REVIEW PAPER

Biotransformation of aromatic compoundsfrom wastewaters containing N and/or S, by nitrification/denitrification: a review

Ricardo Beristain-Cardoso • Anne-Claire Texier •

Elıas Razo-Flores • Ramon Mendez-Pampın •

Jorge Gomez

Published online: 13 October 2009

� Springer Science+Business Media B.V. 2009

Abstract This review presents progress made over

the last decades in the understanding of the metabolic

capabilities of nitrifying and denitrifying microor-

ganisms for the biotransformation of nitrogen, sulfur,

and carbon compounds present in wastewaters. There

are nowadays still many discoveries to be made about

the metabolism, phylogeny and ecological behavior

of bacteria that play an important role in the nitrogen

cycle. The interest of the scientific community in the

biological nitrogen cycle is at present very high,

because it can be linked to either sulfur or carbon

cycles. The connection of biological cycles is of the

utmost technological relevance as it has allowed

the simultaneous elimination of reduced sulfur and

phenolic compounds under nitrifying or denitrifying

conditions. The environmental factors affecting the

nitrification and denitrification biological processes

are described in this review.

Keywords Nitrification � Denitrification �Litho-organotrophic � Phenolic compounds �Sulfide

1 Introduction

Water contamination by carbon-, nitrogen- and sulfur-

containing compounds is a serious environmental

problem. Some industrial wastewaters as those from

the chemical and petrochemical industry (spent

caustic and sour water) represent a challenge for the

treatment before discharge because of their chemical

complexity. These effluents may present high concen-

trations of organic and/or phenolic compounds,

ammonia and sulfide (Olmos et al. 2004). There are

evidences suggesting that phenolic compounds are

toxic, carcinogenic and mutagenic (Autenrieth et al.

1991). The majority of the aromatic compounds

(phenol, cresols, xylene, toluene, etc.) can be used as

carbon and energy sources by microorganisms (Far-

hadian et al. 2008; Van Schie and Young 2000; Wilson

and Bouwer 1997). Thus, the microbiological removal

of these compounds is an essential contribution to the

global carbon cycle as well as to the detoxification

of wastewaters and contaminated soils (Philipp and

Schink 2000).

R. Beristain-Cardoso � R. Mendez-Pampın

Department of Chemical Engineering, University of

Santiago de Compostela, Rua Lope Gomez de Marzoa s/n,

15782 Santiago de Compostela, Spain

R. Beristain-Cardoso (&) � A.-C. Texier � J. Gomez

Departamento de Biotecnologıa, Universidad Autonoma

Metropolitana-Iztapalapa, AP 55-535, 09340 Iztapalapa,

DF, Mexico

e-mail: beristain_3@yahoo.com.mx;

dani@xanum.uam.mx

E. Razo-Flores

Division de Ciencias Ambientales, Instituto Potosıno de

Investigacion Cientıfica y Tecnologica, Camino a la Presa

San Jose No. 2055, Col. Lomas 4a. Seccion, 78216 San

Luis Potosı, SLP, Mexico

123

Rev Environ Sci Biotechnol (2009) 8:325–342

DOI 10.1007/s11157-009-9172-0

The increase of anthropogenic activities has con-

tributed to local unbalances in the natural sulfur cycle,

leading to several serious environmental problems,

including acid rain, odor nuisance from polluted

rivers, landfills or treatment systems, corrosion, heavy

metal and sulfuric acid release from oxygen exposed

mineral ores and soils (Zhang et al. 2008a). Industrial

wastewaters containing sulfur compounds also con-

tribute to the sulfur unbalance (Colleran et al. 1995;

Lens et al. 1998). Sulfide containing waste streams are

generally treated by chemical methods, which involve

high chemical and disposal costs (Cadena and Peters

1988). The ability of autotrophic bacteria to oxidize

sulfide has led to the development of biotechnolog-

ical methods to eliminate sulfide from wastewaters

(Cardoso et al. 2006; Kim et al. 1990; Kleerebezem

and Mendez 2002; Manconi et al. 2007).

On the other hand, wastewater streams containing

nitrogen compounds may cause serious environmen-

tal problems if these compounds are not properly

eliminated before discharge into the receiving water

bodies. A too high nitrogen concentration in the

receiving waters can lead to eutrophication, hypoxia

and loss of biodiversity and habitat (Galloway et al.

2003; Mussati et al. 2002). Nitrification and denitri-

fication are biological processes involved in many

engineering applications for nitrogen removal from

wastewater and groundwater (Hiscock et al. 1991).

There are nowadays still many discoveries to be

made about the metabolism, phylogeny and ecolog-

ical behavior of bacteria that play an important role in

the nitrogen cycle (Francis et al. 2007). The present

interest of the scientific community in the biological

nitrogen cycle is high, because it can be frequently

linked to either sulfur or carbon cycles. In the last

decades, significant increase in the knowledge on

metabolic abilities of nitrifying and denitrifying

bacteria to eliminate simultaneously nitrogen, carbon,

and sulfur compounds has been made. This work

reviews the current knowledge of biological removal

of nitrogen, sulfur, and phenolic compounds by

nitrification and denitrification and describes the

environmental factors that affect these microbial

processes. The importance of carrying out a realistic

evaluation of the final products generated by the

biological processes for the development of environ-

mentally acceptable water treatment processes is

emphasized.

2 Biotransformation of nitrogen compounds

The nitrogen cycle is composed of six major biolog-

ically mediated processes that control the redox

state of nitrogen (Fig. 1) which are: nitrogen fixation,

dissimilatory nitrate reduction to ammonia (DNRA),

ammonification, anaerobic ammonium oxidation

(anammox), nitrification, and denitrification. The

major redox states and nitrogen compounds involved

are -3, -1, 0, ?1, ?2, ?3, ?5 for ammonia (NH3),

hydroxylamine (NH2OH), molecular nitrogen (N2),

nitrous oxide (N2O), nitric oxide (NO), nitrite (NO2-),

and nitrate (NO3-), respectively. Nitrogen fixation,

ammonification, DNRA, anammox and denitrification

are reductive processes while nitrification is an oxida-

tive process. As three of the compounds involved,

Fig. 1 The biogeochemical

cycling of nitrogen. DNRA:

dissimilatory nitrate

reduction to ammonia

326 Rev Environ Sci Biotechnol (2009) 8:325–342

123

ammonia, nitrite, and nitrate, can be taken up for

biological use in proteins and nucleotides, the bio-

chemical capability of the organisms to transform N2

into these chemical forms is vital to life on earth.

In order to protect natural water bodies from

eutrophication, stringent nutrient level is set for the

effluents from the wastewater treatment plants.

Because biological nitrogen removal can be effective

and less expensive, it has been widely adopted

instead of the physical–chemical processes (EPA

1993). Various novel biological nitrogen removal

processes, such as short-cut nitrification and denitri-

fication, anaerobic ammonium oxidation (ANAM-

MOX), completely autotrophic nitrogen removal over

nitrite (CANON) and oxygen-limited autotrophic

nitrification–denitrification (OLAND), have been

proposed as alternatives to the traditional nitrification

and denitrification via nitrate process (Dapena-Mora

et al. 2007; Siegrist et al. 2008; Verstraete and Philips

1998; Volcke et al. 2007). However, recent studies

indicate that nitrification and denitrification processes

have potential for the simultaneous removal of

nitrogen-, carbon-, and sulfur-containing contami-

nants from wastewaters. Thus, these traditional

processes are still of attracting interest due to their

potential use in the treatment of wastewaters with

complex chemical composition.

2.1 Nitrification process

The oxidation of ammonium and nitrite plays a key role

in generating a source of nitrate for denitrifying

bacteria. The coupling of this oxic process (nitrifica-

tion) with an anoxic process (denitrification) leads to

the releasing of nitrous oxide and/or molecular nitro-

gen to the atmosphere (Herbert 1999). The nitrification

is an aerobic respiratory process carried out by two

groups of gram-negative chemolithoautotrophic bac-

teria, phylogenetically unrelated to each other, that are

the ammonium oxidizing and the nitrite oxidizing

bacteria and belong to the Nitrobacteraceae family

(Prosser 1989). The ammonium oxidizing bacteria are

species of the following genera: Nitrosomonas, Nitr-

osospira, Nitrosolobus, Nitrosococcus, and Nitroso-

vibrio, being Nitrosomonas the genus better studied. In

the case of the nitrite oxidizing bacteria the following

genera Nitrobacter, Nitrospina, Nitrococcus, and

Nitrospira, have been found. The genus Nitrobacter

is the better studied (Bock et al. 1991).

In the first step of nitrification, ammonia oxidizing

bacteria oxidize ammonia to nitrite according to

Eq. 1. In the second step (Eq. 2), nitrite is oxidized to

nitrate by the nitrite oxidizing bacteria.

NHþ4 þ 1:5O2 ! NO�2 þ 2Hþ þ H2O

� 274:7 kJ=reactionð1Þ

NO�2 þ 0:5O2 ! NO�3 � 74:1 kJ=reaction ð2Þ

The ammonia oxidation process is mediated by

two enzymes, the ammonia monooxygenase (AMO),

which catalyzes the oxidation of ammonia to hydrox-

ylamine (NH2OH) (Eq. 3), and the hydroxylamine

oxidoreductase (HAO), which catalyzes the oxidation

of NH2OH to nitrite (Eq. 4) (Arp et al. 2002; Fiencke

and Bock 2006; Schmidt et al. 2003).

NHþ4 þ 0:5O2 ! NH2OHþ Hþ � 8:2 kJ=reaction

ð3Þ

NH2OHþ O2 ! NO�2 þ Hþ þ H2O

� 266:5 kJ=reactionð4Þ

As shown by the free energy change (DGo0) values,

the oxidation of NH2OH to NO2- is the main step

where the ammonium oxidizing bacteria obtain

energy. According to the low DGo0 value for the

ammonia oxidation to nitrite process (Eq. 1), it is

possible to predict that the ATP production will be low.

Thus, the growth of the ammonium oxidizing bacteria

will be also very low, because cellular biosynthesis is

limited by the energy availability. Moreover, doubling

times for the ammonium oxidizing species vary

between 7 and 24-h (Bock et al. 1991).

Nitrite oxidation to nitrate (Eq. 2) is catalyzed by

an enzymatic complex named nitrite oxidoreductase

(NOR). As the free energy change is lower for nitrite

oxidation than for ammonia oxidation, it is predict-

able that the nitrite oxidizing bacteria would grow

less than the ammonium oxidizing bacteria. Like-

wise, the doubling time for different species of

Nitrobacter could be longer and varies between 10

and 140-h (Bock et al. 1991). According to these, the

limiting step for nitrification is the nitrite oxidation

process, and then the substrate rate consumption is

determined by:

�dS=dt ¼ qsX ð5Þ

where qs is de specific rate consumption and X the

biomass concentration, which is scarcely produced.

Rev Environ Sci Biotechnol (2009) 8:325–342 327

123

In Table 1 an overview of the main kinetic charac-

teristics of both types of nitrifying microorganisms

and heterotrophic bacteria is presented. Values

reported for all these parameters can vary signifi-

cantly, depending on the environmental conditions

such as influent concentrations, temperature, pH, etc.

2.1.1 Organic compounds oxidation under nitrifying

conditions

The inhibitory effect of organic compounds on

nitrification is well documented, and it is known that

the stability of nitrifying systems in wastewater

treatment can be altered by the presence of toxic and

inhibitory compounds (Schweighofer et al. 1996). In

order to understand the negative effects of organic

matter on nitrification, numerous studies have been

developed with axenic cultures and nitrifying consor-

tia. Some works focused on the growth and inhibition

of nitrifying bacteria, others on the inhibitory effects

on the nitrifying respiratory process (Gomez et al.

2000; Leu et al. 1998) or on AMO enzyme activity

(Keener and Arp 1993; McCarty 1999). It has been

observed that the organic matter affects nitrification in

different magnitude, depending on the chemical

structure of the organic compound, their concentration

and their chemical properties such as hydrophobicity.

Gomez et al. (2000) evaluated the effect of ethanol,

acetate, propionate, and butyrate over nitrification, in

batch cultures. At a concentration of 500 mg l-1, the

nitrification rate of inhibition was different for each

compound in which propionate and butyrate were

the most inhibitory. The authors suggested that the

different degrees of inhibition on the nitrification

process were related to the type of organic matter

added. In microbial consortia, the competition

between heterotrophs and autotrophs for ammonia

and oxygen is another hypothesis commonly men-

tioned for explaining the nitrification inhibition by

organic matter (Hanaki et al. 1990). Okabe et al.

(1996) suggested that the presence of organic matter

under nitrifying conditions induce a competition

between the autotrophs and heterotrophs for dissolved

oxygen, ammonia and space in the aerobic granules,

affecting the nitrification process. Additionally, the

type of culture (axenic or consortium), the origin of the

sludge and solid retention time (SRT) also play a key

role. In spite of the inhibitory effects of organic

compounds on nitrification, it was demonstrated that

in some cases and under controlled experimental

conditions, nitrification processes could successfully

proceed (Texier and Gomez 2007).

Table 1 Some important kinetic parameters of the ammonia oxidizing bacteria (XAOB), nitrite oxidizing bacteria (XNOB), and

heterotrophic bacteria (XHB). (Modified from Pynaert (2003))

Kinetic parameter XAOB XNOB XHB

Maximum specific growth rate (lmax = day-1) 0.3–2.2 0.2–2.5 0.2–7.2a

Biomass yield (Y = g VSS g N-1) 0.04–0.13 0.02–0.08 0.071–0.12b

Maximum specific ammonia-oxidizing activity (g NH4?-N g VSS-1 day-1) 0.079c – –

Maximum specific nitrite-oxidizing activity (g NO2--N g VSS-1 day-1) – 0.082c –

Half saturation constant for ammonia (KNH3 = mg NH3 l-1) 0.06–27.5 – –

Half saturation constant for nitrite (KNO2- = mg NO2

- l-1) – 0.1–15 –

Half saturation constant for oxygen (Ko2= mg O2 l-1) 0.03–1.3 0.3–2.5 0.08f

Inhibition coefficient of ammonium (Ki = mg NH4?-N l-1) 3300c – –

Inhibition coefficient of nitrite (Ki = mg NO2--N l-1) – 325–1400d –

Temperature range (�C) 4.0–42 4–46 5–35e

pH range 4.5–8.5 4.5–9 7–8.2e

a Henze et al. (1996); Hellinga et al. (1999)b Value recalculated considering that biomass has 10% of nitrogen, Rittmann and McCarty (2001)c Carvallo et al. (2002)d Collins et al. (1988); Wett et al. (1998); Carvallo et al. (2002)e Knowles (1982); Lalucat et al. (2006)f Hellinga et al. (1999)

328 Rev Environ Sci Biotechnol (2009) 8:325–342

123

An interesting aspect of the nitrification is that

microbial consortia under nitrifying conditions have

been shown to be able to oxidize simultaneously

ammonia and organic compounds. Cultures with

Nitrosomonas europaea have shown that the enzyme

AMO would be involved in the oxidation of a broad

range of hydrocarbons, including aromatic substances

(Keener and Arp 1994; McCarty 1999). However, in

batch cultures with N. europaea, the oxidation of

aromatic compounds was only partial. No evidence

for ring fission of aromatics by N. europaea was

obtained and aromatic intermediates accumulation in

the culture medium was observed (Keener and Arp

1994). Regarding this, Hyman et al. (1985) reported

that benzene was oxidized to phenol and subse-

quently to hydroquinone by N. europaea. In these

studies, performed in axenic cultures, both interac-

tions and diversity of the bacteria present in microbial

consortia used in wastewater treatment systems were

ignored. Current understanding on the mechanisms of

organic compound degradation and on the involve-

ment of AMO is generally scarce, due to the active

form of AMO have not been isolated. Using a

nitrifying consortium, Zepeda et al. (2003) showed

that benzene was first oxidized to phenol, which was

later oxidized to acetate. The authors suggested that

benzene oxidation with ring fission could have been

possible due to the coexistence and participation of

lithoautotrophic nitrifying bacteria and organo-het-

erotrophic microorganisms in the consortium. These

results have suggested that nitrifying consortium

coupled with a denitrification system may have

promising applications for complete removal of

nitrogen and aromatic compounds from wastewaters.

On the other hand, batch studies have shown that

nitrifying consortium could simultaneously oxidize

ammonia to nitrate and aromatic compounds (tolu-

ene, m-xylene, and p-cresol) to volatile fatty acids

and CO2 (Texier and Gomez 2002; Zepeda et al.

2006, 2007). Yamagishi et al. (2001) reported a

nitrification rate of 200 mg NH4?-N l-1 day-1 in an

activated sludge process where simultaneously phe-

nol was completely removed when the biomass used

for inoculum had been acclimated with phenol for

long time. Amor et al. (2005) evaluated the phenol

oxidation and its effect on the nitrifying process in

a continuous activated sludge reactor. At loading

rates of 14–1,120 mg phenol l-1 day-1 and 140 mg

NH4?-N l-1 day-1, high consumption efficiencies

were obtained for both contaminants. However, the

nitrifying yield severely decreased for phenol loading

rates higher than 600 mg l-1 day-1. It must be noted

that in the majority of the studies with activated

sludge processes, the sludge has not been previously

stabilized under steady-state nitrification. Moreover,

experimental conditions used are generally more

suitable for heterotrophs than for nitrifiers. This can

favor the competition between heterotrophs and

nitrifiers for ammonium and oxygen and contribute

to the instability of nitrifying processes by the

presence of organic matter. In this case, a predictable

decrease in the nitrifying yield due to assimilation of

ammonium by heterotrophs can be observed. In

contrast, Texier and Gomez (2007) used a sludge

produced in steady-state nitrification for inoculating a

sequencing batch reactor (SBR). This reactor was

operated under controlled experimental conditions

specifically favorable for the stabilization of the

nitrifying respiratory process. The nitrifying SBR

operated up to 300 mg p-cresol l-1 day-1, achieving

simultaneously the complete ammonium oxidation

(200 mg NH4?-N l-1 day-1) to nitrate and the com-

plete oxidation of p-cresol. Under these experimental

conditions, microbial growth was low as the process

was clearly dissimilative, and the sludge presented

good settling properties. These results showed that

nitrification as the initial step in the removal of

ammonia from wastewaters might be also used to

oxidize simultaneously ammonia and aromatic com-

pounds, allowing their mineralization or the produc-

tion of intermediates that can be completely oxidized

by denitrification.

2.1.2 Technological applications

Removal of ammonium by biological nitrification,

using activated sludge system, is a process that is

widely used in the treatment of domestic and

industrial wastewater. Unfortunately, the kinetic of

nitrification is slower and more susceptible to envi-

ronmental conditions than organic matter oxidation

by heterotrophs. Generally, simultaneous growth of

nitrifiers and heterotrophs in a single reactor leads to

low nitrification specific rates due to overwhelming

action of heterotrophs, when treating municipal and

industrial wastewaters with a high C/N ratio. In these

cases, the denitrifying reactor can utilize most part

of the organic matter and reduce the influence of

Rev Environ Sci Biotechnol (2009) 8:325–342 329

123

heterotrophs on nitrification. The main problems in

maintaining high nitrification efficiency when treat-

ing low C/N wastewaters are the changes in influent

concentration and flow, which may also affect to the

dissolved oxygen level in the reactor, and pH due to

fluctuating industrial operations (Campos et al. 2007).

Another inconvenient in treating low C/N wastewater

is the necessity to add external organic matter

(methanol, acetic acid, etc.) in order to complete

the denitrification, and the cost treatment is increased.

The combination of nitrification–denitrification for

removing ammonium is favorable when the waste-

water contains a high C/N ratio.

The low growth rate of nitrifying bacteria and the

relatively poor capacity of activated sludge units to

retain nitrifying biomass require large settlers. The

most common problem is the apparition of wash out

(Campos et al. 2000). For these reasons the activated

sludge units can not treat high nitrogen loading rates.

One of the cheapest ways to improve the sludge

retention time (SRT) is the immobilization of micro-

organism. Higher biomass concentrations and com-

pact units are achieved with immobilization. A biofilm

airlift suspension (BAS) reactor is an example of this

kind of reactors (Heijnen et al. 1990). Ammonium

loading rates of 5 g NH4?-N l-1 day-1 and biomass

concentrations of 48 g VSS l-1 were obtained in a

nitrifying BAS (Garrido et al. 1997). Campos et al.

(1999) developed in an activated sludge unit with a

high nitrifying cell density (83 g VSS l-1 particle),

allowing a high SRT and a high biomass concentration

(15 g VSS l-1), reaching a high ammonium loading

rate of 7.5 g NH4?-N l-1 day-1.

Nowadays, the connection of nitrifying and den-

itrifying processes for the treatment of complex

industrial wastewaters has been demonstrated. For

instance, Szpyrkowicz et al. (1991) showed the

simultaneous elimination of sulfide, COD, nitrogen

compounds and chrome using nitrification and deni-

trification processes. The influent was a mixture of

tannery effluent and domestic sewage treated in a

pilot plant. High consumption efficiencies (95%) of

COD, nitrogen and sulfide were obtained. Leta et al.

(2004) also obtained a successful pilot wastewater

treatment plant consisting of a predenitrification–

nitrification process. The reactor was fed with a raw

tannery, and total nitrogen and COD were consumed

up to 98%. In these experiments are not mentioned

the end products formed and special care is necessary

in this aspect, since intermediaries formed could be

more toxic than the original contaminants tested.

Nonetheless, these results showed the potential

application of nitrification coupled to the denitrifica-

tion process for the treatment of complex industrial

wastewaters.

2.2 Denitrification process

Denitrification is the reduction of oxidized nitrogen

compounds like nitrite or nitrate to molecular nitro-

gen. This biological process is performed by various

chemoorganotrophic, lithoautotrophic and photo-

trophic bacteria, and some fungi (Shoun and Tanim-

oto 1991). The enzymes necessaries for the complete

nitrate reduction have been identified in different

genera of facultative respiration, such as Paracoccus

denitrificans, Pseudomonas stutzery, Pseudomana

denitrificans, Alcaligenes faecalis, Escherichia coli,

and Thiosphaera panthotropha, among others (Bau-

mann et al. 1996). Denitrification involves four

enzymatic steps via the sequential formation of the

following intermediates: nitrite, nitric oxide, and

finally nitrous oxide (Zumft 1997). The initial step in

denitrification is catalyzed by the nitrate reductase,

which uses molybdenum as cofactor. The nitrite

reductase that reduces nitrite to nitric oxide can be a

copper enzyme or a cytochrome cd1 with the Hem

group as cofactor (Moura and Moura 2001). The

nitric oxide reductase is also known as cytochrome

bc1 with Heme groups and Fe no-Heme. The last step

of denitrification is catalyzed by the nitrous oxide

reductase, a copper containing metalloenzyme. The

presence of these metallic microelements inside the

wastewater is essential in order to have an efficient

denitrifying process.

The denitrification process can be organotrophic or

lithotrophic depending on the energy source. Easy

consumption compounds, such as methanol, acetate,

ethanol, lactate, and glucose, can serve as electron

donors for the organotrophic denitrification (Akunna

et al. 1993; Cuervo-Lopez et al. 1999; Grabinska-

Loniewska 1991; Tam et al. 1992). Nonetheless, the

kinetic of organotrophic denitrification is strongly

influenced for several factors such as chemical

structure of organic compound, initial concentration

of substrate, origin of the sludge, C/N ratio, HRT,

temperature, pH, etc., as it can be seen in Table 2.

Chemical compounds like aromatic or phenolic

330 Rev Environ Sci Biotechnol (2009) 8:325–342

123

compounds can also be used as energy source (Meza-

Escalante et al. 2007; Sierra-Alvarez et al. 2007). Just

as the organotrophic denitrification allows the simul-

taneous elimination of nitrate and organic com-

pounds, the lithotrophic denitrification can eliminate

simultaneously nitrate and reduced inorganic sulfur

compounds such as sulfide, thiosulfate, and elemental

sulfur (Cardoso et al. 2006; Sierra-Alvarez et al.

2007; Zhang et al. 2008b). Nevertheless, it is possible

to have a litho-organotrophic denitrification where

both organic and inorganic compounds are used as

energy sources (Beristain-Cardoso et al. 2008; Meza-

Escalante et al. 2007; Oh et al. 2001; Reyes-Avila

et al. 2004). Electrons originated from organic matter

and reduced sulfur compounds oxidation are trans-

ferred to nitrate instead of oxygen in order to build up

a proton motive force usable for ATP regeneration

(Schmidt et al. 2003). The quantity of energy

produced by means of denitrification depends on

the type of electron donor. For instance, the stoichi-

ometric expressions for the oxidation of acetate and

sulfide under denitrifying conditions are shown in

Eqs. 6 and 7 with their respective DG�0 values. It can

be seen that both processes are exergonic reactions,

but organotrophic denitrification is more spontaneous

than lithotrophic denitrification.

1:25CH3COOHþ 2NO�3 ! 2:5CO2 þ N2 þ 1:5H2O

þ 2OH�

� 1054:8 kJ=reaction

ð6Þ

S2� þ 1:6NO�3 þ 1:6Hþ ! SO2�4 þ 0:8N2 þ 0:8H2O

� 743:9 kJ=reaction

ð7ÞThe denitrification process can be affected by

several factors, leading to the formation of undesirable

end products as NO2- and N2O. The main factors

affecting the accumulation of intermediates in deni-

trification could be: oxygen concentration, C/N and S/

N molar ratios, nitrite concentration and pH (Cervan-

tes et al. 1998; Hong et al. 1994; Thomsen et al. 1994).

Hernandez and Rowe (1988) demonstrated that oxy-

gen inhibits nitrate uptake instead of nitrate reduction.

Nitrate transport by whole cell suspensions was

completely and reversibly inhibited, whereas nitrate

reduction by cell-free extracts was not affected by

oxygen or was only partially inhibited in some cases.

Bonin et al. (1989) observed that the enzymes

associated with denitrification were affected differ-

ently with respect to oxygen concentration. Nitrate

reductase was less sensitive towards oxygen than

nitrite and nitrous oxide reductases, while nitrate

Table 2 Denitrification specific rates achieved with various organic carbon source

Carbon source Specific denitrification

rate (mg NO3- -N g

VSS-1 day-1)

pH T�C Reference

Methanol 0.130 Nm 23 ± 3 Bilanovic et al. (1999)

Acetate 0.475

Effluent from

anaerobic digestion

0.486

Acetate 1,900 8.3 ± 0.2 30 Reyes-Avila et al. (2004)

Acetic acid 35 6.5 30 Elefsiniotis and Li (2006)

Propionic acid 24

Mixed VFAs 42

Acetate 112.8 7.3 20 Rodriguez et al. (2007)

Urban sewage 103.2

Winery 2

p-cresol 129 ± 5.6 7.2 ± 0.1 30 Meza-Escalante et al. (2007)

Methanol 30.4 9 20 ± 1 Fernandez-Nava et al. (2008)

Phenol 86 ± 2 7 30 Beristain-Cardoso et al. (2009b)

Nm not mentioned

Rev Environ Sci Biotechnol (2009) 8:325–342 331

123

reductase was inhibited at an oxygen concentration

greater than 4.05 mg l-1, compared with 2.15 and

0.25 mg l-1 for nitrite and nitrous oxide reductases,

respectively. The accumulation of nitrite during

denitrification can inhibit the nitrate consumption.

For instance, Almeida et al. (1994) showed that the

denitrification process was inhibited by nitrite con-

centration of 66 lg N l-1. According to Sijbesma

et al. (1996) nitrite acts as a protonophore, an

uncoupler that increases the proton permeability of

membranes by a shuttling mechanism. The effect of

NO and N2O on denitrification process has not been

reported. Other factor affecting the metabolism of

denitrification is the C/N or S/N ratio. The C/N ratio

can modify the denitrifying metabolism, being dis-

similative (when the end product is N2), dissimilatory

nitrate reduction to ammonium or assimilatory nitrate

reduction to ammonium, which ammonium is incor-

porated into the cell (Akunna et al. 1994; Cervantes

et al. 2001; Philips et al. 2002). The S/N ratio has an

important effect on the outcome of sulfo-oxidation

under denitrifying conditions, being the end product

sulfate or elemental sulfur (Cardoso et al. 2006).

Regarding to pH, if it is not controlled above pH 6

the end product could be NO instead of N2 (Wicht

1996). The pH presents a major effect on the activity

of nitrite reductase and nitric oxide reductase (Wu

et al. 1995).

2.2.1 Lithotrophic denitrification

Several chemolithoautotrophic bacteria have the

metabolic capability to anaerobically oxidize reduced

inorganic sulfur compounds, such as sulfide (S2-),

elemental sulfur (S0), thiosulfate (S2O32-), or sulfite

(SO32-), at the expense of the reduction of nitrate or

other oxidized nitrogen compounds such as NO2-

and N2O (Kuenen et al. 1992). The chemolithoauto-

trophic bacteria with these properties are Thiobacillus

denitrificans and Thiomicrospira denitrificans, which

are restricted to an autotrophic mode of growth, as

they use CO2 as only carbon source. While Thioba-

cillus versatus, Thiobacillus thyasiris, Thiosphaera

pantotropha, and Paracoccus denitrificans are facul-

tative chemolithoautotrophics, which present a mixo-

trophic metabolism (Kuenen et al. 1992; Chazal and

Lens 2000). Sulfur utilizing chemolithoautotrophic

denitrifiers play an important role in mineral cycling

by linking sulfur and nitrogen cycles (Cardoso et al.

2006; Korom 1992; Krishnakumar and Manilal 1999;

Sierra-Alvarez et al. 2007). The well known auto-

trophic Thiobacillus denitrificans is distinguished

from all other Thiobacillus species by its ability to

grow as facultative anaerobic chemolithotroph, cou-

pling the oxidation of inorganic sulfur compounds to

the reduction of nitrate, nitrite and other oxidized

nitrogen compounds to molecular nitrogen (Kelly and

Wood 2000). Nowadays, the metabolic pathway of

sulfide oxidation under denitrifying conditions is not

well defined. Visser et al. (1997b) proposed a

hypothetical metabolic pathway of sulfide oxidation

under oxic conditions in Thiobacillus (Fig. 2). It is

proposed that sulfide is oxidized to sulfate via

intermediaries sulfur and sulfite. Electrons enter to

the respiratory chain at the level of cytochrome c and

are coupled to oxygen via a cbb3-type oxidase

(Visser et al. 1997a). Stoichiometry of sulfide oxida-

tion under denitrifying conditions is shown in Eqs. 8

to 11.

S2� þ 1:6NO�3 þ 1:6Hþ ! SO2�4 þ 0:8N2 þ 0:8H2O

DG�0 ¼ �743:9 kJ=reaction ð8Þ

S2� þ 0:4NO�3 þ 2:4Hþ ! S0 þ 0:2N2 þ 1:2H2O

DG�0 ¼ �191:0 kJ=reaction ð9Þ

S2� þ 4NO�3 ! SO2�4 þ 4NO�2

DG�0 ¼ �501:4 kJ=reactionð10Þ

S2� þ NO�3 þ 2Hþ ! S0 þ NO�2 þ H2O

DG�0 ¼ �130:4 kJ=reactionð11Þ

As shown by Eqs. 8 and 9, conversion to elemental

sulfur coupled to complete denitrification consumes

four times less nitrate as compared to complete

oxidation to sulfate. All reactions are exergonic.

However, Eq. 9 is significantly less exergonic than

Fig. 2 Schematic representation of the sulfide-metabolizing

pathway in obligate autotrophic Thiobacillus (Visser et al.,

1997a)

332 Rev Environ Sci Biotechnol (2009) 8:325–342

123

Eq. 8. When denitrification is incomplete (Eqs. 10

and 11), the reactions are less spontaneous.

Oxidation of sulfide by chemolithoautotrophic

denitrifying bacteria can lead to the formation of

elemental sulfur or sulfate, depending on the envi-

ronmental conditions (Beristain-Cardoso et al. 2008;

Cardoso et al. 2006; Krishnakumar et al. 2005; Wang

et al. 2005). Wang et al. (2005) observed that

elemental sulfur production was obtained at sulfide/

nitrate molar ratio in the range of 1.66–2.5 and sulfide

concentrations less than 300 mg l-1. Cardoso et al.

(2006) showed the possibility of controlling the fate

of sulfide oxidation either elemental sulfur or sulfate

by manipulating the nitrate/sulfide ratio in the culture

medium. A sub stoichiometric dose of nitrate could

be used to promote partial oxidation to elemental

sulfur.

2.2.1.1 Respiration and metabolism of Thiobacillus

denitrificans Thiobacillus denitrificans was one of

the first non filamentous bacteria described to be able

to grow with inorganic sulfur compounds as sole

energy source (Kelly and Wood 2000; Kelly et al.

2005). Th. denitrificans has the metabolic ability to

obtain energy from the oxidation of reduced

inorganic sulfur compounds under either aerobic or

denitrifying conditions. Beller et al. (2006) presented

the complete genome of T. denitrificans strain ATCC

25259. Th. denitrificans encodes all the necessary

enzymatic machinery for aerobic respiration and has

all necessary genes encoding the four essential

enzymes that catalyze denitrification, allowing it to

survive under a wide range of redox conditions

(Pereira and Teixeira 2004; Pitcher and Watmough

2004). Genes for all the enzymes of the Krebs

tricarboxylic acid cycle were also identified in the

genome of Th. denitrificans strain ATCC 25259.

It has been shown that the presence of organic

matter may no affect the metabolism of Th. denitrif-

icans. For instance, Sublette and Woolsey (1988)

showed that the H2S oxidation by this species was not

affected by the presence of glutaraldehyde. Beristain-

Cardoso et al. (2009a) through a 16S rRNA gene-

based microbial community analysis have reported

that Th. denitrificans was present in the denitrifying

biofilm from an inverse fluidized reactor fed with

sulfide and organic matter. These researches showed

the extensive enzymatic capabilities and advantages

that this strain could have for wastewater treatment.

Another microorganism of great interest is the

mixotrophic bacterium Thiosphaera panthotropha

which is able to denitrify using reduced sulfur

compounds, hydrogen or a wide range of organic

compounds as electron donors (Kuenen et al. 1992).

It can also nitrify ammonia heterotrophically to

nitrite, and reduce nitrate or nitrite to molecular

nitrogen gas irrespective of the ambient dissolved

oxygen concentration (Gupta 1997). The metabolic

capacity of this bacterium throws open interesting

possibilities for its applications in wastewater

treatment.

2.2.1.2 Technological applications The use of Th.

denitrificans or microbial denitrifying consortia is

of interest for environmental technology because

they can oxidize sulfide and other reduced sulfur

compounds in the absence of oxygen. The use of

nitrate to control sulfide corrosion and odors in sewer

systems has been known for many years and continues

to be of commercial interest (Bentzen et al. 1995).

More recently, the addition of nitrate to sulfide-laden

oil field brines was also shown to be an effective

method to enhance the biological elimination of

sulfide and reduce problems associated with their

toxicity, corrosivity and negative impact on reservoir

permeability (Jenneman et al. 1999; Reinsel et al.

1996). Litoautotrophic denitrification has been also

proposed for H2S removal from biogas (Kleerebezem

and Mendez 2002). The concept has been investigated

for industrial wastewater treatment (Gommers et al.

1988; Reyes-Avila et al. 2004).

Immobilized and free cells of Thiobacillus deni-

trificans have been used for inoculating bioreactors in

order to get high efficiency of sulfide consumption

(Ma et al. 2006; Manconi et al. 2007). Ma et al. (2006)

immobilized Th. denitrificans on granular activated

carbon (GAC). The GAC bioreactor achieved 97%

removal efficiency for sulfide at concentrations from

110 to 120 mg l-1. Zhang et al. (2008b) observed that

Th. denitrificans cells immobilized on polyvinyl

alcohol exhibited faster denitrification and thiosulfate

consumption compared with the control reactor with

free cells.

Biotransformation of sulfide to elemental sulfur

offers interesting opportunities for the removal of this

compound, as the elemental sulfur has very low

solubility in water and can be physically removed

from effluents for reuse (Celis-Garcıa et al. 2008;

Rev Environ Sci Biotechnol (2009) 8:325–342 333

123

Gonzalez et al. 2005; Krishnakumar et al. 2005).

Alternatively, hydrogen sulfide can be oxidized to

sulfate for discharge where sulfate is environmentally

benign (e.g., marine environment). Numerous pro-

cesses have been based on the use of S0 for the

autotrophic denitrification of drinking water (Darbi

et al. 2003; Sierra-Alvarez et al. 2007; Van der Hoek

et al. 1992) or wastewater (Am et al. 2005; Gommers

et al. 1988; Nugroho et al. 2002) due to the high

efficiencies of nitrate consumption that can be

obtained.

2.2.2 Organotrophic denitrification

Organotrophic denitrification processes are the most

studied and most widely applied in the field. While the

nature of the organic compounds may affect the

biomass yield, the choice is generally based on

economic considerations (Soares 2000). Organotro-

phic denitrification is very efficient in terms of nitrate

removal (Flere and Zhang 1999; Zhang and Lampe

1999). However, when organic carbon in the waste-

water is insufficient compared to the nitrogen content,

expensive chemicals, like methanol or similar organic

compounds, must be added. Methanol is the least

expensive of the simple carbon sources, but its use in

the treatment of potable water is not permitted in some

countries (Soares 2000). The organotrophic denitrifi-

cation can be a high rate biological process. For

instance, Cuervo-Lopez et al. (1999) reported an

efficient and a high rate denitrifying process in

presence of acetate and a nitrate loading rate of 2 kg

NO3--N m-3 day-1. In the steady state the nitrate

removal efficiency was 100%, with a denitrifying

yield (Y-N2; g N2 g-1 NO3--N consumed) of 0.9.

Bernet et al. (1996) and Chen et al. (1996) also applied

high nitrate loading rates (above 2.1 kg NO3--

N m-3 day-1), with removal efficiencies around

70%. Methane can be used for denitrification, in spite

of its low solubility. Islas et al. (2004) used methane as

electron source observing high nitrate removal effi-

ciency and a molecular nitrogen yield close to 0.9.

Organotrophic denitrifying bacteria are able to use

a wide variety of organic compounds, such as toluene

and phenolic compounds (Delanghe et al. 1994; Puig-

Grajales et al. 2003; Pena-Calva et al. 2004). Several

strains such as Azoarcus sp., Thauera aromatica

K172 and strain S100 are capable of oxidizing

phenol and phenolic compounds under denitrifying

conditions (Anders et al. 1995; Lack and Fuchs 1992;

Shinoda et al. 2000). The stoichiometric expressions

for the oxidation of acetate and phenol under

denitrifying conditions are shown in Eqs. 12 and

13. It can be seen that the free energy change is

higher with phenol than acetate.

1:25CH3COOHþ 2NO�3 ! 2:5CO2 þ N2 þ 1:5H2O

þ 2OH�

DG�0 ¼ �1054:8 kJ=reaction ð12Þ

C6H5OHþ 5:6Hþ þ 5:6NO�3 ! 6CO2 þ 5:8H2O

þ 2:8N2

DG�0 ¼ �3071:0 kJ=reaction ð13Þ

Considering the common occurrence of nitrate in

many phenolic wastewaters, degradation of some

phenolic compounds by denitrification seems to offer

an attractive option for the wastewater treatment

(Thomas et al. 2002). Under aerobic conditions,

molecular oxygen is used for destabilization and

cleavage of aromatic compounds in oxygenase reac-

tions. In the absence of oxygen, the aromatic ring is

destabilized by a reductive attack (Heider and Fuchs

1997; Schink et al. 1992). The most common and

best studied pathway in anaerobic oxidation is the

benzoyl-CoA pathway (Harwood et al. 1999). Ben-

zoyl-CoA can be regarded in Thauera aromatica as

the central intermediate in the anaerobic oxidation of

many aromatic compounds and likely also in other

microorganisms capable of consuming aromatic

compounds (Dangel et al. 1991; Harwood and Gibson

1997). Initial steps of anaerobic phenol catabolism in

the denitrifying strain T. aromatica are presented in

Fig. 3.

In practice, the parameters strongly influencing the

success of phenolic compounds degradation include

the mode of cultivation (batch, feed-batch or contin-

uous cultures), the presence or absence of other

substrate than the contaminant tested, the type and

size of the inoculum, the stabilization phase, the kind

of electron acceptor (Buitron and Capdeville 1995;

Hu et al. 1998; Razo-Flores et al. 1996; Watson 1993;

Zaidi et al. 1996) and the stoichiometry of the system.

Since the lipid membrane is the only barrier between

the bacterial cytoplasm and the outside world, an

alteration on the membrane structure can readily

cause cell death, and the toxicity correlates well with

the chemical properties of phenolic compounds as the

334 Rev Environ Sci Biotechnol (2009) 8:325–342

123

cell membrane may be the main target of these

antimicrobial agents (Heipieper et al. 1991; Van

Schie and Young 2000). There are reports of bacteria

that have developed mechanisms to resist and survive

to high phenol concentrations. An example is the

isomerization of cis-unsaturated fatty acids to the

trans-configuration, as observed for phenol degrading

Pseudomonas putida P8 (Heipieper et al. 1992). This

might be possible as the chains of trans fatty acids

molecules can align closer together in a biological

membrane than those in the cis-configuration, then a

more rigid membrane is formed (van Schie and

Young 2000).

There are some studies on phenol oxidation under

denitrifying conditions. Tschech and Fuchs (1987)

showed in batch cultures that the phenol oxidation

could be coupled to denitrification, reducing nitrate to

molecular nitrogen. The oxidation of phenol under

denitrifying conditions was also shown by Khoury

et al. (1992) in both batch and continuous cultures.

These authors did not detect organic residuals such as

fatty acids or aromatic intermediates in the batch

cultures. Nevertheless, in a continuous stirrer tank

reactor the increase in the dilution rate (0.02–

0.04 h-1) affected the denitrification process, dimin-

ishing the phenol and nitrate consumption efficiency.

Thomas et al. (2002) calculated the kinetic parame-

ters for phenol oxidation under denitrifying condi-

tions in batch culture with a mixed culture of

Alcaligenes faecalis and Enterobacter species

(8 mg l-1 of initial cell mass). The culture was

capable of consuming high concentrations of phenol

(up to 600 mg l-1). The kinetic constants, maximum

specific growth rate (lmax), inhibition constant (Ki)

and saturation constant (Ks) were determined to be

0.206 h-1, 113 and 15 mg phenol l-1, respectively,

and p-hydroxybenzoic acid was identified as an

intermediate of phenol oxidation. Puig-Grajales

et al. (2003) showed the simultaneous elimination

of phenol and 3,4-dimethylphenol (3,4 DMF) under

denitrifying conditions using an UASB reactor at

different COD/NO3--N ratios. At a COD/NO3

--N

ratio of 4.31 (240 mg l-1 of phenol and 38 mg l -1 of

3,4 DMF), the consumption efficiencies of phenol

and nitrate were of 95%, while the consumption

efficiency of 3,4 DMF was of 70%. Nitrate was

completely reduced to molecular nitrogen. It was

indicated that as nitrate was limited, it was not

enough to oxidize completely the 3,4 DMF. Under

these conditions, methane was detected (40% of

biogas produced) showing that the phenolic com-

pounds were eliminated via denitrification and meth-

anogenesis. At a COD/NO3--N ratio of 2.57, the

consumption efficiencies of phenol and 3,4 DMF

were of 100%, while the consumption efficiency of

nitrate was of 70%. The lower consumption effi-

ciency of nitrate was due to the fact that nitrate was

fed in excess. Methane was not detected under these

last conditions. These evidences showed the compe-

tition between methanogenesis and denitrification for

phenol and 3,4 DMF, however, denitrification was

favored when nitrate was present in excess as electron

acceptor.

2.2.3 Litho-organotrophic denitrification

The litho-organotrophic denitrification process con-

nects the sulfur, carbon and nitrogen cycles each

other. In the last decades it has been demonstrated

that it is possible to remove a second energy source

such as sulfide or thiosulfate in the presence of

organic matter, coupled to the nitrate reduction.

Fig. 3 Intermediates and enzymes involved in the initial steps

of anaerobic phenol metabolism in the denitrifying Pseudo-monas (Lack and Fuchs 1992). (1) and (2), phenol carboxylase

system; (3), 4-hydroxybenzoate-CoA ligase (AMP forming);

(4), 4-hydroxybenzoyl-CoA reductase (dehydroxylating); (5),

benzoyl-CoA reductase (aromatic ring reducing)

Rev Environ Sci Biotechnol (2009) 8:325–342 335

123

Ta

ble

3S

um

mar

yo

fre

sear

ches

rela

ted

tosu

lfu

r,ca

rbo

nan

dn

itro

gen

rem

ov

alb

yli

tho

-org

ano

tro

ph

icd

enit

rifi

cati

on

or

nit

rifi

cati

on

–d

enit

rifi

cati

on

pro

cess

es

Au

tho

rsB

iolo

gic

alp

roce

ss/r

eact

or

con

fig

ura

tio

n

Co

mp

ou

nd

s

(mg

l-1)

Op

erat

ion

al

con

dit

ion

s

Rem

ov

al

effi

cien

cies

(%)

Bio

log

ical

pro

cess

rate

s

En

d

pro

du

cts

Bac

teri

al

com

mu

nit

y

(Szp

yrk

ow

icz

etal

.1

99

1)

Nit

rifi

cati

on

–

den

itri

fica

tio

n/

Co

nti

nu

ou

sst

irre

d

tan

kre

acto

rs

27

74

CO

D

36

7T

KN

-N

16

9N

H4?

-N

45

S2-

32

.1C

r tota

l

Vo

l.re

acto

r:5

50

L

pH

effl

uen

t:9

.2

Flo

w:

28

ld

ay-

1

O2

aero

bic

:4

.3m

gl-

1

O2

ano

xic

:0

.06

mg

l-1

CO

D:

95

NH

4?

:9

8

Nto

tal:

96

S2-

:1

00

Cr t

ota

l:1

00

Den

itri

fica

tio

n:

0.1

mg

N

(mg

ML

VS

S-d

)-1

Nit

rifi

cati

on

:

0.0

8m

gN

(mg

ML

VS

S-d

)-1

No

tm

enti

on

edN

ot

iden

tifi

ed

(Let

aet

al.

20

04

)

Pre

den

itri

fica

tio

n-

nit

rifi

cati

on

/sti

rred

tan

kre

acto

ran

d

aero

bic

acti

vat

ed

slu

dg

eta

nk

92

7-2

14

0

To

tal

N

95

83

-13

51

5C

OD

14

9-1

78

NH

4?

-N

46

6-7

95

S2-

Vo

l.d

enit

rify

ing

reac

tor:

10

0l,

20

ho

fH

RT

Vo

l.n

itri

fyin

gre

acto

r:

20

0l,

40

ho

fH

RT

pH

effl

uen

t:7

.6

To

tal

N:

98

CO

D:

98

NH

4?

:9

5

S2-

:1

00

8m

gN

-NO

3-

(gV

SS

-h)-

1

5.4

mg

NH

4?

-N

(gV

SS

-h)-

1

No

tm

enti

on

edN

ot

iden

tifi

ed

(Rey

es-A

vil

a

etal

.2

00

4)

Den

itri

fica

tio

n/C

on

tin

uo

us

stir

red

tan

kre

acto

r

60

6O

rgan

ic-C

29

4S

2-

41

8.8

NO

3-

-N

Vo

l.re

acto

r:1

.3l

HR

T(h

):4

8

NO

3-

:1

00

S2-

:9

8

Org

anic

-C:

69

0.6

kg

C

(kg

VS

S-d

)-1

8k

gS

2-

(kg

VS

S-d

)-1

1k

gN

-NO

3-

(kg

VS

S-d

)-1

0.0

8k

gN

2

(kg

VS

S-d

)-1

CO

2

S0

N2

No

tid

enti

fied

(Sie

rra-

Alv

arez

etal

.2

00

5)

Mix

otr

op

hic

den

itri

fica

tio

n/

up

flo

wan

aero

bic

slu

dg

eb

ed

87

-14

8p

-cre

sol

10

2-1

47

H2S

86

-10

5N

O3-

-N

Vo

l.re

acto

r:0

.5l

HR

T(h

):1

3.4

NO

3-

:9

9.4

–9

9.5

H2S

:9

8.9

–9

8.3

p-c

reso

l:9

7.7

–9

8.2

1.1

mm

ol

p-c

reso

l

(gV

SS

-d)-

1S

O42-

N2

No

tid

enti

fied

(Mez

a-

Esc

alan

te

etal

.2

00

7)

Lit

ho

-org

ano

tro

ph

ic

den

itri

fica

tio

n/

bat

chre

acto

r

44

p-c

reso

l-C

,

50

NO

3-

-N

20

S2-

Vo

lum

enb

atch

reac

tor:

16

0m

l

pH

:7

.2

S2-

:1

00

p-c

reso

l:1

00

NO

3-

:1

00

26

gN

-NO

3-

(gV

SS

-d)-

1C

O2

SO

42-

N2

No

tid

enti

fied

(Ber

ista

in-

Car

do

so

etal

.2

00

8)

Lit

ho

-org

ano

tro

ph

ic

den

itri

fica

tio

n/i

nv

erse

flu

idiz

edb

edre

acto

r

20

9N

O3-

-N

30

6ac

etat

e-C

64

S2-

Vo

l.re

acto

r:1

.7l.

HR

T(h

):2

2

NO

3-

:1

00

S2-

:1

00

acet

ate:

10

0

No

tca

lcu

late

dN

2

CO

2

S0

No

tid

enti

fied

(Ch

enet

al.

20

08

)

Den

itri

fica

tio

n/E

GS

B

reac

tor

73

8ac

etat

e

80

0S

2-

38

6N

O3-

-N

Vo

l.re

acto

r:4

.0l

HR

T(h

):6

.4

Ace

tate

:9

0

S2-

:10

0

NO

3-

:9

9

No

tca

lcu

late

dC

O2

S0

N2

Des

ulf

om

icro

biu

mn

orv

egic

um

Th

au

era

sp.

Den

itro

mo

na

sin

do

licu

m

Clo

stri

diu

msp

.

336 Rev Environ Sci Biotechnol (2009) 8:325–342

123

There is still little information about this biological

process (Table 3). Nowadays, there is no evidence

about one denitrifying strain with litho-organotrophic

metabolism, that is to say, with the capability to use

sulfur and carbon compounds as energy source.

Therefore, the evidences suggest that the simulta-

neous oxidation either sulfur or carbon compounds

under denitrifying conditions might be carry out by

mixed cultures of lithotrophic and organotrophic

denitrifiers. The following studies show the litho-

organotrophic denitrification process, where the inor-

ganic sulfur compounds as well as the organic matter

were coupled to the nitrate reduction. Gommers et al.

(1988) observed the simultaneous oxidation of sulfide

and simple organic matter such as acetate under

denitrifying conditions in an upflow fluidized bed

reactor. During the steady state, the consumption

efficiencies of acetate, sulfide and nitrate were of

100%. The end products formed were CO2, SO42-,

N2 and NO2-. In this case it is relevant to diminish

the formation of undesirable products such as NO2-

because it may cause health problems. Kim and Son

(2000) carried out batch cultures in order to study the

effect of COD/N/S ratio on the denitrification

process. The authors worked with a mixed culture

of sulfate reducing bacteria and sulfur denitrifying

bacteria, using acetate and thiosulfate as energy

sources, and nitrate as electron acceptor. At a COD/

N/S ratio of 0.8/1/3.3, nitrate, thiosulfate and acetate

were consumed in less than 6 h, while at a COD/N/S

ratio of 3.3/1/3.3 the consumption for nitrate and

thiosulfate was faster and the consumption of acetate

inhibited, probably due to a competition between

thiosulfate and acetate for the electron acceptor.

Reyes-Avila et al. (2004) showed the simultaneous

elimination of acetate and sulfide by a denitrifying

consortium using a continuous stirred tank reactor.

The authors worked with a C/N ratio of 1.40 and a S/

N ratio of 1.43. Under steady state denitrification, the

consumption efficiencies of sulfide and nitrate were

of 100%, while the consumption efficiency for

acetate was of 65%. The end products were CO2,

N2 and S0. These authors also showed in batch

cultures that, in the presence of sulfide, acetate, and

nitrate, the specific rate of sulfide oxidation to

elemental sulfur was higher than the specific rate of

acetate oxidation to carbon dioxide, and the slowest

reaction was the elemental sulfur oxidation to sulfate.

In this kind of reactor, elemental sulfur wasTa

ble

3co

nti

nu

ed

Au

tho

rsB

iolo

gic

alp

roce

ss/r

eact

or

con

fig

ura

tio

n

Co

mp

ou

nd

s

(mg

l-1)

Op

erat

ion

al

con

dit

ion

s

Rem

ov

al

effi

cien

cies

(%)

Bio

log

ical

pro

cess

rate

s

En

d

pro

du

cts

Bac

teri

al

com

mu

nit

y

(Ber

ista

in-

Car

do

so

etal

.2

00

9a)

Lit

ho

-org

ano

tro

ph

ic

den

itri

fica

tio

n/

inv

erse

flu

idiz

ed

bed

reac

tor

15

1p

hen

ol-

C

34

S2-

15

1N

O3-

-N

Vo

l.re

acto

r:1

.7l

HR

T(h

):2

1

NO

3-

:1

00

S2-

:1

00

ph

eno

l:1

00

No

tca

lcu

late

dC

O2

SO

42-

N2

Th

iob

aci

llu

sd

enit

rifi

can

s

Th

iob

aci

lllu

ssa

jan

ensi

s

Th

iob

aci

llu

ssp

.

Th

au

era

aro

ma

tic

(Li

etal

.2

00

9)

Des

ulf

uri

zati

on

-

den

itri

fica

tio

n/

anae

rob

icat

tach

ed-

gro

wth

bio

reac

tor

33

.6g

luco

se-C

20

0S

2-

52

NO

3-

-N

20

NO

2-

-N

Vo

l.re

acto

r:

3.5

l

pH

effl

uen

t:7

.5

HR

T(h

):2

.6

S2-

:9

9.9

C:

91

.8

NO

3-

:9

9.9

NO

2-

:9

9.9

No

tca

lcu

late

dS

O42-

S0

No

tid

enti

fied

Rev Environ Sci Biotechnol (2009) 8:325–342 337

123

accumulated and this could change the metabolic

outcome of sulfide oxidation. For this reason, a

special system is required for the elemental sulfur

separation in continuous mode. The inverse fluidized

bed reactor presents this characteristic, working

successfully for the separation of solids in continuous

mode (Celis-Garcıa et al. 2008; Gallegos-Garcıa et al.

2009; Krishnakumar et al. 2005). Beristain-Cardoso

et al. (2008) worked with an inverse fluidized bed

reactor for the elemental sulfur separation in contin-

uous mode under denitrifying conditions. The ele-

mental sulfur formation was controlled by acetate/

nitrate molar ratio being CO2 and N2 the end

products. These authors showed that C/N ratio was

an important factor affecting the fate to either S0 or

SO42-. These different works showed that there are

several factors affecting the litho-organotrophic deni-

trification, such as: stoichiometry of the reactions,

initial substrates concentrations, and consumption

specific rates, among others.

Sierra-Alvarez et al. (2005) showed the possibility

to eliminate simultaneously sulfide and p-cresol.

Sulfide (102–147 mg l-1), p-cresol (87–148 mg l-1)

and NO3--N (86–105 mg l-1) were completely elim-

inated at a hydraulic retention time of 13-h employ-

ing a UASB reactor. Meza-Escalante et al. (2007)

also studied the simultaneous oxidation of p-cresol

and sulfide under denitrifying conditions. The authors

showed that p-cresol and sulfide consumption was

coupled to the nitrate reduction, being the end

products CO2, SO42- and N2, respectively. These

investigations show the advantage of using a micro-

bial consortium to eliminate p-cresol and sulfide

under denitrifying conditions, independently of the

sludge origin. As p-cresol, phenol is another persis-

tent and toxic compound. However, under denitrify-

ing conditions, it was shown to be completely

oxidized to CO2 in the presence of sulfide, while

nitrate was totally reduced to molecular nitrogen

(Beristain-Cardoso et al. 2009a). These results clearly

show that the denitrification allows the complete

oxidation of p-cresol, phenol and sulfide with a high

recovering of nitrate as molecular nitrogen.

3 Conclusions

The results presented in this review show that

the connection of nitrification and denitrification

processes could be a feasible treatment for the

recovery of effluents contaminated with nitrogen,

sulfur and carbon compounds. Nevertheless, the

mechanisms that allow understanding of the phenom-

enon are not still properly described and more studies

are required in order to get insight about the nitrifi-

cation and denitrification in biological reactors, in

particular with different organic and inorganic reduc-

ing sources, including recalcitrant compounds, alone

or in mixtures. Moreover, additional studies in batch

cultures about physiological and kinetic aspects are

necessary in order to control the sludge metabolic

capacity, as well as studies in biological reactors

combining physiology, ecology and engineering

information.

Acknowledgments This work was financed by NSF–

CONACYT Project 35982-U. R. Beristain received a

Postdoctoral fellowship from CONACYT.

References

Akunna JC, Bizeau C, Moletta R (1993) Nitrate and nitrite

reductions with anaerobic sludge using various carbon

sources: glucose, glycerol, acetic acid, lactic acid and

methanol. Water Res 27:1303–1312

Akunna JC, Bizeau C, Molleta R (1994) Nitrate reduction by

anaerobic sludge using glucose at various nitrate con-

centrations: ammonification, denitrification and metha-

nogenic activities. Environ Technol 15:41–49

Almeida JS, Julio SM, Reis MAM, Carrondo MJT (1994)

Nitrite inhibition of denitrification by Pseudomonas fluo-rescens. Biotechnol Bioeng 46:194–201

Am J, Bum M, Kim S, Ahn Y, Kim IS, Bishop PL (2005)

Assessment of characteristics of biofilm formed on auto-

trophic denitrification. J Microbiol Biotechnol 15:455–460

Amor L, Eiroa M, Kennes C, Veiga MC (2005) Phenol bio-

degradation and its effect on the nitrification process.

Water Res 39:2915–2920

Anders HJ, Kaetzke A, Kampfer P, Ludwig W, Fuchs G (1995)

Taxonomic position of aromatic-degrading denitrifying

pseudomonad strains K 172 and KB 740 and their

description as new members of the genera Thauera, as

Thauera aromatica sp. nov., and Azoarcus, as Azoarcus

evansii sp. nov., respectively, members of the beta sub-

class of the Proteobacteria. Int J Syst Bacteriol 45:

327–333

Arp D, Sayavedra-Soto LA, Hommes NG (2002) Molecular

biology and biochemistry of ammonia oxidation by Nitr-osomonas europaea. Arch Microbiol 178:250–255

Autenrieth RL, Bonner JS, Akgerman A, Okaygum M,

McCreary EM (1991) Biodegradation of phenolic wastes.

J Hazard Mater 28:29–53

Baumann B, Snozzi M, Zehnder A, Roelof J, van der Meer JR

(1996) Dynamics of denitrification activity of Paracoccus

338 Rev Environ Sci Biotechnol (2009) 8:325–342

123

denitrificans in continuous culture during aerobic-anaer-

obic changes. J Bacteriol 178:4367–4374

Beller HR, Letain TE, Chakicherla A, Kane SR, Legler TC,

Coleman MA (2006) Whole-genome transcriptional

analysis of chemolithoautotrophic thiosulfate oxidation by

Thiobacillus denitrificans under aerobic versus denitrify-

ing conditions. J Bacteriol 188:7005–7015

Bentzen G, Smith AT, Bennet D, Webster NJ, Reinholt FES,

Hobson J (1995) Controlled dosing of nitrate for preven-

tion of H2S in a sewer network and the effects on the

subsequent treatment processes. Water Sci Technol

31:293–302

Beristain-Cardoso R, Texier AC, Razo-Flores E, Sierra-Alva-

rez R, Field J, Gomez J (2008) Simultaneous sulfide and

acetate oxidation under denitrifying conditions using an

inverse fluidized bed reactor. J Chem Technol Biot

83:1197–1203

Beristain-Cardoso R, Anne-Claire T, Alpuche-Solis A, Gomez

J, Razo-Flores E (2009a) Phenol and sulfide oxidation in a

denitrifying biofilm reactor and its microbial community

analysis. Process Biochem 44:23–28

Beristain-Cardoso R, Texier A-C, Sierra-Alvarez R, Razo-

Flores E, Field JA, Gomez J (2009b) Effect of initial

sulfide concentration on sulfide and phenol oxidation

under denitrifying conditions. Chemosphere 74:200–205

Bernet N, Delgenes N, Moletta R (1996) Denitrification by

anaerobic sludge in piggery wastewater. Environ Technol

17:293–300

Bilanovic D, Battistoni P, Cecchi F, Pavan P, Mata-Alvarez J

(1999) Denitrification under high nitrate concentration and

alternating anoxic conditions. Water Res 33:3311–3320

Bock E, Koops HP, Harms H, Ahlers B (1991) In variations in

autotrophic life. In: Barton JMSLL (ed) The biochemistry

of nitrifying organisms. Academic Press, San Diego, pp

171–200

Bonin P, Gilewicz M, Bertrand JC (1989) Effects of oxygen on

each step of denitrification on Pseudomonas nautica. Can

J Microbiol 35:1061–1064

Buitron G, Capdeville B (1995) Enhancement of the biodeg-

radation activity by the acclimation of the inoculum.

Environ Technol 16:1175–1184

Cadena F, Peters RW (1988) Evaluation of chemical oxidizers

for hydrogen sulfide control. J Water Pollut Control Fed

60:1259–1263

Campos JL, Garrido-Fernandez JM, Mendez R, Lema JM

(1999) Nitrification at high ammonia loading rates in an

activated sludge unit. Bioresour Technol 68:141–148

Campos JL, Mendez R, Lema JM (2000) Operation of a

nitrifying activated sludge airlift (NASA) reactor without

biomass carrier. Water Sci Technol 41:113–120

Campos JL, Garrido JM, Mosquera-Corral A, Mendez R

(2007) Stability of a nitrifying activated sludge reactor.

Biochem Eng J 35:87–92

Cardoso RB, Sierra-Alvarez R, Rowlette P, Flores ER, Gomez

J, Field JA (2006) Sulfide oxidation under chemolitho-

autotrophic denitrifying conditions. Biotechnol Bioeng

95:1148–1157

Carvallo L, Carrera J, Chamy R (2002) Nitrifying activity

monitoring and kinetic parameters determination in a

biofilm airlift reactor by respirometry. Biotechnol Lett

24:2063–2066

Celis-Garcıa LB, Gonzalez-Blanco G, Meraz M (2008) Removal

of sulfur inorganic compounds by a biofilm of sulfate

reducing and sulfide oxidizing bacteria in a down-flow

fluidized bed reactor. J Chem Technol Biot 83:260–268

Cervantes F, Monroy O, Gomez J (1998) Accumulation of

intermediates in a denitrifying process at different copper

and high nitrate concentration. Biotechnol Lett 20:

959–961

Cervantes FJ, De la Rosa D, Gomez J (2001) Nitrogen removal

from wastewaters at low C/N ratios with ammonium and

acetate as electron donors. Bioresour Technol 79:165–170

Chazal M, Lens P (2000) Interactions between the sulfur and

nitrogen cycle: microbiology and process technology. In:

Lens PNL, Hulshoff Pol L (eds) Environmental technol-

ogies to treat sulfur pollution principles and engineering.

International Water Association, London, pp 415–447

Chen SD, Chen CY, Shen YC, Chui CM, Cheng HJ (1996)

Treatment of high-strength nitrate wastewater by biolog-

ical methods-operational characteristics study. Water Sci

Technol 34:269–276

Chen C, Ren N, Wang A, Yu Z, Lee DJ (2008) Simultaneous

biological removal of sulfur, nitrogen and carbon using

EGSB reactor. Appl Microbiol Biotechnol 78:1057–1063

Colleran E, Finnegan S, Lens P (1995) Anaerobic treatment of

sulphate-containing waste streams. Antonie van Lee-

uwenboek 67:24–46

Collins AG, Clarkson WW, Vrona M (1988) Fixed-film bio-

logical nitrification of a strong industrial waste. J Water

Pollut Control Fed 60:499–504

Cuervo-Lopez F, Martinez F, Gutierrez-Rojas M, Loyola RA,

Gomez J (1999) Effect of nitrogen loading rates and

carbon source on denitrification sludge settles ability in

upflow anaerobic sludge blanket (UASB) reactor. Water

Sci Technol 40:123–130

Dangel W, Brackmann R, Lack A, Mohamed M, Koch J,

Oswald B, Seyfried B, Tschech A, Fuchs G (1991) Dif-

ferential expression of enzyme activities initiating anoxic

metabolism of various aromatic compounds via benzoyl-

CoA. Arch Microbiol 155:256–262

Dapena-Mora A, Fernandez I, Campos JL, Mosquera-Corral A,

Mendez R, Jetten MSM (2007) Evaluation of activity and

inhibition effects on Anammox process by batch tests

based on the nitrogen gas production. Enzyme Microb

Tech 40:859–865

Darbi A, Viraraghavan T, Butler R, Corkal D (2003) Column

studies on nitrate removal from potable water. Water Air

Soil Poll 150:235–254

Delanghe B, Nakamura F, Myoga H, Magara Y (1994) Bio-

logical denitrification with ethanol in a membrane biore-

actor. Environ Technol 15:61–70

Elefsiniotis P, Li D (2006) The effect of temperature and

carbon source on denitrification using volatile fatty acids.

Biochem Eng J 28:148–155

EPA (1993) Nitrogen control manual. In: Environmental Pol-

lution Agency of United States (ed) Washington, DC

Farhadian M, Duchez D, Vachelard C, Larroche C (2008)

Monoaromatics removal from polluted water through

bioreactors—A review. Water Res 42:1325–1341

Fernandez-Nava Y, Maranon E, Soons J, Castrillon L (2008)

Denitrification of wastewater containing high nitrate and

calcium concentrations. Bioresour Technol 99:7976–7981

Rev Environ Sci Biotechnol (2009) 8:325–342 339

123

Fiencke C, Bock E (2006) Immunocytochemical localization of

membrane-bound ammonia monooxygenase in cells of

ammonia oxidizing bacteria. Arch Microbiol 185:99–106

Flere JM, Zhang TC (1999) Nitrate removal with sulfur-lime-

stone autotrophic denitrification processes. J Environ Eng

Asce 125:721–729

Francis CA, Beman JM, Kuypers MMM (2007) New processes

and players in the nitrogen cycle: the microbial ecology of

anaerobic and archaeal ammonia oxidation. ISME J 1:

19–27

Gallegos-Garcıa M, Celis LB, Rangel-Mendez R, Razo-Flores E

(2009) Precipitation and recovery of metal sulfides from

metal containing acidic wastewater in a sulfidogenic down-

flow fluidized bed reactor. Biotechnol Bioeng 102:91–99

Galloway JM, Aber JD, Erisman JW, Seitzinger SP, Howarth

RW, Cowling EB, Cosby BJ (2003) The nitrogen cascade.

BioSciene 53:341–356

Garrido JM, van Benthum WAJ, van Loosdrecht MCM,

Heijnen JJ (1997) Influence of dissolved oxygen concen-

tration on nitrite accumulation in a biofilm airlift sus-

pension reactor. Biotechnol Bioeng 53:168–178

Gomez J, Mendez-Pampin R, Lema JM (2000) Kinetic study of

the addition of volatile organic compounds on a nitrifying

sludge. Appl Biochem Biotechnol 87:189–202

Gommers PJF, Bijleveld W, Kuenen JG (1988) Simultaneous

sulfide and acetate oxidation in a denitrifying fluidized

bed reactor. I: start-up and reactor performance. Water

Res 22:1075–1083

Gonzalez A, Alcantara S, Razo-Flores E, Revah S (2005)

Oxygen transfer coefficient and consumption in a thio-

sulfate oxidizing bioreactor with sulfur production. Let-

ters Appl Microbiol 41:141–146

Grabinska-Loniewska A (1991) Denitrification unit biocenosis.

Water Res 25:1565–1573

Gupta AB (1997) Thiosphaera pantotropha: a sulphur bacte-

rium capable of simultaneous heterotrophic nitrification

and aerobic denitrification. Enzyme Microb Tech 21:

589–595

Hanaki K, Wantawin C, Ohgaki S (1990) Effects of the activity

of heterotrophs on nitrification in a suspended-growth

reactor. Water Res 24:289–296

Harwood CS, Gibson J (1997) Shedding light on anaerobic

benzene ring degradation: a process unique to prokary-

otes? J Bacteriol 179:301–309

Harwood CS, Burchhardt G, Herrmann H, Fuchs G (1999)

Anaerobic metabolism of aromatic compounds via the

benzoyl-CoA pathway. FEMS Microbiol Rev 22:439–458

Heider J, Fuchs G (1997) Anaerobic metabolism of aromatic

compounds. Eur J Biochem 243:577–596

Heijnen JJ, Mulder A, Weltevrede R, Hols PH, van Leeuwen

HLJM (1990) Large-scale anaerobic\aerobic treatment of

complex industrial wastewater using immobilized bio-

mass in fluidized bed and air-lift suspension reactor.

Chem Eng Technol 13:202–208

Heipieper HJ, Keweloh H, Rehm HJ (1991) Influence of phe-

nols on growth and membrane permeability of free and

immobilized Escherichia coli. Appl Environ Microbiol

57:1213–1217

Heipieper HJ, Diefenbach R, Keweloh H (1992) Conversion of

cis unsaturated fatty acids to trans, a possible mechanism

for the protection of phenol-degrading Pseudomonas

putida P8 from substrate toxicity. Appl Environ Microbiol

58:1847–1852

Hellinga C, Loosdrecht MCM, Heijnen JJ (1999) Model based

design of a novel process for nitrogen removal from con-

centrated flows. Math Comp Model Dynam Syst 5:351–371

Henze M, Harremoes P, Arvin E, Cour Jansen J (1996) Waste

water treatment. Lyngby, Springer, Berlin

Herbert RA (1999) Nitrogen cycling in coastal marine eco-

systems. FEMS Microbiol Rev 23:563–590

Hernandez D, Rowe JJ (1988) Oxygen inhibition of nitrate

uptake is a general regulatory mechanism in nitrate res-

piration. J Biol Chem 263:7937–7939

Hiscock K, Lloyd J, Lerner D (1991) Review of natural and

artificial denitrification of groundwater. Water Res 25:

1099–1111

Hong Z, Hanaki K, Matsuo T (1994) Greenhouse gas-N2O

production during denitrification in wastewater treatment.

Water Sci Technol 28:203–207

Hu HY, Nozawa K, Fujie K, Makabe T, Urano K (1998)

Analysis of microbial acclimation to refractory chemicals

in wastewater using respiratory quinone profiles. Water

Sci Technol 37:407–411

Hyman MR, Samsone-Smith AW, Shears JH, Wood PM

(1985) A kinetic study of benzene oxidation to phenol by

whole cells of Nitrosomonas europaea and evidence for

the further oxidation of phenol to hydroquinone. Arch

Microbiol 143:302–306

Islas S, Thalasso F, Gomez J (2004) Evidence of anoxic

methane oxidation coupled to denitrification. Water Res

38:13–16

Jenneman GE, Moffitt PD, Bala GA, Webb RH (1999) Sulfide

removal in reservoir brine by indigenous bacteria. Soc Pet

Eng Prod Facilities 14:219–225

Keener WK, Arp DJ (1993) Kinetic studies of AMO inhibition

in Nitrosomonas europaea by hydrocarbons and haloge-

nated hydrocarbons in an optimizer whole-cell assay.

Appl Environ Microbiol 59:2501–2510

Keener WK, Arp DJ (1994) Transformations of aromatic

compounds by Nitrosomonas europaea. Appl Environ

Microbiol 60:1914–1920

Kelly DP, Wood AP (2000) Confirmation of Thiobacillus deni-trificans as a species of the genus Thiobacillus, in the beta-

subclass of the Proteobacteria, with strain NCIMB 9548 as

the type strain. Int J Syst Evol Microbiol 50:547–550

Kelly DP, Wood AP, Stackebrandt E (2005) Genus II. Thio-bacillus Beijerinck. In: Garrity IGM (ed) Bergey’s manual

of systematic bacteriology, 2nd edn. Springer, New York,

pp 764–769

Khoury N, Dott W, Kampfer P (1992) Anaerobic degradation

of phenol in batch and continuous cultures by a denitri-

fying bacterial consortium. Appl Microbiol Biotechnol

37:524–528

Kim I, Son J (2000) Impact of COD/N/S ratio on denitrification

by the mixed cultures of sulfate reducing bacteria and

sulfur denitrifying bacteria. Water Sci Technol 42:69–76

Kim BW, Kim IK, Chang HN (1990) Bioconversion of

hydrogen sulfide by free and immobilized cells of Chlor-obium thiosulfatophilum. Biotechnol Lett 12:381–386

Kleerebezem R, Mendez R (2002) Autotrophic denitrifica-

tion for combined hydrogen sulfide removal from biogas

and post-denitrification. Water Sci Technol 45:349–356

340 Rev Environ Sci Biotechnol (2009) 8:325–342

123

Knowles R (1982) Denitrification. Microbiol Rev 46:43–70

Korom SF (1992) Natural denitrification in the saturated

zone—A review. Water Resour Res 28:1657–1668

Krishnakumar B, Manilal BV (1999) Bacterial oxidation of

sulphide under denitrifying conditions. Biotechnol Lett

21:437–440

Krishnakumar B, Majumdar S, Manilal BV, Hardas A (2005)

Treatment of sulphide containing watewater with sulphur

recovery in a novel reverse fluidized loop reactor (RFLR).

Water Res 39:639–647

Kuenen JG, Robertson LA, Tuovinen OH (1992) The genera

Thiobacillus, Thiomicrospira, and Thiosphaera. In: Ba-

lows A, Truper HG, Dworkin M, Harder W, Schleifer

K-H (eds) The prokaryotes, 2nd edn. Springer, New York,

pp 2638–2657

Lack A, Fuchs G (1992) Carboxylation of phenylphosphate by

phenol carboxylase, an enzyme system of anaerobic

phenol metabolism. J Bacteriol 174:3629–3636

Lalucat J, Bennasar A, Bosch R, Garcıa-Valdes E, Palleroni NJ

(2006) Biology of Pseudomonas stutzeri. Microbiol Mol

Biol R 70:510–547

Lens P, van de Bosch M, Hulshoff Pol L, Lettinga G (1998)

Effect of staging on volatile fatty acid degradation in

a sulfidogenic granular sludge reactor. Water Res 32:

1178–1192

Leta S, Assefa F, Gumaelius L, Dalhammar G (2004) Bio-

logical nitrogen and organic matter removal from tannery

wastewater in pilot plant operations in Ethiopia. Environ

Biotechnol 66:333–339

Leu HG, Lee CD, Ouyang CF, Tseng HT (1998) Effects of

organic matter on the conversion rates of nitrogenous

compounds in a channel reactor under various flow con-

ditions. Water Res 32:891–899

Li W, Zhao Q-l, Liu H (2009) Sulfide removal by simultaneous

autotrophic and heterotrophic desulfurization–denitrifica-

tion process. J Hazard Mater 162:848–853

Ma Y-L, Yan B-L, Zhao J-L (2006) Removal of H2S by

Thiobacillus denitrificans immobilized on different

matrices. Bioresour Technol 97:2041–2046

Manconi I, Carucci A, Lens P (2007) Combined removal of

sulfur compounds and nitrate by autotrophic denitrifica-

tion in bioaugmented activated sludge system. Biotechnol

Bioeng 98(3):551–560

McCarty GW (1999) Review article: modes of action of

nitrification inhibitors. Biol Fert Soils 29:1–9

Meza-Escalante ER, Anne-Claire T, Cuervo-Lopez F, Gomez

J, Cervantes F (2007) Inhibition of sulfide on the simul-

taneous removal of nitrate and p-cresol by a denitrifying

sludge. J Chem Technol Biot 83:372–377

Moura I, Moura J (2001) Structural aspects of denitrifying

enzymes. Curr Opin Chem Biol 5:168–175

Mussati M, Gernaey K, Gani R, Jørgensen SB (2002) Perfor-

mance analysis of a denitrifying wastewater treatment

plant. Clean Techn Environ Policy 4:171–183

Nugroho R, Takanashi H, Hirata M, Hano T (2002) Denitrifi-

cation of industrial wastewater with sulfur and limestone

packed column. Water Sci Technol 46:99–104

Oh SE, Yoo YB, Young JC, Kim IS (2001) Effect of organics

on sulfur-utilizing autotrophic denitrification under

mixotrophic conditions. J Biotechnol 92:1–8

Okabe S, Oozawa Y, Hirata K, Watanabe Y (1996) Relation-

ship between population dynamics of nitrifiers in biofilms

and reactor performance at various C:N ratios. Water Res

30:1563–1572

Olmos A, Olguin P, Fajardo C, Razo-Flores E, Monroy O

(2004) Physicochemical characterization of spent caustic

from the OXIMER process and source waters from

Mexican oil refineries. Energy and Fuels 18:302–304

Pena-Calva A, Olmos-Dichara A, Viniegra-Gonzalez G, Cu-

ervo-Lopez FM, Gomez J (2004) Denitrification in pres-

ence of benzene, toluene and m-xylene. Appl Biochem

Biotechnol 119:195–208

Pereira MM, Teixeira M (2004) Proton pathways, ligand

binding and dynamics of the catalytic site in haem-copper

oxygen reductases: a comparison between the three fam-

ilies. Biochim Biophys Acta 1655:340–346

Philipp B, Schink B (2000) Two distinct pathways for anaer-

obic degradation of aromatic compounds in the denitri-

fying bacterium Thauera aromatica strain AR-1. Arch

Microbiol 173:91–96

Philips S, Laanbroek JH, Verstraete W (2002) Origin, causes

and effects of increased nitrite concentrations in aquatic

environments. Rev Environ Sci Biotechnol 1:115–141

Pitcher RS, Watmough NJ (2004) The bacterial cytochrome

cbb3 oxidases. Biochim Biophys Acta 1655:388–399

Prosser JI (1989) Autotrophic nitrification in bacteria. Adv

Microb Physiol 30:125–181

Puig-Grajales L, Rodrıguez-Nava O, Razo-Flores E (2003)

Simultaneous biodegradation of a phenol and 3, 4-

dimethylphenol mixture under denitrifying conditions.

Water Sci Technol 48:171–178

Pynaert K (2003) Nitrogen removal in wastewater treatment by

means of oxygen-limited autotrophic nitrification–deni-

trification. PhD thesis, Ghent University

Razo-Flores E, Svitelskaya A, Donlon B, Field J, Lettinga G

(1996) The effect of granular sludge source on the

anaerobic biodegradability of aromatic compounds. Bi-

oresour Technol 56:215–220

Reinsel MA, Sears JT, Stewart PS, Mc Inerney MJ (1996)

Control of microbial souring by nitrate, nitrite or glutar-

aldehyde injection in a sand-stone column. J Ind Micro-

biol Biotechnol 17:128–136

Reyes-Avila J, Razo-Flores E, Gomez J (2004) Simultaneous

biological removal of nitrogen, carbon and sulfur by

denitrification. Water Res 38:3313–3321

Rittmann BE, McCarty PL (2001) Environmental biotechnol-

ogy principles and applications. McGraw-Hill, New York

Rodriguez L, Villasenor J, Fernandez FJ (2007) Use of agro-

food wastewater for the optimization of the denitrification

process. Water Sci Technol 55:63–70

Schink B, Brune A, Schnell S (1992) Anaerobic degradation of

aromatic compounds. In: Winkelmann G (ed) Microbial

degradation of natural compounds. VCH, Weinheim, pp

219–242

Schmidt I, Sliekers O, Schmid M, Bock E, Fuerst J, Kuenen

JG, Jetten MS, Strous M (2003) New concepts of micro-

bial treatment processes for the nitrogen removal in

wastewater. FEMS Microbiol Rev 27:481–492

Schweighofer P, Nowak O, Svardal K, Kroiss H (1996) Steps

towards the upgrading of a municipal WWTP affected by

Rev Environ Sci Biotechnol (2009) 8:325–342 341

123

nitrification inhibiting compounds—a case study. Water

Sci Technol 33:39–46

Shinoda Y, Sakai Y, Ue M, Hiraishi A, Kato N (2000) Isolation

and characterization of a new denitrifying spirillum

capable of anaerobic degradation of phenol. Appl Environ

Microbiol 66:1286–1291

Shoun H, Tanimoto T (1991) Denitrification by the fungus

Fusarium oxysporum and involvement of cytochrome

P-450 in the respiratory nitrite reduction. J Biol Chem

266:11078–11082

Siegrist H, Salzgeber D, Eugster J, Joss A (2008) Anammox

brings WWTP closer to energy autarky due to increased

biogas production and reduced aeration energy for

N-removal. Water Sci Technol 57:383–388

Sierra-Alvarez R, Guerrero F, Rowlette P, Freeman S, Field J

(2005) Comparison of chemo-, hetero- and mixotrophic

denitrification in laboratory-scale UASBs. Water Sci

Technol 52:337–342

Sierra-Alvarez R, Beristain-Cardoso R, Salazar M, Gomez J,

Razo-Flores E, Field JA (2007) Chemolithotrophic deni-

trification with elemental sulfur for groundwater treat-

ment. Water Res 41:1253–1262

Sijbesma WFH, Almeida JS, Reis MAM, Santos H (1996)

Uncoupling effect of nitrite during denitrification by

Pseudomonas fluorescens: an in vivo 31P-NMR study.

Biotechnol Bioeng 52:176–182

Soares MIM (2000) Biological denitrification of groundwater.

Water Air Soil Poll 123:183–193

Sublette KL, Woolsey ME (1988) Sulfide and glutaraldehyde

resistant strains of Thiobacillus denitrificans. Biotechnol

Bioeng 34:565–569

Szpyrkowicz S, Rigoni-Stern S, Zilio Grandi F (1991) Nitrifi-

cation and denitrificaton of tannery wastewaters. Water

Res 25:1351–1356

Tam NFY, Wong YS, Leung G (1992) Effect of exogenous

carbon sources on removal of inorganic nutrient by

the nitrification–denitrification process. Water Res 26:

1229–1236

Texier A-C, Gomez J (2002) Tolerance of nitrifying sludge to

p-cresol. Biotechnol Lett 24:321–324

Texier A-C, Gomez J (2007) Simultaneous nitrification and

p-cresol oxidation in a nitrifying sequencing batch reactor.

Water Res 41:315–322

Thomas S, Sarfaraz S, Mishra LC, Iyengar L (2002) Degradation

of phenol and phenolic compounds by a defined denitrifying

bacterial culture. World J Microb Biot 18:57–63

Thomsen J, Geest T, Cox R (1994) Mass spectrometric studies

of the effect of pH on the accumulation of intermediates in

denitrification by Paracoccus denitrificans. Appl Environ

Microbiol 60:536–541

Tschech A, Fuchs G (1987) Anaerobic degradation of phenol

by pure culture of newly isolated denitrifying Pseudo-monads. Arch Microbiol 148:213–217

Van der Hoek JP, Kappelhof JWNM, Hijnen WAM (1992)

Biological nitrate removal from ground-water by sulfur

limestone denitrification. J Chem Technol Biot 54:197–200

Van Schie PM, Young LY (2000) Biodegradation of phenol:

mechanisms and applications. Bioremediation J 4:1–18

Verstraete W, Philips S (1998) Nitrification–denitrification

processes and technologies in new contexts. Environ

Pollut 102:717–726

Visser JM, De Joung GAH, de Vries S, Robertson LA, Kuenen

JG (1997a) cbb (3)-type cytochrome oxidase in the obli-

gately chemolithoautotrophic Thiobacillus sp. FEMS

Microbiol Ecol 147:127–132

Visser JM, Robertson LA, van Verseveld HW, Kuenen JG

(1997b) Sulfur production by obligately chemolithoauto-

trophic Thiobacillus species. Appl Environ Microbiol

63:2300–2305

Volcke EI, Loccufier M, Noldus EJ, Vanrolleghem PA (2007)

Operation of a SHARON nitritation reactor: practical

implications from a theoretical study. Water Sci Technol

56:145–154

Wang AJ, Du DZ, Ren NQ, van Groenestijn JW (2005) An

innovative process of simultaneous desulfurization and

denitrification by Thiobacillus denitrificans. J Environ Sci

Health A Tox Hazard Subst Environ Eng 40:1939–1949

Watson HM (1993) A comparison of the effects of two

methods of acclimation on anerobic biodegradability.

Environ Toxicol Chem 12:2023–2030

Wett B, Rostek R, Rauch W, Ingerle K (1998) pH-controlled

reject-water-treatment. Water Sci Technol 37:165–172

Wicht H (1996) A model for predicting nitrous oxide pro-

duction during denitrification in activated sludge. Water

Sci Technol 34:99–106

Wilson LP, Bouwer EJ (1997) Biodegradation of aromatic

compounds under mixed oxygen/denitrifying conditions:

a review. J Ind Microbiol Biot 18:116–130

Wu Q, Knowles R, Chan YK (1995) Production and con-

sumption of nitric oxide by denitrifying Flexibactercanadensis. Can J Microbiol 41:585–591

Yamagishi T, Leite J, Ueda S, Yamaguchi F, Suwa Y (2001)

Simultaneous removal of phenol and ammonia by an

activated sludge process with cross-flow filtration. Water

Res 35:3089–3096

Zaidi BR, Imam SH, Greene RV (1996) Accelerated biodeg-

radation of high and low concentrations of p-nitrophenol

(PNP) by bacterial inoculation in industrial wastewater:

the role of inoculum size on acclimation period. Curr

Microbiol 33:292–296

Zepeda A, Texier A-C, Gomez J (2003) Benzene transformation

in nitrifying batch cultures. Biotechnol Progr 19:789–793

Zepeda A, Texier A-C, Gomez J (2006) Kinetic and metabolic

study of benzene, toluene and m-xylene in nitrifying batch

cultures. Water Res 40:1643–1649

Zepeda A, Texier A-C, Gomez J (2007) Batch nitrifying cul-

tures in presence of mixtures of benzene, toluene, and

m-xylene. Environ Technol 28:355–360

Zhang TC, Lampe DG (1999) Sulfur:limestone autotrophic

denitrification processes for treatment of nitrate-contami-

nated water: batch experiments. Water Res 33:599–608

Zhang L, De Schryver P, De Gusseme B, De Muynck W, Boon

N, Verstraete W (2008a) Chemical and biological tech-

nologies for hydrogen sulfide emission control in sewer

systems: a review. Water Res 42:1–12

Zhang Z, Lei Z, He X, Zhang Z, Yang Y, Sugiura N (2008b)

Nitrate removal by Thiobacillus denitrificans immobilized

on poly(vinyl alcohol) carriers. J Hazard Mater 163:1090–

1095

Zumft WG (1997) Cell biology and molecular basis of deni-

trification. Microbiol Mol Biol R 61:533–616

342 Rev Environ Sci Biotechnol (2009) 8:325–342

123