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 León, 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 metha-
nogens, tolerated fluoride at very high concentrations (>500 mg/L).
ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction Fluoride is ubiquitous in the environment as it is a compo-

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).
nent 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 brick-
making industries, and coal power plants (Fuge and Andrews,

* Corresponding author. Tel.: þ1 520 626 2896; fax: þ1 520 621 6048.
E-mail address: rsierra@email.arizona.edu (R. Sierra-Alvarez).
0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2009.04.032
3178 water research 43 (2009) 3177–3186
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 addi-
tion, 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 under-
standing 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 microorgan-
isms by inorganic fluoride, resulting in increased accumula-
tion of soil organic matter, has also been reported in several
studies (Rao and Pal, 1978; Wilke, 1987; Tscherko and Kand-
eler, 1997). In one study microbial biomass and enzymatic
activity in soil were decreased substantially at water-
extractable 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 pop-
ulations 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 anaer-
obic 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 waste-
water 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 Recla-
mation Facility, Tucson, AZ) and contained 4.8% VSS. An anaer-
obic 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) sup-
plemented 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 concen-
trated stock solutions of sodium fluoride. Flasks lacking fluo-
ride 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. Subse-
quently, 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.
 water research 43 (2009) 3177–3186 3179

Table 1 – Summary of experimental conditions utilized in the various inhibition batch bioassays.
Inhibition Substrateb Substrate Inoculum Fluoride Basal Headspace Monitoring
bioassaya concn. concn. concn. mediumc contentd
(g/L-liquid) (g VSS/L) (g F/L)

Methane production H2 0.222 1.5 0–1500 BM-4 H2/CO2 (80/20 v/v) CH4
Acetate 1.88* 1.5 0–300 N2/CO2 (80/20 v/v)

Propionate- Propionate 1.32* 1.5 0–539 BM-4 N2/CO2 (80/20 v/v) Propionate, acetate,
degradation 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 Glucose 1.00 1.5 0–539 BM-3 Air Glucose

degradation

Nitrification NHþ
4 0.055 0.50 0–300 BM-1 Air Ammonium

Denitrification Acetate 0.45 1.00 0–800 BM-2 N2/CO2 (80/20 v/v) Nitrate
Nitrate 1.01

* 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 glucose-
degraded/(g VSS d)), nitrifying (mg NHþ 4 -consumed/(g VSS d)),
denitrifying (mg NO 3 /(g VSS d)) and methanogenic (mg CH4-
COD/(g VSS d)) activities were calculated from the slope of the
glucose consumption, ammonium concentration, nitrate
concentration and cumulative methane production; respec-
tively, 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 temper-
ature 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 calcu-
lated by interpolation in the graph plotting the inhibition
observed (expressed as percent) as a function of the inhibitor
concentration. Unless otherwise indicated, reported inhibi-
tory 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
3180
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 fluoride-
selective 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).
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%), pro-
pionic 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
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 con-
taining 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
60
NH4+ concentration (mg/l)
40
20
0 Fig. 2 – Inhibitory effect of sodium fluoride on the specific
0 40 80
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.
water research 43 (2009) 3177–3186
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 concen-
tration is plotted in Fig. 2A. The same procedure was utilized
to calculate the microbial activities of the different microor-
ganisms 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 pop-
ulation to another nitrifying strain which is less sensitive to
the contaminant.
A 100
Activity (% of control)
80
60
40
20
0
0 100 200 300 400 500 600
Fluoride (mg/l)
B 100
Activity (% of the control)
80
60
40
20
0
0 200 400 600 800
Fluoride (mg/l)
120 160 200
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) 2nd feeding (mg/L)

IC20 IC50 IC80 IC20 IC50 IC80

Mesophilic methanogens
Acetate Anaerobic Methanogenesis Eerbeek granular sludge 40.0 160.0 500.0 15.0 30.3 60.0
Acetate Anaerobic Methanogenesis Ina Road sludge 17.5 34.5 93.0 12.4 25.8 35.7

water research 43 (2009) 3177–3186

H2 Anaerobic Methanogenesis Eerbeek granular sludge 815.0 >815.0 >815.0 520.0 645.0 795.0
H2 Anaerobic Methanogenesis Ina Road sludge 30.0 82.0 390.0 805.0 1005.0 1125.0
Thermophilic methanogens
Acetate Anaerobic Methanogenesis Hyperion sludge 7.2 18.1 39.2 29.5 42.6 62.9
H2 Anaerobic Methanogenesis Hyperion sludge 218.6 432.6 >600.0 >600.0 >600.0 >600.0
Anaerobic propionate-utilizing microorganisms
Propionate Anaerobic Propionate degradation Eerbeek granular sludge 10.5 36.5 62.0
Anaerobic butyrate-utilizing microorganisms
Butyrate Anaerobic Butyrate degradation Eerbeek granular sludge 17.5 25.5 34.8
Denitrification
Nitrate Anoxic Nitrate reduction Eerbeek granular sludge >800.0b >800.0b >800.0b
Nitrification
Ammonium Aerobic NHþ 4 oxidation Randolph Park I 104.3 148.8 179.8
Heterotrophic aerobes
Glucose Aerobic Glucose degradation Randolph Park II >539.0c >539.0c >539.0c
Glucose fermenters
Glucose Anaerobic Glucose degradation Aviko granular sludge 150.5 >539.0 >539.0
Glucose Anaerobic Glucose degradation Enrichment culture 87.0 325.0 539.0

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.

3181
3182 water research 43 (2009) 3177–3186

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 concen-
trations 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 signifi-
cant 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
assay inoculated with the dispersed enrichment culture,
microbial growth has a minor impact on the glucose utiliza-
tion rate of the granular sludge due to the high biomass
concentrations utilized in the latter assay (1.5 g VSS/L). Inhi-
bition 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 metabo-
lism 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).
3.1.4. Anaerobic propionate- and butyrate-degrading
microorganisms
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
Activity (% of control)

biological wastewater treatment. However, fluoride in

soils (up to 3,700 mg/kg) has been reported to affect nitrate 80
reduction, but the inhibitory effect varied considerably
depending on the soil characteristics (Ottow and Kottas, 1984; 60
Wilke, 1987).
These results suggest that biological processes for the 40
removal of nitrogen from wastewater are not expected to
be affected by fluoride unless the concentrations are exceed- 20
ingly high.

0
3.1.2. Aerobic heterotrophs 0 100 200 300 400 500 600
Sodium fluoride did not display any inhibitory effect towards Fluoride (mg/l)
glucose-utilizing bacteria in aerobic sewage sludge at fluoride
concentrations of up to 540 mg/L (Fig. 3A). In agreement with B
our results, the only other study assessing the impact of 100

fluoride (50–100 mg/L) on the removal of organic matter

Activity (% of control)

(ethanol and acetate) in the activated sludge process reported 80

no significant effect on cell growth and chemical oxygen
demand (COD) removal efficiency (Singh and Kar, 1989). In the 60
latter study, the presence of fluoride resulted in deterioration
of the sludge settling properties as indicated by a 100 to 200%
40
increase of the sludge volume index. The mechanisms
contributing to the deterioration of the sludge settling ability
20
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 0
0 100 200 300 400 500
et al., 2001; Cao and Doyle, 2002; Marquis et al., 2003).
Fluoride (mg/l)

3.1.3. Glucose fermenting bacteria
Fluoride was inhibitory to glucose-fermenting microorgan-
isms 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
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.

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 waste-
water 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
A 100
80
Activity (% of Control)
60
40
20
0 ature reports concerned with the inhibitory effects of fluoride
0 100 200 300
Fluoride (mg/l)
B 100
80
Activity (% of Control)
60
40
20
0
0 100 200 300
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.
water research 43 (2009) 3177–3186 3183
the first substrate feeding were 34.5 and 18.1 mg/L, respec-
tively. Mesophilic acetoclastic methanogens in granular
sludge appeared to be more tolerant to fluoride as demon-
strated 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 limi-
tations can impact the transport of solutes in anaerobic bio-
films (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 incu-
bation 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 hydro-
genotrophic methanogens with incubation time are unclear.
To the best of our knowledge, there are no previous liter-
400 500 600 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 meth-
anogenic 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.
400 500 600
The mechanisms responsible for the microbial toxicity of
fluoride towards acetate, propionate and butyrate-utilizing
microorganisms in anaerobic reactors are not well under-
stood. 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
3184 water research 43 (2009) 3177–3186

A C
100
120

Activity (% of control)

Activity (% of control)
80 100

60 80

60
40
40
20
20

0 0
0 50 100 150 200 250 300 0 250 500 750 1000 1250 1500
Flouride (mg/l) Fluoride (mg/l)

B D 100
100
Activity (% of control)

Activity (% of control)
80
80
60
60
40
40

20 20

0 0
0 50 100 150 200 250 300 0 100 200 300 400 500 600 700

Flouride (mg/l) Fluoride (mg/l)

Fig. 5 – Inhibitory effect of fluoride on the specific acetoclastic and hydrogenotrophic activity of mesophilic anaerobically-
digested 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 hydro-
genotrophic 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, Meth-
anococcus jannaschii, have shown that prior exposure to
specific metallic ions (e.g., Mn2þ or Co2þ) increased the toler-
ance 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 meth-
anogenic trophic groups to fluoride, suggesting the possibility
that microbial methylation of fluoride might have occurred
leading to the formation of toxic CH3F. However, this biotrans-
formation 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 concentra-
tions 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 inhibi-
tion by fluoride indicates that measures to reduce the
concentration of this ion (e.g., pretreatment of the wastewater
 water research 43 (2009) 3177–3186
using ion exchange or precipitation with calcium(II), waste-
water dilution, etc.) may be required to prevent microbial
inhibition during the anaerobic treatment of fluoride-con-
taining 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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