Toxicity of fluoride to microorganisms in biological

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May 3, 2009 - were obtained from Spectrum Chemicals and Laboratory. Products (Gardena, CA, USA), respectively. Sodium fluoride. (99.0%), sodium nitrite ...
water research 43 (2009) 3177–3186

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Toxicity of fluoride to microorganisms in biological wastewater treatment systems Valeria Ochoa-Herrera, Qais Banihani, Glendy Leo´n, Chandra Khatri, James A. Field, Reyes Sierra-Alvarez* Department of Chemical and Environmental Engineering, University of Arizona, P.O. Box 210011, Tucson, AZ 85721-0011, USA

article info

abstract

Article history:

Fluoride is a common contaminant in a variety of industrial wastewaters. Available

Received 8 December 2008

information on the potential toxicity of fluoride to microorganisms implicated in biological

Received in revised form

wastewater treatment is very limited. The objective of this study was to evaluate the

19 April 2009

inhibitory effect of fluoride towards the main microbial populations responsible for

Accepted 20 April 2009

the removal of organic constituents and nutrients in wastewater treatment processes. The

Published online 3 May 2009

results of short-term batch bioassays indicated that the toxicity of sodium fluoride varied widely depending on the microbial population. Anaerobic microorganisms involved in

Keywords:

various metabolic steps of anaerobic digestion processes were found to be very sensitive to

Fluoride

the presence of fluoride. The concentrations of fluoride causing 50% metabolic inhibition

Microbial inhibition

(IC50) of propionate- and butyrate-degrading microorganisms as well as mesophilic and

Wastewater treatment

thermophilic acetate-utilizing methanogens ranged from 18 to 43 mg/L. Fluoride was also inhibitory to nitrification, albeit at relatively high levels (IC50 ¼ 149 mg/L). Nitrifying bacteria appeared to adapt rapidly to fluoride, and a near complete recovery of their metabolic activity was observed after only 4 d of exposure to high fluoride levels (up to 500 mg/L). All other microbial populations evaluated in this study, i.e., glucose fermenters, aerobic glucose-degrading heterotrophs, denitrifying bacteria, and H2-utilizing methanogens, tolerated fluoride at very high concentrations (>500 mg/L). ª 2009 Elsevier Ltd. All rights reserved.

1.

Introduction

Fluoride is a widespread environmental contaminant and is estimated to be the 13th most abundant element on the earth’s crust (Mason and Moore, 1982). The public health benefits and risks of fluoride from drinking water and other sources has been a source of controversy in recent years. Although fluoride is beneficial to human health at low concentrations (0.7–1.2 mg/L) by affording protection against dental caries, at concentrations exceeding the U.S. federal drinking water standard (4 mg/L), it has been reported to cause skeletal and dental fluorosis (US-EPA, 2006).

Fluoride is ubiquitous in the environment as it is a component of most types of soils. Concentrations of inorganic fluoride in unpolluted surface water generally range from 0.01 to 0.30 mg/L, but considerably higher concentrations may be found in regions impacted by geothermal or volcanic activity (Camargo, 2003). Human activities can also contribute to increase the concentration of fluoride in aquatic environments. Fluoride is often present in a variety of untreated industrial effluents, including those from chemical plants manufacturing organofluorine compounds, aluminum smelters, phosphate fertilizers, semiconductor manufacturing, glass and brickmaking industries, and coal power plants (Fuge and Andrews,

* Corresponding author. Tel.: þ1 520 626 2896; fax: þ1 520 621 6048. E-mail address: [email protected] (R. Sierra-Alvarez). 0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.04.032

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1988; Skjelkvale, 1994; Sujana et al., 1998; Camargo, 2003; Shen et al., 2003). The concentrations of fluoride in untreated industrial wastewaters vary widely, and concentrations as high as 500 to 2,000 mg/L have been reported in effluents from semiconductor industry operations in Taiwan (Huang and Liu, 1999; Hu et al., 2005). In contrast, fluoride levels in municipal wastewaters receiving fluoride-bearing industrial effluents are generally low because industrial effluents will be diluted by wastewaters from residential and industrial sources. In addition, significant removal of fluoride can occur by precipitation with calcium(II) (CaF2, Ksp ¼ 3.9  1011), which is a common wastewater contaminant. Although fluoride is often present as a wastewater contaminant, published data on its inhibitory effect to microbial populations present in wastewater treatment systems are very scarce. Nitrification is the only process that has been studied in some detail (Whang, 1985; Clarkson et al., 1989; Carrera et al., 2003). In contrast with the limited understanding of the potential inhibitory effects of fluoride on wastewater treatment microorganisms, the impact of fluoride on oral bacteria of interest to dentistry is well documented by a vast body of literature (Marquis et al., 2003; Wiegand et al., 2007). Oral bacteria are inhibited by fluoride at concentrations in the range of 10–1600 mg/L. Inhibition of soil microorganisms by inorganic fluoride, resulting in increased accumulation of soil organic matter, has also been reported in several studies (Rao and Pal, 1978; Wilke, 1987; Tscherko and Kandeler, 1997). In one study microbial biomass and enzymatic activity in soil were decreased substantially at waterextractable fluoride concentrations exceeding 20 mg/g soil (Tscherko and Kandeler, 1997). These findings suggest that, if present at sufficient concentration, fluoride might have a negative impact on biological wastewater treatment systems. The objective of this study is to evaluate the inhibitory effect of inorganic fluoride towards the main microbial populations involved in organic matter and nitrogen nutrient removal in wastewater treatment plants.

2.

Materials and methods

2.1.

Sludge sources

Three different mesophilic inocula were evaluated in this study, including two types of methanogenic granular sludge (Eerbeek and Aviko sludge) and anaerobically digested sewage sludge (Ina Road sludge). Eerbeek sludge was obtained from an industrial anaerobic sludge blanket (UASB) reactor treating recycle paper effluent (Industriewater, Eerbeek, The Netherlands), and the Aviko sludge from a UASB reactor treating potato processing wastewater (Aviko, Steenderen, The Netherlands). Both inocula were washed and sieved to remove fine particles and they were stored under nitrogen gas  at 4 C. The content of volatile suspended solids (VSS) in the Eerbeek and Aviko sludges was 12.9% and 11.5%, respectively. The Ina Road sludge (1.5% VSS) was obtained from an anaerobic sewage sludge digester at the Ina Road municipal wastewater treatment plant, in Tucson, AZ. A thermophilic inoculum (Hyperion sludge, 1.6% VSS) was also utilized in the

methanogenic studies which was obtained from an anaerobic sludge digestor operating at the Hyperion municipal wastewater treatment plant, Los Angeles, CA. The inoculum utilized in the nitrification assays, Randolph Park sludge I, was obtained from the nitrification stage of a sewage treatment facility (Randolph Park Wastewater Reclamation Facility, Tucson, AZ) and contained 4.8% VSS. An anaerobic glucose-degrading enrichment cultures was developed from Aviko sludge by successive transfer of the culture supernatant to fresh glucose containing basal medium at a rate of 5% (v/v) upon glucose depletion. The aerobic sewage sludge, Randolph Park sludge II (0.13% VSS), was obtained from the aeration tank of the Randolph Park Wastewater Reclamation Facility. Sludge samples  were stored under N2 gas in a refrigerator at 4 C.

2.2.

Culture media

The composition of the basal mineral medium supplied in the nitrification bioassays (BM-1) was (in mg/L): NaH2PO4 (1500); and Na2HPO4 (894). The basal mineral medium employed in the denitrification bioassays (BM-2) contained (in mg/L): K2HPO4 (250); (NH4)HCO3 (417); NaHCO3 (2678); and yeast extract (10). The basal mineral medium utilized in both the aerobic heterotrophic toxicity and fermentation bioassays (BM-3) contained (in mg/L): NH4Cl (280); NaHCO3 (3000); K2HPO4 (250); KH2PO4 (2050); CaSO4 $ 2H2O (10), MgSO4 $ 7H2O (100), and yeast extract (50). The basal mineral medium used in all other anaerobic bioassays (BM-4) contained (in mg/L): K2HPO4 (37); CaCl2 $ 2H2O (10); MgSO4 $ 7H2O (10); MgCl2 $ 6H2O (78); NH4Cl (669); NaHCO3 (3000); and yeast extract (20). All media were supplemented with 1 mL/L of a trace element solution containing (in mg/L): H3BO3 (50), FeCl2 $ 4H2O (2000), ZnCl2 (50), MnCl2 $ 4H2O (50),(NH4)6Mo7O24 $ 4H2O (50), AlCl3 $ 6H2O (90), CoCl2 $ 6H2O (2000), NiCl2 $ 6H2O (50), CuCl2 $ 2H2O (30), NaSeO3 $ 5H2O (100), EDTA (1000), resazurin (200) and 36% HCl (1 mL/L), and they were adjusted to pH 7.2 with HCl or NaOH, as required.

2.3.

Batch microbial toxicity assays

The experimental conditions utilized in the various toxicity bioassays are summarized in Table 1. Batch experiments were conducted in triplicate using glass serum flasks (165 mL) supplemented with 50 mL of medium, unless otherwise indicated. The desired amount of fluoride was added to flasks containing the growth medium and inoculum using neutralized concentrated stock solutions of sodium fluoride. Flasks lacking fluoride were also included and served as uninhibited controls. In anaerobic and anoxic bioassays, flasks were sealed with butyl rubber stoppers and aluminum crimp seals and, subsequently, the headspace was flushed with a N2/CO2 mixture (80:20, v/v) to exclude oxygen from the assays. The headspace of the aerobic heterotrophic and nitrification bioassays was atmospheric air, and it was replenished daily to prevent oxygen limitation. In the methanogenic assays, the methane content in the headspace of each flask was determined periodically until 80% or more of the substrate in the controls was depleted. Subsequently, a second feeding of the substrate (acetate or H2/CO2) was supplied in some assays to test the possible impact of extended exposure to fluoride on the methanogenic activity of the anaerobic sludge.

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Table 1 – Summary of experimental conditions utilized in the various inhibition batch bioassays. Inhibition bioassaya Methane production

Substrateb

Substrate concn. (g/L-liquid)

Inoculum concn. (g VSS/L)

Fluoride concn. (g F/L)

Basal mediumc

Headspace contentd

Monitoring

H2 Acetate

0.222 1.88*

1.5 1.5

0–1500 0–300

BM-4

H2/CO2 (80/20 v/v) N2/CO2 (80/20 v/v)

CH4

Propionate

1.32*

1.5

0–539

BM-4

N2/CO2 (80/20 v/v)

Propionate, acetate, and CH4

Butyrate degradation

Butyrate

0.98*

1.5

0–539

N2/CO2 (80/20 v/v)

Butyrate, propionate, acetate and CH4

Glucose fermentation

Glucose

1.00–3.00

1.5

0–539

BM-3

N2/CO2 (80/20 v/v)

Glucose

Aerobic glucose degradation

Glucose

1.00

1.5

0–539

BM-3

Air

Glucose

NHþ 4

0.055

0.50

0–300

BM-1

Air

Ammonium

Acetate Nitrate

0.45 1.01

1.00

0–800

BM-2

N2/CO2 (80/20 v/v)

Nitrate

Propionatedegradation

Nitrification Denitrification

* These concentrations are equivalent to 2 g COD/L.   a All bioassays were incubated at 30  2 C, with the exception of thermophilic methanogenic assays, which were conducted at 55  2 C. Assays were incubated in an orbital shaker (110 rpm). b Acetate, propionate and butyrate were added as the corresponding sodium salts, nitrate as KNO3, and ammonium as NH4(HCO3). c The composition of the basal media is detailed in Section 2. d The headspace pressure in all the bioassays using a N2/CO2 or air atmosphere was 102.3 kPa. When using a H2/CO2 atmosphere the pressure was 121.6 kPa.

The maximum specific glucose consumption (mg glucosedegraded/(g VSS d)), nitrifying (mg NHþ 4 -consumed/(g VSS d)), denitrifying (mg NO 3 /(g VSS d)) and methanogenic (mg CH4COD/(g VSS d)) activities were calculated from the slope of the glucose consumption, ammonium concentration, nitrate concentration and cumulative methane production; respectively, and biomass concentration versus time (d), as the mean value of triplicate or duplicate assays. In each case, the maximum specific activity at a given fluoride concentration was determined during the time period when the fluoride-free control displayed maximum specific activity. The inhibition observed was calculated as shown below. The initial concen-

headspace of the serum flasks was determined by gas chromatography using an HP5290 Series II system (Agilent Technologies, Palo Alto, CA) equipped with a flame ionization detector (GC-FID). The GC was fitted with a DB-FFAP column (J&W Scientific, Palo Alto, CA) capillary column. The temperature of the column, the injector port and the detector was  140, 180 and 275 C, respectively. The carrier gas was helium at a flow rate of 9.3 mL/min and a split flow of 32.4 mL/min. Samples for measuring methane content (100 mL) in the headspace were collected using a pressure-lock gas syringe. Sugars were determined colorimetrically (Dubois et al., 1956). Briefly, 0.5 mL of centrifuged sample was transferred into

  Maximum Specific Activity at the Tested Concentration Inhibition,ð%Þ ¼ 100  100, Maximum Specific Activity of the Control

trations of fluoride causing 20%, 50% and 80% reduction in activity compared to an uninhibited control were referred to as IC20, IC50 and IC80, respectively. These values were calculated by interpolation in the graph plotting the inhibition observed (expressed as percent) as a function of the inhibitor concentration. Unless otherwise indicated, reported inhibitory concentrations are average values of triplicate assays and corresponding standard deviations.

2.4.

Analytical methods

The concentration of acetate, propionate, and butyrate in liquid samples as well as the methane content in the

a test tube and then 0.5 mL of 5% (v/v) phenol and 2.5 mL of concentrated sulfuric acid was added. The sample was vortexed and allowed to rest for 7 min. Subsequently, the tube  was heated at 45 C for 20 min. The solution was allowed to cool down and then the concentration of glucose in the bioassays was determined by measuring the color intensity of the sample at 490 nm. Nitrate, nitrite and fluoride were analyzed by suppressed conductivity ion chromatography using a Dionex 3000 system (Sunnyvale, CA, USA) fitted with a Dionex IonPac AS18 analytical column (4  250 mm) and an AG18 guard column  (4  40 mm). The column was maintained at 35 C. The eluent used was 10 mM KOH at a flow rate of 1.0 mL/min, and the

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2.5.

Chemicals

Potassium nitrate (>99.0% purity) and sodium acetate (99.0%) were obtained from Spectrum Chemicals and Laboratory Products (Gardena, CA, USA), respectively. Sodium fluoride (99.0%), sodium nitrite (>99.5%), acetic acid (99.7%), propionic acid (99.5%) and butyric acid (99.0%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ammonium bicarbonate was obtained from MP Biomedicals (Solon, OH, USA). Phenol (99.0% ACS grade) was obtained from EMD Chemicals (Gibbstown, NJ). D-Glucose anhydrous was purchased from Mallinckrodt Chemical (Paris, KY).

3.

Results and discussion

3.1.

Microbial toxicity assays

3.1.1.

Nitrification and denitrification

A

100 80

60

40

20

60 40 20 0

0

100

200

300

400

500

600

Fluoride (mg/l)

B Activity (% of the control)

NH4+ concentration (mg/l)

An illustrative example of the time course of ammonium consumption in nitrifying activity assays amended with fluoride concentrations ranging from 0 to 300 mg/L is shown in Fig. 1. The specific nitrifying activities in treatments containing fluoride were normalized based on the activity of the control treatment lacking fluoride. In each case, the nitrifying activity was determined during the time period when the

control displayed maximum ammonium oxidizing activity (i.e., time 17–91 h). The normalized activity of nitrifying microorganism as a function of the initial fluoride concentration is plotted in Fig. 2A. The same procedure was utilized to calculate the microbial activities of the different microorganisms evaluated in this study. The IC20, IC50 and IC80 values determined are summarized in Table 2. Fluoride was found to inhibit nitrifying microorganisms in aerobic sewage sludge. Nonetheless, nitrifying bacteria appeared to become acclimated rapidly to this contaminant. The metabolic activity determined in assays that were initially inhibited by fluoride increased substantially with exposure time (Fig. 1). Assays with fluoride concentrations of 230 mg/L and higher were completely inhibited during the initial 100 h of exposure, but their activity increased sharply thereafter reaching levels close to those observed in the fluoride-free controls. The observed recovery may be due to physiological acclimatization to fluoride or to a shift in the microbial population to another nitrifying strain which is less sensitive to the contaminant.

Activity (% of control)

injection volume was 25 mL. Before measurement, all samples were either, centrifuged (10,000 rpm) for 10 min or passed through a membrane filter (0.45 mm). Routine analyses of fluoride were conducted using a VWR SympHony fluorideselective combination electrode. Ammonium was determined using an Orion Thermo combination ion-selective electrode. The pH was determined immediately after sampling with an Orion model 310 PerpHecT pH-meter with a PerpHecT ROSS glass combination electrode. Volatile suspended solids and other analytical parameters were determined according to standard methods APHA (1998).

100

80 60 40 20 0

0

200

400

600

800

Fluoride (mg/l) 0

0

40

80

120

160

200

Time (hours) Fig. 1 – Time course of ammonium consumption by a mixed microbial culture obtained form the nitrification stage of a municipal wastewater treatment plant in the presence of increasing fluoride concentrations (in mg/L): (C) 0,(B) 50,(J) 90,(-) 130,(,) 180,(6) 230, and (:) 300.

Fig. 2 – Inhibitory effect of sodium fluoride on the specific ammonium-oxidizing activity of a mixed microbial culture obtained from the nitrification stage of a full-scale municipal wastewater treatment plant (A); and on the denitrifying specific activity of an industrial anaerobic granular sludge as determined by nitrate depletion (B). Error bars (shown if larger than the symbols) represent standard deviations of duplicate assays.

Table 2 – Inhibitory effect of sodium fluoride on the key microbial populations in biological wastewater treatment systems. IC20, IC50 and IC80 are the concentrations of fluoride causing 20%, 50% and 80% decrease in the activity of the target microbial population, respectively. Substrate

Redox conditions

Inhibitory effect

Inoculuma

1st feeding (mg/L) IC20

Methanogenesis Methanogenesis

Anaerobic Methanogenesis H2 Anaerobic Methanogenesis H2 Thermophilic methanogens Acetate Anaerobic Methanogenesis Anaerobic Methanogenesis H2 Anaerobic propionate-utilizing microorganisms Propionate Anaerobic Propionate degradation Anaerobic butyrate-utilizing microorganisms Butyrate Anaerobic Butyrate degradation Denitrification Nitrate Anoxic Nitrate reduction Nitrification Ammonium Aerobic NHþ 4 oxidation Heterotrophic aerobes Glucose Aerobic Glucose degradation Glucose fermenters Glucose Anaerobic Glucose degradation Glucose Anaerobic Glucose degradation

IC50

IC80

IC20

IC50

IC80

Eerbeek granular sludge Ina Road sludge

40.0 17.5

160.0 34.5

500.0 93.0

15.0 12.4

30.3 25.8

60.0 35.7

Eerbeek granular sludge Ina Road sludge

815.0 30.0

>815.0 82.0

>815.0 390.0

520.0 805.0

645.0 1005.0

795.0 1125.0

Hyperion sludge Hyperion sludge

7.2 218.6

18.1 432.6

39.2 >600.0

29.5 >600.0

42.6 >600.0

62.9 >600.0

Eerbeek granular sludge

10.5

36.5

62.0

Eerbeek granular sludge

17.5

25.5

34.8

>800.0b

>800.0b

>800.0b

Randolph Park I

104.3

148.8

179.8

Randolph Park II

>539.0c

>539.0c

>539.0c

150.5 87.0

>539.0 325.0

>539.0 539.0

Eerbeek granular sludge

Aviko granular sludge Enrichment culture

water research 43 (2009) 3177–3186

Mesophilic methanogens Acetate Anaerobic Acetate Anaerobic

2nd feeding (mg/L)

a All inocula used consisted of dispersed biomass unless otherwise indicated. b Not toxic at the highest concentration tested, i.e., 800 mg/L. c Not toxic at the highest concentration tested, i.e., 539 mg/L.

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Anaerobic microorganisms that utilize propionate and butyrate were very sensitive to fluoride as evidenced by the relatively low IC50 values determined for these microbial

A 100

Glucose fermenting bacteria

Fluoride was inhibitory to glucose-fermenting microorganisms in different microbial consortia (Fig. 3B). The somewhat higher inhibition observed in the assay with the enrichment culture as compared to the anaerobic granular sludge suggests a greater impact of fluoride on the growth as compared to the metabolic activity of glucose fermenters. In contrast with the

80 60 40 20 0

Aerobic heterotrophs

Sodium fluoride did not display any inhibitory effect towards glucose-utilizing bacteria in aerobic sewage sludge at fluoride concentrations of up to 540 mg/L (Fig. 3A). In agreement with our results, the only other study assessing the impact of fluoride (50–100 mg/L) on the removal of organic matter (ethanol and acetate) in the activated sludge process reported no significant effect on cell growth and chemical oxygen demand (COD) removal efficiency (Singh and Kar, 1989). In the latter study, the presence of fluoride resulted in deterioration of the sludge settling properties as indicated by a 100 to 200% increase of the sludge volume index. The mechanisms contributing to the deterioration of the sludge settling ability in the presence of fluoride are unknown. Several studies from the dental literature have reported that fluoride can inhibit the formation of biofilms and other cell aggregates (Embleton et al., 2001; Cao and Doyle, 2002; Marquis et al., 2003).

3.1.3.

3.1.4. Anaerobic propionate- and butyrate-degrading microorganisms

0

100

200

300

400

500

600

Fluoride (mg/l)

B Activity (% of control)

3.1.2.

assay inoculated with the dispersed enrichment culture, microbial growth has a minor impact on the glucose utilization rate of the granular sludge due to the high biomass concentrations utilized in the latter assay (1.5 g VSS/L). Inhibition of fermentation in anaerobic wastewater treatment sludge has not been reported in the literature. Nonetheless, there are numerous studies that confirm the toxic action of fluoride on glucose incorporation and carbohydrate metabolism by oral bacteria (Marquis et al., 2003; Wiegand et al., 2007). The minimum inhibitory concentrations reported to impair carbohydrate degradation by Streptococcus species and other oral bacteria vary widely from 5 to 1600 mg/L (Maltz and Emilson, 1982; Eisenberg et al., 1991; Lenander-Lumikari et al., 1997; Ekenback et al., 2001; Bradshaw et al., 2002).

Activity (% of control)

Literature data indicates that fluoride exerts relatively low inhibition towards nitrifying bacteria in activated sludge systems. Complete oxidation of ammonium (400 mg/L) in a synthetic coal gasification wastewater was observed in batch assays amended with 300 mg F/L (Whang, 1985). CSTR experiments inoculated with a nitrifying culture enriched from municipal sewage sludge showed that fluoride concentrations of up to 2430 mg /L did only exert moderate inhibition (39%) of nitrification activity (Clarkson et al., 1989). Carrera et al. (2003) reported that elevated fluoride levels (630 mg/L) were required to cause 50% inhibition of the nitrifying activity of biomass in a lab-scale activated sludge reactor treating a high-strength ammonium wastewater (546 mg NHþ 4 /L). The wide variation observed among the various studies in the toxicity response of nitrification to fluoride may be due to differences in the experimental conditions, including ammonia concentration and other medium components, as well as variations in the microbial community structure of the inoculum, among others. Regarding the toxic action of fluoride on denitrifying microorganisms, the contaminant did not exert any significant inhibitory effect on the activity of the mixed culture at concentrations as high as 800 mg/L (Fig. 2B). We are not aware of any previous study from the literature reporting on the inhibitory effect of fluoride on denitrifying bacteria during biological wastewater treatment. However, fluoride in soils (up to 3,700 mg/kg) has been reported to affect nitrate reduction, but the inhibitory effect varied considerably depending on the soil characteristics (Ottow and Kottas, 1984; Wilke, 1987). These results suggest that biological processes for the removal of nitrogen from wastewater are not expected to be affected by fluoride unless the concentrations are exceedingly high.

100 80 60 40 20 0

0

100

200

300

400

500

Fluoride (mg/l) Fig. 3 – Inhibitory effect of sodium fluoride on the specific glucose-degrading activity of aerobic sewage sludge (A); glucose fermentation by different mixed anaerobic culture (B). Legends for panel B: Anaerobic granular sludge (-), and an anaerobic glucose-degrading enrichment culture (C). Error bars (shown if larger than the symbols) represent standard deviations of triplicate assays.

water research 43 (2009) 3177–3186

populations, 36.5 and 25.5 mg/L, respectively (Table 2). As shown in Fig. 4A and B, the utilization rate of VFA steeply decreased with increasing fluoride concentrations. This decrease was accompanied by a reduction in the rate of methanogenesis. The high toxicity of fluoride towards anaerobic microbial populations involved in VFA degradation is a concern because anaerobic digestion is commonly applied for the management of excess sludge in municipal wastewater treatment systems (Chen et al., 2008). The inhibition of acetogenic microorganisms would prevent the effective degradation of VFA (e.g. propionate, butyrate, etc.) into acetate and, therefore, compromise the anaerobic digestion process.

3.1.5.

Methanogens

Acetoclastic methanogens were particularly susceptible to the inhibitory effect of fluoride. Fig. 5A and B reveal a sharp decrease in the specific acetoclastic methanogenic activity of mesophilic and thermophilic microorganisms in digested sewage sludge with increasing fluoride concentrations. The IC50 values determined for those microbial populations during

Activity (% of Control)

A

100

80

60

40

20

0 0

100

200

300

400

500

600

500

600

Fluoride (mg/l)

Activity (% of Control)

B

100

80

60

40

20

0

0

100

200

300

400

Fluoride (mg/l) Fig. 4 – Inhibitory effect of fluoride on the maximum specific activity of propionate- (A) and butyrate-degrading microorganisms (B) in a mesophilic anaerobic consortium. Error bars (shown if larger than the symbols) represent standard deviations of triplicate assays.

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the first substrate feeding were 34.5 and 18.1 mg/L, respectively. Mesophilic acetoclastic methanogens in granular sludge appeared to be more tolerant to fluoride as demonstrated by the IC50 value of 160 mg/L during the same feeding (Table 2). The impact of extending the time of exposure on the methanogenic inhibition of fluoride was investigated by supplying a second feeding of substrate to the bioassay once that the initially supplied substrate had been depleted. The inhibitory concentrations determined for the mesophilic granular sludge following the second substrate feeding were lower and relatively similar to those determined for the mesophilic sewage sludge (Table 2). These results suggest that time was required to enable for fluoride penetration in the thick anaerobic granular sludge biofilm and, thus, for effective exposure of the methanogens to the toxicant. It is well established in the literature that internal mass transfer limitations can impact the transport of solutes in anaerobic biofilms (Dolfing, 1985; Pavlostathis and Giraldo-Gomez, 1991; Wu et al., 1995; Gonzalez-Gil et al., 2001). In the case of H2-utilizing methanogens, fluoride caused a decrease in the rate of methane production by mesophilic and thermophilic anaerobically-digested sludges but only when present at very high concentrations (Fig. 5C and D), and the inhibitory effect decreased sharply with extended incubation time. The IC50 values determined for those microbial populations during the first substrate feeding were 433 mg/L or higher (Table 2). On the other hand, hydrogenotrophic methanogens in mesophilic anaerobically-digested sludge were more sensitive to the presence of fluoride during the first feeding, but halogen ion caused little methanogenic inhibition during the second feeding at concentrations below 900 mg/L (Fig. 5C). The mechanisms responsible for the substantial decrease in the inhibitory effects of fluoride towards hydrogenotrophic methanogens with incubation time are unclear. To the best of our knowledge, there are no previous literature reports concerned with the inhibitory effects of fluoride towards methanogenic microorganisms. The relatively low fluoride inhibiting values observed in this study are of particular concern in view of the slow growth kinetics of acetate-utilizing methanogens (doubling times range from 1 to 7 d (Mara and Horan, 2003)). An active acetoclastic methanogenic population is essential for the degradation of acetate which, in turn, is required to attain adequate removal of biodegradable organic matter during wastewater treatment. In practice, this means that fairly long time periods might be required for the recovery from an incidental toxicity exposure. For instance, Zayed and Winter (2000) reported that recovery of methanogenesis following exposure to 10–20 mg/L of copper was attained after 35 or 47 d after the toxic shock. The apparent tolerance of H2-utilizing methanogens to fluoride does not preclude inhibition of anaerobic sludge digestion by this ion, since only about one-third of the electron equivalents in complex organic matter are channeled through H2 gas. The mechanisms responsible for the microbial toxicity of fluoride towards acetate, propionate and butyrate-utilizing microorganisms in anaerobic reactors are not well understood. Nonetheless, fluoride can bind to many microbial enzymes, including heme-containing enzymes, copper-based oxidases and other metalloenzymes, affecting metabolism (Hamilton and Ellwood, 1978; Marquis et al., 2003). Fluoride

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C

100

Activity (% of control)

Activity (% of control)

A

80 60 40 20 0

0

50

100

150

200

250

120 100 80 60 40 20 0

300

0

250

Flouride (mg/l)

750 1000 1250 1500

Fluoride (mg/l)

B

D 100 100

Activity (% of control)

Activity (% of control)

500

80 60 40 20 0

0

50

100

150

200

250

80 60 40 20 0

300

0

100

200

300

400 500

600

700

Fluoride (mg/l)

Flouride (mg/l)

Fig. 5 – Inhibitory effect of fluoride on the specific acetoclastic and hydrogenotrophic activity of mesophilic anaerobicallydigested sludge, (panels A and C, respectively), and on the specific acetoclastic and hydrogenotrophic activity of thermophilic anaerobically-digested sludge (panels B and D, respectively). Legend: First substrate feeding (C), and second substrate feeding (B). Error bars (shown if larger than the symbols) represent standard deviations of triplicate assays.

can form complexes with metals such as aluminum or beryllium, leading to compounds that can mimic phosphate  (e.g. AlF 4 and BeF3 ), and interfere with a variety of enzymes, e.g., phosphatases and pyrophosphatases (Marquis et al., 2003). Fluoride is a potent inhibitor of pyrophosphatase (PPase) in various microorganisms (Tominaga and Mori, 1977; Smirnova and Baykov, 1983). PPase plays an important role in the energy metabolism of methanogens (Roth and Bachofen, 1994; Smith and Ingram-Smith, 2007), and variation in the susceptibility to fluoride of PPase enzymes from acetoclastic and hydrogenotrophic methanogens might have contributed to the considerable differences observed in the response of these methanogens to fluoride. A PPase has been isolated from Methanothrix soehngenii, a common acetoclastic methanogen in anaerobic reactors that is not inhibited by fluoride (Jetten et al., 1992). The composition of the medium could also affect inhibition by fluoride. Studies with the methanogen, Methanococcus jannaschii, have shown that prior exposure to specific metallic ions (e.g., Mn2þ or Co2þ) increased the tolerance against inhibition by sodium fluoride, to which the enzyme was otherwise very sensitive (Kuhn et al., 2000). The inhibitory effect of organic compounds containing fluoride such as methyl fluoride (CH3F) is well documented. CH3F is known to be a specific inhibitor of acetoclastic methanogens and anaerobic mixed cultures that produce acetate as an intermediate, but this organofluorine compound is not or only mildly inhibitory to hydrogenotrophic methanogens

(Frenzel and Bosse, 1996; Janssen and Frenzel, 1997; Conrad and Klose, 1999). The different sensitivity of acetate- and H2-utilizing methanogens towards CH3F is in agreement with the toxic response observed in this study for these two methanogenic trophic groups to fluoride, suggesting the possibility that microbial methylation of fluoride might have occurred leading to the formation of toxic CH3F. However, this biotransformation has not been reported earlier (O’Hagan et al., 2002; Murphy et al., 2003).

4.

Conclusions

Fluoride is inhibitory towards microbial populations involved in various metabolic steps in anaerobic digestion processes, i.e., mesophilic and thermophilic acetoclastic methanogens, as well as propionate- and butyrate-degraders, at concentrations lower than those found in some fluoride-containing industrial effluents. In contrast, very high concentrations of soluble fluoride (>500 mg/L) can be tolerated by microbial communities involved in the aerobic activated sludge and in denitrification processes without significant inhibitory impact. Nitrification processes are somewhat more sensitive but they appear to acclimate rapidly to fluoride. In conclusion, the high susceptibility of key microbial populations involved in the anaerobic metabolism of volatile fatty towards inhibition by fluoride indicates that measures to reduce the concentration of this ion (e.g., pretreatment of the wastewater

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using ion exchange or precipitation with calcium(II), wastewater dilution, etc.) may be required to prevent microbial inhibition during the anaerobic treatment of fluoride-containing streams.

Acknowledgements This study was conducted with financial support from the Semiconductor Research Corporation/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing.

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