain insufficient buffer for complete nitrificationof the influent ammonium concentration (Fig. 1b). In the sameperiod, the pH dropped from 7.2 to 4.6, and the carbonate alkalinityfell to just below the H₂CO₃* (H₂CO₃ = H₂CO₃ + CO₂ [aqueous])equivalence point (–0.04 meq · liter^–1),indicating that all of the influent buffer was destroyed andonly acidity species were present. At this point, the nitrificationrate was 24% of the original rate. The nitrogen load was increasedfor 10 days, and the nitrification rate recovered to 3.2 g ofN oxidized · liter of reactor volume^–1 ·day^–1 in spite of the low average pH (4.3 ± 0.1)and alkalinity (–0.04 ± 0.03 meq · liter^–1).The reactor was maintained at a nitrification rate of about3.0 g of N oxidized · liter of reactor volume^–1· day^–1 and recorded the same average pH and negativealkalinity values for a further 30 days except for a few transientpH peaks observed during this period (days 34, 35, 40, and 43)caused by malfunctions in the oxygen supply system. From day48 to day 66, the influent load was increased, and a very highnitrification rate of 5.6 g of N oxidized · liter ofreactor volume^–1 · day^–1 was achieved. Theaverage pH during this period was only slightly higher at 4.4± 0.1. The biomass concentration in the reactor increasedby about 50% to 10.1 ± 0.4 g of VSS · liter^–1,with a specific nitrification rate of 0.55 g of N · gof biomass^–1 · day^–1, similar to the valueobtained at the end of reactor start-up at pH 7 and with otherhigh-rate nitrification reactors operating at pH 7 reportedin the literature (3, 9).

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FIG. 1. (a) Nitrifying biofilm reactor pH. (b) Performance of a nitrifying biofilm reactor at low pH. l, liter.

Approximately 94% of the influent ammonium was converted tonitrate, while the average nitrite concentration was 0.2 ±0.3 mg of N · liter^–1 and rarely higher than 1.0mg of N · liter^–1 (Fig. 2). The average ammoniumconcentration in the reactor was low, 9.3 ± 3.3 mg ofN · liter^–1, and the corresponding free ammoniaconcentration at low pH was negligible, 0.01 ± 0.006µM NH₃. Daily nitrogen mass balance based on ammoniumfed, nitrate and nitrite produced, and alkalinity balance (wherefor every mole of NO₃^– produced, 2 mol of alkalinity wasdestroyed) showed no apparent nitrogen loss due to denitrification.This is to be expected from reactor systems operating at highconcentrations of oxygen (>8 mg · liter^–1 inthe effluent) and a feeding solution consisting only of inorganicsalts.

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FIG. 2. Ammonium, nitrite, and nitrate concentrations in the nitrifying biofilm reactor (influent [Inf.] NH₄⁺, •; effluent [Eff.] NH₄⁺, ${circ}$ ; effluent NO₂^–, ${diamondsuit}$ ; effluent NO₃^–, ${square}$ ). l, liter.

After continuous operation of the reactor for more than 250days at low pH and nitrification rates greater than 2 g of Noxidized · liter of reactor volume^–1 · day^–1,the microbial composition of the biofilm reactor was characterizedby using fluorescent rRNA targeted probes (Table 1). The bacterialcells showed high viability, since more than 95% of the DAPI-stainedcells were positive for the eubacterial probe. Sixty-five percentof the eubacterial population was identified as AOB, while 25%was from the genus Nitrospira, a NOB. These values are higherthan those with a similar control pH 7 nitrifying biofilm reactorshowing 40% AOB and 20% NOB and reported values of dedicatednitrifying reactors using fluorescently labeled oligonucleotideprobes (10, 15, 17, 23, 29). The low-pH reactor also presenteda more uniform morphology of detected nitrifiers, with onlysmaller AOB cell clusters (about 10 µm), while a morediverse colony structure with various bigger AOB cell clusters(from 10 to 25 µm) could be observed in the pH 7 reactor.Coccoid Nitrospira cells were grouped in very tight and mostlysmall clusters. In the biofilm of the nitrifying reactors, AOBand Nitrospira clusters formed coaggregated microcolonies. Withuse of more-specific oligonucleotide probes, all the AOB wererevealed to be from the N. oligotropha lineage. These knowngroups of nitrifiers are not considered acid-tolerant bacteria,although N. oligotropha is reported to have very low K_s valuesof 1.9 to 4.2 µM NH₃ (18). With use of selective inhibitors(19, 26, 37, 39), no signs of either bacterial or fungal heterotrophicnitrification were found. The exclusive presence of Nitrosomonas-typebacteria after long-term reactor operation is noteworthy, becausetolerance to low pH has been previously attributed mostly tothe monophyletic genus Nitrosospira (8, 32). This suggests thatautotrophic nitrification at low pH is more widespread thanpreviously thought.

If indeed the bacteria identified in the low-pH reactor arenot acid tolerant and the biofilm's glass carrier provides noalkaline or ion exchange properties, it still could be conjecturedthat somehow the biofilm provides a favorable pH microenvironment.Previous microelectrode measurements profiling pH and oxygenconcentrations in a nitrifying biofilm grown at low pH revealedan active biofilm with a pH similar to the bulk pH and evenlower (36). Furthermore, modeling nitrifying biofilm based onknown kinetic parameters and diffusion constants supported themicrosensor's measurements (35). Summarizing, a proton-producingbiofilm cannot possibly have a favorable pH microenvironment,with the corresponding increase in NH₃ concentration stronglysuggesting that autotrophic nitrifying bacteria have an efficientuptake mechanism for the ammonium cation. The phenomenon ofhigh-rate nitrification at low pH was repeated in several otherbiofilm reactors with inocula from different sources (36), andpH values as low as 3.75 were observed (results not shown).

Suspended-biomass reactor.
To further prove the abilities of known chemolithotrophic bacteriato nitrify at a high rate and a low pH, the research study wasextended to include a suspended-biomass reactor. Comparisonbetween biofilm and suspended-biomass reactors is important,since biofilms and aggregates were reported to facilitate nitrificationat low pH (1, 7).

The low-pH nitrifying suspended-biomass reactor was inoculatedwith biomass taken from a fixed-bed reactor already acclimatedto low pH (pH 5). Protons released by nitrification in the continuouslyfed suspended-biomass reactor destroyed all of the influentalkalinity (10 to 28 meq · liter^–1 with a pH of7.9 to 8.5) during the 100-day experimental period and resultedin a very low reactor pH, varying between 3.2 and 4.5, withan average pH of 3.8 ± 0.3 (Fig. 3a). Interestingly,the average pH in the suspended-biomass reactor was significantlylower by a half a unit than the average pH in the biofilm reactor.Although the bacteria could have operated at a higher pH simplyby oxidizing a smaller amount of the influent ammonium concentration(resulting in the formation of less acidity), the bacteria seemedto be indifferent to the bulk pH as long as it was higher than3.2. The measured alkalinity was significantly lower than thatin the biofilm reactor and well below the H₂CO₃* equivalencepoint (–0.26 ± 0.16 meq · liter^–1),i.e., no carbonate species with proton-accepting capacity werepresent in the suspended-biomass reactor, and the phosphatespecies concentration was low (1 to 2 mg · liter^–1as PO₄^–). A temporary increase in the pH was observedon days 23, 59 and 78 (Fig. 3a) due to a malfunction in oxygensupply (days 59 and 78) and a too-fast increase in the alkalinityand ammonium loading relative to the nitrification rate (day23). During the entire experimental period, the nitrite concentrationwas always close to zero. As in the case of the nitrifying biofilmreactor, mass balance on both nitrogen and alkalinity showedthat no denitrification occurred in the suspended-biomass reactor.

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FIG. 3. (a) Bulk pH in nitrifying suspended-biomass reactor. (b) Performance of a suspended biomass nitrifying reactor at low pH. l, liter.

The working procedure ensured that the ammonium concentrationin the reactor was always low, the average concentration being6.2 ± 3 mg · liter^–1 for N-NH₄⁺. The correspondingfree ammonia concentration was negligible, with an average concentrationof 0.002 ± 0.0015 µM NH₃. Concentrations as lowas 0.0002 µM NH₃ were observed, which is four orders ofmagnitude lower than the lowest reported K_s for nitrifying bacteria(18).

The loading rate was gradually increased over the 100-day period.Increasing the loading rate was carried out only when lowervalues of reactor pH were observed, which indicated an increasein nitrification activity. At the end of the experimental period,a volumetric nitrification rate of 1.1 g of N oxidized^–1· liter of reactor volume^–1 · day^–1(Fig. 3b) and a specific nitrification rate of 0.24 g if N oxidized· g of biomass^–1 · day^–1 was attainedbased on nitrate produced. The gradual increase in the nitrificationrate was probably due to continued cell growth (see below) andnot to adaptation, since the bacteria were already acclimatedto a low pH from the beginning of the experiment.

Using fluorescently labeled oligonucleotide probes, the microbialcomposition of the suspended-biomass reactor was characterized(Table 2). As in the biofilm reactor, the bacterial cells showedhigh viability, since more than 95% of the DAPI-stained cellswere positive for the eubacterial probe. Remarkably, 80% ofthe total eubacterial population was the nitrite oxidizer Nitrospira,while only 2% was identified as AOB. Using more-specific DNAprobes, 1.6% of the AOB were identified as N. oligotropha- andN. mobilis-related groups in equal concentrations. The remaining0.4% of AOB was undetected by any of the more specific probesused. The Nitrospira cells showed a uniform shape, coccoid cells,which were observed in small as well as large clusters. Theidentified ammonia oxidizers showed a diverse morphology. Densecell clusters not greater than 10 µm in size belongingto the N. mobilis group and loosely packed cell "swarms" belongingto the N. oligotropha lineage were observed attached to largerNitrospira clusters, forming large aggregates. Dense N. mobilisclusters were also observed separate from the large aggregates.Single AOB cells were hardly detected. No signs of either bacterialor fungal heterotrophic nitrification were found using selectiveinhibitors (19, 26, 37, 39) or specific molecular probes forthe major heterotrophic nitrifying groups.

Based on total biomass concentration, a constant specific nitrificationrate of 0.23 g of N oxidized · g of dry biomass^–1· day^–1 was observed from day 25. This value isabout one order of magnitude lower than reported maximal nitrificationrates at pH values of between 7 and 8 (27, 30, 38) (values basedon total biomass concentrations). Although this value is remarkablyhigh for such a low pH, it is actually even much higher whenbased on AOB instead of the total biomass concentration becauseof the extremely small fraction of these bacteria.

Growth in the suspended-biomass reactor was shown by an increasein the ¹⁵N isotope content of the biomass with use of a feedingsolution enriched with ¹⁵N-labeled ammonium chloride in theearly part of the experiment, from day 7 to day 33. Growth ofbacteria was also confirmed as an increase in the reactor drybiomass concentration (from 2.7 to 4.1 g · liter^–1)and protein concentration (from 0.9 to 1.5 g · liter^–1)from day 25 to day 95, in spite of biomass losses caused byweekly sampling and daily filtration (Fig. 4). Although thesemethods do not differentiate between autotrophic and heterotrophicgrowth, there are several points which support the assumptionthat the increase in reactor protein, dry biomass, and ¹⁵N assimilationis mainly due to autotrophic growth: (i) the feeding solutioncontained only inorganic salts and no direct organic substrateto support substantial heterotrophic growth; (ii) a highly activenitrifying population was maintained for the whole experimentalperiod, lasting 100 days, and the reactor nitrification rateeven increased; (iii) the gradual increase in biomass concentrationthroughout the experiment was accompanied by a proportionalrise in the nitrification rate in the reactor; and (iv) theincremental increase in biomass concentration was much greaterthan the measured share of the heterotrophs in the reactor.As for possible heterotrophic growth of the NOB population,this has been shown only for Nitrobacter, which was absent inthe reactor, and not for Nitrospira, the only NOB found in thesuspended-biomass reactor.

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FIG. 4. Biomass concentration (g of protein · liter^–1; •) and specific ammonium oxidation rate (g of N oxidized · g of protein^–1; ${circ}$ ) in the suspended-biomass reactor.

The ¹⁵N results showed a yield of 0.1 g of dry biomass ·g of N oxidized · day^–1. The yield coefficientobserved in the suspended-biomass reactor is in the lower rangeof reported cell yield coefficients for nitrifying bacteriaat optimal pH (0.05 to 0.33 g of dry biomass · g of N-NH₄⁺oxidized^–1) (14, 27, 28, 38). Typical kinetic constantsof nitrifying bacteria indicate that the AOB concentration shouldbe higher than that of NOB, as was the case in the control pH7 and low-pH biofilm reactors mentioned above. The abundanceof NOB in the suspended-biomass reactor was found to be 40 timeshigher than that of AOB, and this suggests that the observedyield is mainly that of NOB alone.

To create such a large difference in population sizes, the yieldof AOB may have been much lower due to the suspended-biomassreactor's lower pH (at least half a pH unit of difference betweenthe biofilm and the suspended-biomass reactors) or the resultof the filtration technique used daily to prevent biomass washout.In the first case, energy-consuming mechanisms for maintaininghomeostatic pH and for ammonium uptake to overcome the problemof a negligible free ammonia concentration at low pH may havebeen responsible for the disproportionate populations. However,a normal population ratio of AOB to NOB under negligible freeammonia concentrations and low pH was already observed in thebiofilm reactor. It can be speculated that at the very low pHconditions prevailing in the suspended-biomass reactor, theenzymatic system for AOB may require more energy to maintainhomeostasis because of its more elaborate nature. In the secondcase, glass microfiber filter paper with 1.6-µm pores(Whatman GFA) used in filtration may have selectively retainedthe much larger NOB aggregates while smaller embryonic AOB clustersor free cells passed the filter barrier and washed out of thesystem. However, results from a number of filtrate samples showedthat the protein and biomass concentration in the effluent wasnegligible. Regardless of the reasons for the disproportionatelylow AOB-to-NOB ratio, the fact that a high ammonium oxidationrate was accomplished by only 2% of the bacterial populationis further evidence for the existence of an efficient ammoniumuptake mechanism and the means to cope with low pH in autotrophicnitrifying bacteria.

In this paper, successful operation of high-rate nitrificationat low pH in both attached (biofilm) and suspended-biomass reactorsby chemolithotrophic bacteria is reported under conditions ofno special NH₃-generating mechanisms. The results from thisresearch clearly contradict those in many other previously publishedstudies on high-rate nitrification in reactor systems. In spiteof the unique and carefully designed reactor operation proceduresused here to overcome the specific sensitivities of the chemolithotrophicnitrifying bacteria, it is still perplexing why the phenomenonof high-rate nitrification at low pH was not reported beforein engineered and laboratory systems. In engineered systems,CO₂ limitation due to excessive degassing by conventional airaeration is probably the main reason why high-rate nitrificationat low pH has not been observed (12). With regard to microbialwork in this field, it is usually carried out on pure culturesin batch reactor systems equipped with vigorous air aeration,using the recommended American Type Culture Collection mediumcontaining a high ammonium concentration of 48 mM (33). We canonly surmise that using a heterogeneous nitrifying populationin a continuous-flow reactor aerated with pure oxygen contributedto the successful results by preventing the accumulation oftoxic HNO₂ and decreasing degassing of essential CO₂ occurringat low pH. In addition, the working procedure minimized bacterialloss that is vital to prevent washout of the extremely slow-growingammonia-oxidizing bacteria. Finally, it is possible that growingthe nitrifying population at low ammonium concentrations mayhave induced an efficient ammonium uptake mechanism, as opposedto cultivation in high-ammonium concentrations, which may repressit (16).

FOOTNOTES

* Corresponding author. Mailing address: Faculty of Civil and Environmental Engineering, Technion, Haifa 32000, Israel. Phone: 972-4-829-3479. Fax: 972-4-829-5696. E-mail: agmgreen{at}tx.technion.ac.il.

REFERENCES

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