Beruflich Dokumente
Kultur Dokumente
INDUSTRIAL WASTEWATER
Nitrification in Saline Industrial
Wastewater
DISSERTATION
Submitted in fulfilment of the requirements of
the Board for Doctorates of Delft University of Technology
and of the Academic Board of the UNESCO-IHE Institute for Water Education
for the Degree of DOCTOR
to be defended in public
on Monday, 29 March 2004 at 10:30 hours
in Delft, The Netherlands
by
This research was sponsored by BTS Senter (BTS99130), Shell Global Solutions International,
The Hague, Heiploeg Shrimp Processing, Zoutkamp and Ecco Tannery, Dongen. The project was
carried out at the departments of Environmental Resources, (UNESCO-IHE, Delft) and of
Biotechnology (Delft University of Technology).
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Symboles vii
Summary x
Chapter1 Introduction 4
Chapter2 Improved method for determination of ammonia and nitrite oxidation 32
activities in mixed bacterial cultures
Chapter3 Short term effects of various salts on ammonia and nitrite oxidisers in 48
enriched bacterial cultures
Chapter4 Long Term Effects of Salt on Activity, Population Structure and Floc 69
Characteristics in Enriched Bacterial Cultures of Nitrifiers
Chapter5 Modelling Nitrification, Heterotrophic growth and Predation in 95
Activated Sludge
Chapter6 Nitrification activities in full-scale treatment plants with varying salt 124
loads
Chapter7 Model-based evaluation of the upgrading of a full-scale industrial 140
wastewater treatment plant
Chapter8 Evaluation and Outlook 156
Nitrogen fixation
Fixation of nitrogen (physical, chemical or biological) means the incorporation of inert,
gaseous nitrogen into chemical compounds that eventually can be used by living
organisms. Biological fixation of N2 is prominently accomplished by specialized
microorganisms: cyanobacteria, symbiotic and free-living bacteria. Lightning also
indirectly transforms atmosphere nitrogen into nitrate, which rains onto soil. Finally, N2
can be fixed industrially by the Haber-Bosch process, invented in 1913. At present the
industrial fixation of nitrogen into ammonia plays a significant role, because it is
responsible for 30% of the total nitrogen influx into the biosphere (Gijzen and Mulder
2001).
Ammonification
In most ecosystems nitrogen is preliminary stored in living and dead organic mater.
Ammonification is the process responsible for the change of organic nitrogen compounds
into the ammonia form. In general, ammonification occurs during decomposition of
animal and plant tissue and animal faecal matter by bacteria; after hydrolysis of the
proteins, the amino acids are either reused or the amino groups are converted into
ammonia. Also the nitrogen present in urine is—via urea—converted into ammonia.
Nitrification
Nitrification is the biological oxidation of ammonium. This is done in two steps, first to
the nitrite form, then to the nitrate form. Both steps can be carried out by different genera,
both using CO2 as their source of cellular carbon. These transformation reactions are
generally coupled and proceed rapidly to the nitrate form; under normal conditions nitrite
levels are usually very low. The produced nitrate is used either by plants in the
assimilation process or reduced by denitrification to N2.
Denitrification
Denitrification is the biological reduction of nitrate to nitrogen gas. It can proceed
through several steps in the biochemical pathway, with the ultimate production of
nitrogen gas. A fairly broad range of heterotrophic bacteria is involved in the process,
requiring an organic carbon source for energy (Kuenen and Robertson 1994; Schmidt et
al 2003).
ANAMMOX
The denitrifying bacteria (as described above) are not the only bacteria producing
nitrogen gas. Ammonia can be oxidized under anaerobic conditions also leading to N2
and it became clear that slow growing autotrophic bacteria belonging to the order of the
Planctomycetales are carrying out this process. This process, in which both ammonia and
nitrite are converted to N2, is called ANAMMOX, an acronym for ANaerobic AMMonia
OXidation (Mulder et al 1995; Schmidt et al 2003).
Assimilation
Assimilation is the process in autotrophic organisms in which nitrogen compounds
(NH4+, NO3−) are incorporated into cell material for growth, a biochemical mechanism
that uses ammonia or nitrate. Animals and other heterotrophic organisms require protein
from plants and other animals as their nitrogen source. They are not capable of
transforming inorganic nitrogen into an organic nitrogen form.
The influx and efflux of N in the biosphere has been kept in balance by nature. Several
decades ago this balanced situation started to undergo a radical change mainly due to
binding of atmospheric nitrogen gas for the manufacturing fertilisers (the invention of
ammonia synthesis by Fritz Haber).
The first commercial ammonia factory started its operations in 1913 in Germany, but
production levels at a global scale remained low until the process became more energy
efficient due to technological innovations in the 1960s. Since then the production of
industrial nitrogen fertiliser via the so-called Haber-Bosch process showed a sharp
increase. This process has removed the fundamental restriction on food production and
therefore on population growth. Indeed, the doubling of the world population over the
last 40 years would not at all have been possible without the intensive agriculture and
animal production systems which primarily depend on nitrogen fertiliser. The increase in
production of nitrogen fertiliser has been much faster than population increase. While
population doubled between 1960 and 2000, the annual production of fertiliser nitrogen
increased nine-fold from 1×1010 to 9×1010 kg. Current production is equivalent to about
37% of the total amount of nitrogen input achieved via terrestrial and marine biological
N2 fixation (about 24×1010 kg per year). There is probably no other elemental cycle
where the human impact has been so dramatic as the case for nitrogen (Gijzen and
Mulder 2001).
The massive introduction of reactive forms of nitrogen into the environment over a
relatively short period of time has numerous deleterious consequences, causing
environmental and public health problems, both locally and at a global scale (Scheible
and Heidman, 1994; Vitousek et al 1997; Wiesmann 1994):
• The formation of blooms of toxic cyanobacteria in fresh waters is of considerable
concern with respect to human and animal health (e.g. potable water supply, fish
production). Eventually the produced cyanobacteria, algal and plant biomass will die
Introduction 7
When analysing the nitrogen pollution, care must be taken that the source of pollution is
identified, in order to take measures to solve the problem. Different sources can
contribute to the nitrogen problem, differing from location to location. The challenge is to
find out the most effective approach to tackle the most urgent and most important one.
Moreover, the approach for the same problem in one specific location may not be
applicable to another.
Not only the nitrogen concentration, but also the amount of water polluted with nitrogen
compounds is playing a role. The quantities of the three main sources of pollution
(agriculture, industry and municipalities) are presented in Table 1.2. It illustrates that
70% of the total water use is for agriculture, 20% for industry and only 10% for domestic
purposes. Industries generate more wastewater with higher quantities of nitrogen than
municipalities.
Introduction 9
The fast population growth, urbanisation and industrialisation, all of which impose high
demands on local water resource quality and quantity, while simultaneously generating
pollution, which affects the very same water resource. The response to increasing
pollution problems in receiving waters, and the growing concern about water quality
protection, necessitated the promulgation of effluent standards for nutrients, especially
for sensitive areas. In this framework environmental legislation in most countries
includes stringent limitations for nitrogen to be discharged. National technology-based
standards were established, moving all wastewater treatment facilities to secondary level
at minimum.
Considering N-removal, in 2002 the removal efficiency averaged 69.4%. It is not certain,
whether the 75% target for nitrogen removal will be achievable by 2006, taking the
limited planned investments into consideration.
In 2002, 40% of all sewage treatment plants failed at some time to meet the individual
discharge requirements. Rapid improvement of the nitrogen removal in the next few
years (before 2006) is a point of attention (Postma et al. 2003).
Due to the successful implementation of the Directive in many EU states European
waters have started to change. Reports by the European Environment Agency (1998,
1999a) clearly show improvements. The number of heavily polluted rivers has declined
significantly, in particular as the pressure from organic matter and phosphates in urban
wastewater has decreased. While progress has been made in many areas, others are still in
a deplorable situation. After 25 years of European water legislation, Europe’s waters are
in need of more protection, in need of increased efforts to get them clean or to keep them
clean. This is a demand not only from the scientific community and other experts, but
also to an ever-increasing extent from citizens and environmental organisations.
ANAMMOX in a single, aerated reactor. This process has been tested extensively on
laboratory scale (Slikers et al 1998, 2003). Although ANAMMOX requires strict anoxic
conditions, nitrifiers and ANAMMOX organisms are able to coexist under oxygen-
limited conditions. Therefore, CANON would need process control to prevent nitrite
build-up by oxygen excess under ammonia limitation (fluctuation of ammonia load).
1.4.1 Background
Industry is an essential engine of economic growth worldwide and requires adequate
resources of good quality water as a key raw material. Global annual water use by
industry is expected to rise from an estimated 725 km3 in 1995 to about 1,170 km3 by
2025, by which time industrial water usage will represent 24% of all water abstractions.
Industrial use of water increases with country income, ranging from 10% for low-and
middle-income countries to 59% for high-income countries (World Bank, 2001).
A number of industrial categories (petroleum refining, coke processing, dairy,
chemical production, tannery, fish processing) contain significant amounts of nitrogen in
their wastewater (Wiesmann 1994). Before discharge to the water body (according to the
stricter regulations), almost full nitrogen removal is necessary. This has led to increasing
activities in the field of development and optimisation of biological nitrogen removal.
Biological nitrogen removal is conventionally achieved by making use of processes of the
natural nitrogen cycle, namely through nitrification in an aerobic environment followed
by denitrification in an anoxic environment. Biological nitrification-denitrification is the
most common processes for nitrogen removal from wastewater; nitrification is the rate-
limiting step in biological nitrogen removal, because nitrifying bacteria are growing
slowly and are very sensitive to environmental factors (e.g. temperature, pH, dissolved
oxygen concentration, toxic and inhibitory compounds) (Antonious et al 1990; Wagner et
al 1996; Wagner and Loy 2002).
Studies so far have concentrated mainly on domestic wastewater treatment and the
results obtained may not be directly applicable to industrial wastewater due to their
specific composition (high temperatures, sub-optimal pH values, presence of toxic
compounds or high salinity). Thus, special attention for and concern with the design and
operation of nitrogen removal for industrial wastewater treatment systems is necessary.
The effect of salts on nitrogen removal is a major concern, especially in industrial
wastewater treatment. Industries such as pickling, cheese manufacturing, seafood
processing, tanning, productions of chemicals and Pharmaceuticals, oil and gas recovery,
produce high inorganic salt concentrations in their wastewater. Other sources of saline
wastewater include infiltration of subsurface water in the coastal areas into the sewer
system, landfill leachates and contaminated ground water and ballast water for marine
vessels or offshore installations. In future, waste minimisation practices are expected to
generate brines via effective water reuse and recycling schemes. Also the use of saline
water for toilet flushing due to the scarcity of fresh water will increase the wastewater
salinity that reaches the treatment plants (Campos et al 2002; Dahl et al 1997; Woolard
and Irvine 1995; Yu et al 2002).
Nitrification is the bottleneck of the nitrogen removal process under salt stress, while
denitrification has proved to be more stable under salt stress (Vredenbregt et al 1997;
Dahl et al 1997). Studies on the effect of salt on nitrification are contradictory and
difficult to be interpreted.
Introduction 15
1.4.2 Nitrification
The nitrification process—an important process in the nitrogen cycle in nature—is
defined as the biological transformation of reduced forms of nitrogen into nitrite and
subsequently to nitrate. Generally, two absolutely different types of nitrification must be
distinguished (Schmidt et al 2003):
• Lithotrophic nitrification, in which the oxidation of inorganic, reduced nitrogen
compounds serves as energy source for growth. Lithotrophic nitrification is carried
out by two groups of bacteria, the ammonium-oxidizers and nitrite-oxidizers
• Heterotrophic nitrification, in which nitrification is a co-oxidation and does not
serve as an energy source. It is carried out by diverse groups of microorganisms
(bacteria, fungi, and algae).
In natural environments, the chemolithotrophic nitrifiers are the only group of
microorganisms producing considerably high amounts of nitrite and nitrate from
ammonia. The heterotrophic nitrifiers’ specific activity is estimated to be around 103– 104
times lower than that of lithotrophic nitrifiers and therefore heterotrophic nitrification is
of minor ecological significance (Kuenen and Robertson 1994; Richardson et al 1998).
Originally, the lithoautotrophic nitrifying bacteria altogether were grouped within one
family, named Nitrobacteraceae and composed of two physiologically distinct groups of
bacteria that are not phylogenetically related (ammonia-oxidizing bacteria and nitrite-
oxidizing bacteria). However, phylogenetic investigations made evident that a lot of
distinct groups of organisms exist, which are not closely related to each other (Koops and
Röser 2001). Cells of both groups are able to aggregate in clusters (flocs), which is
common in wastewater treatment plants (Stalely et al 1989).
Although the basic metabolism is more or less uniform within the physiologically defined
groups of lithoautotrophic ammonia oxidizing bacteria, ecophysiological differences exist
between the distinct representatives. Different members of these genera have been found
to dominate different wastewater treatment plants or natural ecosystems, but general
relationships between the ecological niche and evolutionary position are often still
obscure (Schmidt et al 2003).
Salt requirement is an ecophysiologically relevant discrimination factor. All isolates of
the two species of Nitrosococcus (γ subclass of the Proteobacteria), Nitrosococcus oceani
and Nitrosococcus halophilus, are obligately halophilic. The group located in the β
subclass of the Proteobacteria, comprises obligately halophilic species and moderately
halophilic or halotolerant species, respectively, together with species missing salt
requirement or being salt sensitive. Within the genus Nitrosomonas, these differences are
well reflected by the pronounced formation of phylogenetic lineages (Koops and Röser
2001).
Physiology
The physiology of conventional, ‘aerobic’ ammonia oxidizers is not completely
understood. Only recently, it was discovered that these organisms also have an anaerobic
metabolism. The proteobacterial ammonia oxidizers can obtain their energy for growth
from both aerobic or anaerobic ammonia oxidation. Most likely ammonia (NH3) and not
ammonium (NH4+) is the substrate for the oxidation process (Suzuki et al 1974; Bock et
al 1991). The main products are nitrite under oxic conditions (DO> 0.8 mg O2/L), while
under anoxic conditions (DO<0.8 mg O2/L) nitrogen gas, nitrite and nitric oxide are the
main products (Schmidt and Bock 1997).
Physiology
For nitrite oxidising bacteria the oxidation of nitrite to nitrate is the energy generation
process. There is some evidence that Nitrospira is the more specialized nitrite oxidizer.
The other genera are more versatile, are all able to use organic energy sources beside the
major source nitrite, being facultative autotrophs and anaerobes, able to grow on
heterotrophic substrates such as pyruvate and also capable of the first step of
denitrification (the reduction of nitrate to nitrite) (Schmidt et al 2003; Koops and Röser
2001).
Nitrification in saline industrial wastewater 18
Supclass of Nitrobacter
Proteobacteria hamburgensis
γ Nitrococcus obligatory halophilic
mobilis
Nitrospina gracilis obligatory halophilic marine
environment
δ Nitrospira marina obligatory halophilic
Nitrospira no salt requirement freshwater
moscoviesis
Nitrococcus mobilis and Nitrospina gracilis are both obligatory halophilic, all isolates
originate from marine habitats. The genus Nitrospira comprises obligatory halophilic
species (N. marina) together with nonhalophilic species (N. moscoviensis). With
Nitrobacter-isolates, obligate salt requirement has not been observed, although some
strains were isolated from marine environments or from soda lakes (Koops and Röser
2001).
The application of molecular methods revealed that yet uncultured Nitrospira-like
microorganisms and not Nitrobacter spp., are the dominating nitrite oxidisers in most
WWTPs. Nitrospira-like nitrite oxidisers are also of major importance in other
ecosystems like drinking water distribution systems or soil (Wagner and Loy 2002). A
recent study on the ecophysiology of the uncultured Nitrospira-like nitrite oxidisers in
activated sludge has shown that these bacteria are able to fix bicarbonate and to
simultaneously take up pyruvate (Daims et al 2001). Nitrospira-like nitrite oxidisers are
probably K-strategists (with high substrate affinities and low maximum activity or growth
rate) for oxygen and nitrite and thus outcompete Nitrobacter under substrate-limiting
conditions in WWTPs. This hypothesis would also explain why Nitrobacter and
Nitrospira co-exist in reactors with temporarily higher nitrite concentrations (Wagner and
Loy, 2002).
Introduction 19
(1.1)
The term total ammonia refers to the sum of NH3 and NH4+. The concentration of NH3 is
dependent on a number of factors in addition to total ammonia concentration. Most
important among these are pH and temperature; the concentration of ammonia increases
with increasing pH and with increasing temperature (Emerson et al 1975). The
stoichiometric dissociation constant (Ka) is defined as:
Ka=(NH3)(H+)/(NH4+)
Where the brackets represent molar concentrations and the following equation was used
to calculate Ka at all temperatures:
pKa=0.09018+2729.92/T
Where
pKa the negative log of stoichiometric dissociation constant and
T the temperature in °k
The percentage of unionised ammonia (NH3%) can be calculated from the solution pH
and pKa by the following equations:
NH3%=100(1+10(pKa−pH))−1
Ionic strength of a solution has a noticeable effect on the percent of unionised ammonia.
The fraction of ammonia in the unionised form decreases with increasing ionic strength
in hard water and saline water. In most natural fresh water systems the reduction of
unionised ammonia attributable to dissolved solids is negligible. In saline or very hard
waters there will be a small but noticeable reduction in unionised ammonia fraction
(Emerson et al 1975; Johansson and Wedborg 1980; Clegg and Whitfield 1995).
Adaptation mechanisms
Mechanisms for survival and growth in high-osmolarity conditions must aim at
maintaining osmotic equilibrium across the membrane (physiological means). Adaptation
to a saline cytoplasm usually covers only a relatively narrow range of physiological
reactions, whereas compatible solute producers have developed a more flexible type of
adaptation.
It is also apparent that seemingly salt-sensitive organisms may display this phenotypic
response only because of the lack of suitable solute. Hence the composition of the growth
medium has a great influence on the individual salt tolerance of bacteria. This is
especially pronounced with media containing complex components that may serve as a
source for compatible solutes. It should be noted that osmoadaptation at higher levels is
determined not only by the composition of the cytoplasm. It also requires major changes
in the composition of the membrane structure and arrangement (structural adaptation),
and although the external phase of the cytoplasmic membrane as well as the periplasmic
space are always in contact with a saline environment it still requires the necessary
adjustments. Most of the mechanisms known for salinity adaptation are by physiological
means (Galinski and Truper 1994).
bioenergetic point of view for chemoautotrophic nitrifying bacteria living at high salinity
(Oren 1999).
Studies on the effect of salt on nitrification show a decline in activity for ammonia and
nitrite oxidisers. However, it does not give a clear answer on the maximum acceptable
salt level and which nitrifying group is most sensitive to salt stress: ammonia oxidisers
(Campos et al 2002; Hunik et al 1992, 1993) or nitrite oxidisers (Catalan-Sakairi et al
1996; Dincer and Kargi 1999; Furumai et al 1988; Oren 1999; Vredenbregt et al 1997).
Moreover, results have a wide range, are difficult to compare and even show
contradictory effects (Table 1.8). Reasons for these contradictions might be:
• the system configuration and instability in the experimental conditions with respect
to temperature, pH, presence of inhibitory compounds or factors;
• the way of salt introduction to the system, as a pulse or by gradual increase;
• the species involved, use of pure or mixed cultures and of adapted or non-adapted
bacteria.
Table 1.8 Reported results on the impact of salt on
the nitrification activity and settling characteristics
in various systems and under different
environmental conditions.
The increasing pollution problems in receiving waters and the growing interest in water
quality protection necessitate more stringent effluent criteria. Especially with respect to
the discharge of nutrients like nitrogen effluent criteria are becoming more stringent.
Biological nitrification-denitrification is one of the most promising methods to remove
nitrogen from wastewater. However, nitrification, the rate limiting step in biological
nitrogen removal, proved to be one of the most difficult processes to design and control
in wastewater treatment plants, because nitrifying bacteria are slow-growing and very
sensitive to environmental factors (temperature, pH, dissolved oxygen concentration,
toxic and inhibitory compounds). Researchers so far have concentrated mainly on
nitrification in domestic wastewater treatment and achieved broad knowledge and
practical experience about the process. The result is that biological nitrogen removal is
widely and successfully applied for municipal wastewater. However, these experiences
are not directly applicable to industrial wastewater because of the specific composition of
the wastewater. Several industries are dealing with a high salt concentration in their
wastewater. Also the policy of more economic use of water and water reuse will result in
an increase of salt content of the ultimately produced wastewater. High salt levels may
negatively affect nitrification, demonstrating the need for improved understanding of the
precise effects of salt on nitrification.
The main focus of this dissertation is on the effect of salinity on nitrification. The
objective of the research was to generate an understanding, based on laboratory scale
experiments, modelling and full-scale investigation, of the sensitivity of the nitrification
process to sub-optimal salt concentrations in combination with other sub-optimal
environmental conditions expected to occur at full scale treatment plants. The dissertation
is organized in 8 chapters.
Chapter 1 introduces the subject and presents a general literature review on the global
nitrogen cycle and nitrogen control technologies with special emphasis on the
nitrification process. The contradictory results available on the impact of salt on
nitrification are discussed.
Chapter 2 presents a new method to measure the activity of ammonia and nitrite
oxidisers in mixed bacterial cultures. The developed method allows measuring of the
short-term effect of an inhibitor on both the ammonia and nitrite oxidisers in one test
without the use of additional metabolic inhibitors. This method was applied in the
research as a standard method to determine the inhibition effects of salt on ammonia and
nitrite oxidisers.
Chapter 3 presents the effect of various types of salt on the activity of ammonia and
nitrite oxidisers using enriched cultures of nitrifiers obtained from two lab-scale
sequencing batch reactors (SBRs) operated at different sludge ages. Basic equations for
the impact of salts on the activities of ammonia and nitrite oxidisers were proposed.
The long-term effects of salt on the activity and composition of nitrifiers are presented
in Chapter 4. The long-term effects of salt on the flock characteristics of nitrifying sludge
using salt-adapted (one year) and non-adapted enriched cultures of nitrifiers are also
presented.
Introduction 25
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Introduction 27
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Chapter 2
Improved Method for Determination of
Ammonia and Nitrite Oxidation Activities in
Mixed Bacterial Cultures
Previously published as:
Moussa MS, Lubberding HJ, Hooijmans CM, van Loosdrecht MCM, Gijzen HJ
(2003) Improved method for determination of ammonia and nitrite oxidation activities in
mixed bacterial cultures. Appl. Microbiol. Biotechnol. 63:217–221.
Abstract
A simple and reliable method to measure the activity of ammonia and
nitrite oxidisers in mixed bacterial cultures was developed. This method
considers nitrification as a two-step process and can also be applied to
measure the effects of specific inhibitors on the activity of nitrifiers. It
allows measuring of the short-term effect of an inhibitor on both the
ammonia and nitrite oxidisers in one test under controlled environmental
conditions (pH, temperature).
The developed method differentiates between the ammonia and nitrite
oxidisers by consecutive injection of NO2− and NH4+. The main advantage
of this method is avoiding the use of metabolic inhibitors for ammonia or
nitrite oxidisers, as used by other methods.
The method was applied in two different procedures, both using an
enriched culture of nitrifiers. In the first procedure a small reactor of 10
mL in which the oxygen consumption rate (OUR) was used to determine
the ammonia and nitrite oxidisers activities. This procedure only takes a
few minutes per sample and therefore is suitable for screening of a large
number of inhibitors in a short time period. In the second procedure a
reactor of 500 ml was used in which the ammonia and nitrite consumption
rate was determined. This procedure takes several hours. The advantage
compared to the other procedure is, however, that the obtained substrate
consumption rate can be used to determine the kinetic parameters of the
ammonia and nitrite oxidisers.
Both procedures were used to determine the inhibitory effects of salt
(NaCl up to 15 g Cl−/L) on an enriched culture of nitrifying bacteria at
lab-scale. The results obtained with the small reactor of 10 mL and the
500 mL reactor in the case of NaCl are very similar and in agreement with
the results obtained for pure cultures of ammonia and nitrite oxidisers.
The results of the method demonstrate its potential to accurately
determine the individual activities of nitrite and ammonia oxidisers.
2
Improved method for determination of
ammonia and nitrite oxidation activities in
mixed bacterial cultures
2.1 Introduction
were used to completely and instantaneously inhibit ammonia and nitrite oxidisers,
respectively, without affecting other activities of for instance, endogenous respiration,
ammonia oxidisers, and heterotrophic bacteria. However, potential interaction of a
selective metabolic inhibitor for ammonia or nitrite with the other toxic compounds to be
tested makes these methods unreliable. Such interaction might lead to unrealistic effects
of the toxicant on the nitrification activity. Therefore the aim of this research was to
develop a reliable, simple, robust and quick procedure to measure shock-load effects of
specific inhibitors on the activity of ammonia and nitrite oxidisers separately for a mixed
culture.
2.2.2 Media
Synthetic medium prepared with demineralized water had the following composition:
(NH4)2CO3 857.95 mg/L as ammonia source, NaH2PO4.H2O 167.5 mg/L, MgSO4.7H2O
90 mg/L, CaCl2.H2O 14 mg/L, KCl 36 mg/L, yeast extract 1 mg/L, nutrient solution 0.3
mL/L. The nutrient solution was added to the medium in order to enhance the microbial
growth and was prepared with the following chemicals mixed in one liter of
demineralized water: 1.5g of FeCl3.6H2O, 0.15g of H3BO3, 0.03g of CuSO4.5H2O, 0.18g
of KI, 0.12g of MnCl2.4H2O, 0.06g of Na2MoO4.2H2O, 0.12 g of ZnSO4.7H2O, 0.15 g of
CoCl.6H2O, and 10g of EDTA. The very low COD in the influent medium (10 mg/L
mainly due to the result of the yeast extract) was to enhance the growth of nitrifiers over
heterotrophs.
possible to obtain the separate activities. The method was implemented and tested in two
different procedures. In the first one, oxygen uptake rate (OUR) measurements were
used, in which it takes a few minutes to quantify the nitrification activity under normal or
inhibition conditions in a small sample volume (10-mL). In the second one, ammonia and
nitrite removal rate measurements were used to assess the nitrification activity in a 500-
mL sample volume. The ammonia and nitrite measurements will take several hours in the
bigger reactor but it allows the quantification of the kinetic parameters of nitrification
under normal or inhibition conditions (short-term effects).
consequently cause an increase in pH). The dissolved oxygen concentration was kept
above 4.0 mg/L. Temperature was controlled at 30°C by means of a water bath. When the
testing condition was stable (pH 7.5; DO above 4 mg/L; T 30 °C), the oxygen probe was
sealed in the BOM vessel in such a way that no air bubbles remained in the liquid. The
decrease in oxygen concentration was monitored and recorded by a computer. The
endogenous respiration rate of the nitrifiers was determined first. Hereafter, NaNO2 was
injected as substrate for the nitrite oxidisers and after 3 minutes NH4Cl was injected as
substrate for the ammonia oxidisers. The recorded OUR in these three phases, no
substrate, after nitrite injection and after ammonia injection are used to calculate the
nitrite and ammonia oxidisers activity (Figure 2.2). The biomass content of the tested
samples was determined as dry weight (g VSS/L) and the activity was expressed as
specific oxygen consumption rate (mg O2. (g VSS)−1.h−1)
The optimal initial concentration of NaNO2 to be added was determined. The
concentration should not be below the substrate affinity constant of nitrite (KNO2) and not
as high as to inhibit ammonia oxidisers (Anthonisen et al 1976; Antoniou et al 1990;
Sharma and Ahlert 1977). Ammonia oxidising activities were measured with different
NO2− concentrations (up to 50 mg-N/L) to determine the highest NO2− concentration
without inhibition. Moreover, the developed procedure was compared with another
method, in which 24 of µM azide were used to completely and instantaneously inhibit
nitrite oxidisers (Ginestet et al 1998).
(2.1)
(2.2)
where:
SNH4, SNO2 ammonia and nitrite concentration (mg-N/L)
XNH4, XNO2 ammonia and nitrite oxidisers (mg-COD/L)
maximum specific growth rates for ammonia and nitrite oxidisers (L/h)
2.3 Results
ionic strength.
(1)
ionic strength=0.5∑ (Ci Zi2), where Ci is concentration of ionic species (M) and Zi is the charge
of species
(2)
pH change refers to the change in the pH within the test time (5 minutes) due to the H+
production
(3)
Mind that all NH4+ is converted into NO3−, the ionic strength increased due to addition of NaOH
for pH control.
The oxygen uptake measurements using the BOM resulted in profiles shown in Figure
2.2. The initial slope is used for determination of the endogenous respiration. From the
next part of the OUR profile the nitrite and ammonia activity can be calculated. A
concentration of 10 mg-N/L was chosen since this had no effect on the ammonia
oxidisers and is higher than the KNO2 (0.5−1 mg-N/L). The ammonia concentration used
was similar to the concentration in the SBR cycle (100 mg-N/L) and high enough to
avoid any ammonia limitation. The coefficient of variation of the measured activity is
between 10–20%, which illustrates the reliability of the developed procedure.
The results were compared with an alternative method, using azide as metabolic
inhibitor for nitrite oxidisers. Lower nitrite oxidation rates were observed. Therefore, the
inhibitory effect of azide on the nitrite oxidiser was tested. NaNO2 was used as a
substrate for the nitrite oxidisers. The endogenous OUR was compared with the OUR
after injecting azide in the presence of NaNO2 (Figure 2.4). The instantaneous inhibition
of 24 µM azide to the nitrite oxidisers was about 80–90% (8 samples). The test was
repeated (6 samples) with a higher azide concentration of 48 µM, but full inhibition of the
nitrite oxidisers could not be reached.
2.4 Discussion
agreement with the value reported by Ginestet et al (1998). The ammonia concentration
(100 mg-N/L) used was higher than the ones reported by Ginestet et al (1998), Kong et al
(1996) and Nowak and Svardal (1993) which were 50, 2.5 and 20 mg-N/1, respectively.
The ammonia concentration used in this study (100 mg-N/l of NH4+) appeared to have no
toxic effect on both ammonia and nitrite oxidisers. The enriched bacterial culture was
adapted to this concentration because it was the same as used for cultivation in the pulse
fed SBR systems, which were running for more than two years at the start of the
experiments.
2.5 Conclusions
A simple and reliable method to measure the activity of ammonia and nitrite oxidisers in
mixed bacterial cultures was developed. The main advantage of this method is avoiding
the use of metabolic inhibitors for ammonia or nitrite oxidisers, as used by other methods.
The method was successfully applied to determine the inhibitory effects of salt (NaCl
up to 15 g Cl−/L) on nitrifying bacteria at lab-scale in two different procedures: a small
reactor of 10 mL in which the oxygen consumption rate (OUR) was determined and a
reactor of 500 mL in which the ammonia and nitrite consumption rate was determined.
The results obtained from both procedures are very similar and in agreement with the
results obtained for pure cultures of ammonia and nitrite oxidisers.
The developed method could be extended to determine the kinetics of other
environmental samples containing nitrifiers, such as samples from activated sludge
systems and biofilms provided the following precautions are taken:
• Select a proper washing/re-suspending medium (in case of BOM), which has the same
ionic strength as the growth medium and a high buffering capacity to prevent pH shift;
• Avoid having an activity faster than the response time of the oxygen electrode (in case
of BOM) and take into account the required corrections for the calibration of the of
oxygen electrode (pressure, temperature, salt or any other interference);
• Test the suitable NO2− and NH4+ concentration to be used (maximal activity without
toxicity);
• Inject consecutively NO2− and NH4+ to fully aerated samples and to control the
environmental conditions (pH, temperature) during the test time period.
References
Anthonisen AC, Loher RC, Prakasam TBS, Srinath EG (1976) Inhibition of nitrification by
ammonia and nitrous acid. J. Water Pollut. Control Fed. 48:835–852.
Antoniou P, Hamilton J, Koopman B, Jain R, Holloway B, Lyberatos G, Svoronos SA (1990)
Effect of temperature and pH on the effective maximum specific growth rate of nitrifying
bacteria. Water Res. 24:97–101.
APHA (1998) Standard methods for the examination of water and wastewater, 20th edn. American
Public Health Association/American Water Works Association/Water Environment Federation,
Washington D.C.
Nitrification in saline industrial wastewater 44
Belser LW, Mays EL (1980) Specific inhibition of nitrite oxidation by chlorate and its use in
assessing nitrification in soil and sediments. Appl. Environ. Microbiol. 39:505–510.
Ginestet P, Audic JM, Urbain V, Block JC (1998) Estimation of nitrifying bacterial activities by
measuring oxygen uptake rate in the presence of the metabolic inhibitor allylthiouria and azide.
Appl. Environ. Microbiol. 64:2266–2268.
Ginkel CG van, Tramper J, Luyben KChAM, Klapwijk A (1983) Characterisation of Nitrosomonas
europaea immobilised in calcium alginate. Enzyme Microb. Technol. 5:297–303.
Hellinga C, Schellen AAJC, Mulder JW, van Loosdrecht MCM, Heijnen JJ (1998) The SHARON
process: an innovative method for nitrogen removal from ammonia-rich wastewater. Water Sci.
Technol. 37:135–142
Hunik JH (1993) Engineering aspects of nitrification with immobilised cells. PhD Thesis.
Wageningen Agricultural University, The Netherlands.
Hynes RK, Knowles R (1983) Inhibition of chemoautotrophic nitrification by sodium chlorate and
sodium chlorite: a re-examination. Appl. Environ. Microbiol. 45:1178–1182.
Kong Z, Vanrolleghem P, Willems P, Verstraete W (1996) Simultaneous determination of
inhibition kinetics of carbon oxidation and nitrification with a respirometer. Water Sci. Technol.
30:825–836.
Leenen EJTM, Boogert AA, van Lammeren AAM, Tramper J, Wijffels RH (1997) Dynamic of
artificially immobilised Nitrosomonas europaea: effect of biomass decay. Biotechnol. Bioeng.
55:630–641.
Nowak O, Svardal K (1993) Observations on the kinetics of nitrification under inhibiting
conditions caused by industrial wastewater compounds. Water Sci. Technol. 28:115–123.
Nowak O, Svardal K, Schweighofer P (1995) The dynamic behaviour of nitrifying activated sludge
systems influenced by inhibiting wastewater compounds. Water Sci. Technol. 31:115–124.
Reichert P, Ruchti J, Simon W (1994) Aquasim 2.0. Swiss Federal Institute For Environmental
Science and Technology (EAWAG), Dübendorf, Switzerland.
Smolders GJF, van Loosdrecht MCM, Heijnen JJ (1994) Stoichiometric model of the aerobic
metabolism of the biological phosphorus removal process. Biotechnol. Bioeng. 44:837–848.
Spanjers H, Vanrolleghem P, Olsson G, Dold PL (1998) Respirometry in control of the activated
sludge process: principles. Int. Water Assoc. Q, Scientific and Technical Report 7.
Sumacz-Gorska J, Gernaey K, Demuynck C, Vanrolleghem P, Verstraete W (1995) Nitrification
process control in activated sludge using oxygen uptake measurements. Environ. Technol.
16:569–577.
Sumacz-Gorska J, Gernaey K, Demuynck C, Vanrolleghem P, Verstraete W (1996) Nitrification
monitoring in activated sludge by oxygen uptake (OUR) measurements. Water Res. 30:1228–
1236.
van Loosdrecht MCM, Jetten MSM (1998) Microbiological conversions in nitrogen removal.
Water Sci. Technol. 38:1–7.
Chapter 3
Short Term Effects of Various Salts on
Ammonia and Nitrite Oxidisers in Enriched
Bacterial Cultures
Submitted as:
Moussa MS, Song Y, Lubberding HJ, Hooijmans CM, van Loosdrecht MCM, Gijzen
HJ (2003) Short-term effects of various salts on ammonia and nitrite oxidisers in enriched
bacterial culture Appl. Microbiol. Biotechnol.
Abstract
The effect of various types of salt on the activity of ammonia and nitrite
oxidisers was investigated. Enriched cultures of nitrifiers obtained from
two lab-scale sequencing batch reactors operated at sludge age (SRT) of
30 and 100 days were used in this study. The fluorescent in situ
hybridisation technique was used to identify the presence of ammonia and
nitrite oxidisers in both reactors. Respiration activity tests were used to
determine the shock load effects of salt on ammonia and nitrite oxidisers
under controlled conditions (pH 7.5, T: 30°C).
At the same molar concentration the divalent cations (CaCl2, MgCl2)
have a stronger inhibitory effect than monovalent cations both on
ammonia and nitrite oxidisers. The effect of different salt ions was
evaluated and quantified. Based on this a basic equation for the impact of
salts on nitrification processes was proposed.
SRT has no effect on the tolerance of ammonia oxidisers for shock
loads of salt, nor on the type of ammonia oxidisers present. Moreover, the
different ammonia oxidising species seem to have a similar response to
salt stress at similar environmental conditions. In contrast, SRT has a
significant impact on salt tolerance of nitrite oxidisers: the longer the
sludge age the stronger the inhibition. The results demonstrate that
Nitrobacter agilis is more resistant to shock loads of salt than Nitrospira
under the same environmental conditions.
3
Short term effects of various salts on
ammonia and nitrite oxidisers in enriched
bacterial cultures
3.1 Introduction
Nitrifying bacteria and the process of nitrification are sensitive to environmental factors
such as temperature, dissolved oxygen concentration, pH, available substrate, product
inhibition and inhibitory compounds (Antoniou et al 1990; Hellinga et al 1998; Sharma
and Ahlert 1977). Understanding the effect of these factors will lead to better design and
operation of wastewater treatment plants (WWTP). Keller et al (2002) have summarised
the existing literature on nitrification and stressed the use of new technologies and the use
of modelling. Additionally, a better understanding of the ecology and physiology of the
prevailing organisms in nitrifying reactors may also help to improve the treatment
efficiency (Dabert et al 2002; Wagner et al 2002).
The effect of salts on nitrification is a major concern nowadays, especially in
industrial wastewater treatment. Industries such as pickling, cheese manufacturing,
seafood processing, tanning, chemicals and pharmaceuticals productions, oil and gas
recovery produce high inorganic salt concentrations in their wastewater. Other sources of
saline wastewater include infiltration of subsurface water in the coastal areas, landfill
leachates and contaminated ground water. Waste minimisation practices are expected to
generate brines in future via effective water reuse and recycling schemes. Also the use of
saline water for flushing due to the scarcity of fresh water will increase the wastewater
salinity that reaches treatment plants (Campos et al 2002; Dahl et al 1997; Woolard and
Irvine 1995; Yu et al 2002).
It is not clear what the maximum acceptable salt level is and which nitrifying group is
most sensitive to salt stress: ammonia oxidisers (Campos et al 2002; Hunik et al 1992,
1993) or nitrite oxidisers (Catalan-Sakairi et al 1996; Dincer and Kargi 1999; Furumai et
al 1988; Vredenbregt et al 1997). Contradictions from literature reports on this issue can
be explained by the involvement of different microbial species, the use of either pure
culture or mixed culture, differences in the environmental conditions, type of salt and the
way of salt application.
This study aims at qualifying and quantifying the short-term effect of different types
of salt on ammonia and nitrite oxidisers. Short-term effects represent the sudden increase
of salt concentration for non-salt adapted species, which is similar to salt fluctuation
occurring in practice. In addition, the influence of sludge age on the salt inhibiting effect
will be assessed. Long sludge age is commonly applied in industrial WWTPs as safety
approach to avoid the washout of nitrifiers and to reduce the sludge production. For this
Short term effects of various salts 49
purpose stable enrichment cultures of nitrifying bacteria were subjected to various salts
and salt concentrations. The respiration activities were monitored to assess salt effects.
3.2.2 Media
Synthetic medium prepared with de-mineralised water had the following composition:
(NH4)2CO3 857.95 mg/L as ammonia source, NaH2PO4.H2O 167.5 mg/L, MgSO4.7H2O
90 mg/L, CaCl2.H2O 14 mg/L, KCl 36 mg/L, yeast extract 1 mg/L, nutrient solution 0.3
mL/L. The nutrient solution was added to the medium in order to enhance the microbial
Nitrification in saline industrial wastewater 50
3.3 Results
shown in Figure 3.1. Ammonia was consumed within 95 and 115 minutes at SRTs of 30
and 100 days, respectively. Accumulation up to 40 mg/L of NO2–N was observed in the
reactor operated at 30 days, while only 4 mg/L NO2–N was detected at an SRT of 100
days. Full oxidation of ammonia and nitrite occurred within 2 h, the rest of the cycle was
a starvation period for the ammonia and nitrite oxidisers. Biomass concentrations of 1140
mg MLVSS/L and 3268 mg MLVSS/L were measured in the reactor operated at SRT of
30 days and 100 days, respectively.
3.3.4 Effect of different types of salt on nitrite oxidisers (at 30 days SRT)
A similar reduction in the activity of nitrite oxidisers was observed as for ammonia
oxidisers (Figure 3.3). The chloride salts with divalent cations have a stronger effect on
nitrite oxidisers (Ca++, Mg++: 50% inhibition at 183 mM) than with monovalent cations
(Na+, K+: 50% inhibition at 350 mM) (Figure 3a).
The effect of other anions (I−, F−, SO4−−) is shown in Figure 3b. Sodium sulphate,
potassium sulphate and potassium iodide were more inhibitory (Na2SO4, K2SO4, KI: 50%
inhibition at 230 mM) than sodium fluoride (NaF: 50% inhibition at 470 mM).
Nitrification in saline industrial wastewater 54
3.3.5 Influence of sludge age on the salt tolerance of ammonia and nitrite
oxidisers
With respect to ammonia oxidisers no significant difference in salt tolerance was
observed between the two reactors operated at 30 and 100 days SRT (Figure 3.4).
However, the nitrite oxidisers in the 100 days reactor were significantly more inhibited
than the ones in the 30 days reactor (Figure 3.5).
Short term effects of various salts 55
3.4 Disscusion
In this study the impact of different types of salt on activities of ammonia and nitrite
oxidisers was explored in two nitrifying SBR reactors differing in sludge age. The
composition of the nitrifying community was monitored using FISH aiming to correlate
this composition with the observed activities.
(3.1)
The inhibition effects of NaCl, KCl, Na2SO4 and K2SO4 found in this study are in
agreement with the formula reported by Hunik et al (1992). However, the proposed
formula failed to describe the inhibition effect of CaCl2, MgCl2, NaF and KI, salts not
included in their study. Our results showed that at the same molarity, the divalent cations
have a higher inhibitory effect than the monovalent cations. Thus the inhibitory effect of
salts on ammonia oxidisers was likely due to the ionic strength rather than to molar
concentration.
The inhibition of salt on ammonia oxidisers cultivated at different sludge ages (30 and
100 days) is in agreement with the inhibition in pure culture (Hunik et al 1992) as shown
in Figure 3.6. This suggests that Nitrosomonas (dominant in our systems and in most
wastewater treatment plants) responds in a similar way to salt stress at similar
environmental conditions. This hypothesis could also explain the results of Catalan-
Sakairi et al (1997); Dahl et al (1997); Vredenbregt et al (1997), who worked in different
systems, but found similar inhibition effects.
Other researchers claim different responses of ammonia oxidisers to salt stress
(Campos et al 2002; Dincer and Kargi 2001; Panswad and Anan 1999 a, b). This
disagreement is probably caused by differences in the environmental conditions, the way
of introducing salt to the system, and the presence of destabilising factors. The sensitivity
Nitrification in saline industrial wastewater 58
of ammonia oxidisers as well as the presence of destabilising factors during the tests
(uncontrolled pH, Temperature) might lead to unrealistic inhibition behaviour (Moussa et
al 2003).
(30°C, pH 7.5) was investigated by Hunik et al (1993). Similar to ammonia oxidisers they
concluded that osmotic pressure could explain the inhibition in activity. They also
proposed a formula that describes the inhibition of salt on nitrite oxidisers as a function
of salt concentration (equation 3.2).
(3.2)
The inhibition effects of the NaCl, KCl and NaF on nitrite oxidisers in the 30 days SRT
reactor agrees with this formula. However, again the proposed formula failed to describe
the inhibition effect of CaCl2, MgCl2, Na2SO4, and KI. The results of these salts show
that at the same molarity, the divalent cations have a higher inhibitory effect than the
monovalent cations. So also for nitrite oxidisers the inhibitory effect of salt is likely due
to the ionic strength rather than to the osmotic pressure.
A significant difference was observed between sludge cultivated at 100 days on the
one hand and sludge cultivated at 30 days and pure culture of Nitrobacter agilis used by
Hunik et al (1993) on the other hand. Higher sludge age led to more inhibition (Figure
3.6). These results suggest that an increase of the sludge age resulted in reduction of
Nitrobacter sp. and consequently in a reduction of resistance of nitrite oxidisers to shock
loads of salt. So at similar environmental conditions Nitrobacter sp. might be more
resistant to shock load effects of salt than Nitrospira sp. It remains unclear, whether the
uncultured Nitrospira sp. has also less resistance to salt stress than Nitrobacter sp. under
prolonged salt stress. More research is needed to correlate the microbial population with
the physiology of nitrite oxidisers.
(3.3)
Nitrification in saline industrial wastewater 60
(3.4)
The constants in equation (3.4) are estimated to be 92 and (95% reliability interval of 71
to 112) and −227 (95% reliability interval of −454 to −139).
MgCl2, Na2SO4, K2SO4, NaF, and KI on nitrite oxidiser activities operated at 30 days
SRT resulted in equation (3.5).
(3.5)
(3.6)
The first constant is estimated to be 91.6 and with 95% reliability interval of 82 to 100.8
and the second one is estimated to be −218.9 and with 95% reliability interval of (−254 to
−183.74).
Ammonia oxidisers: All the inhibitory effects of various salts on ammonia oxidisers
were explained as a function of ionic strength of salt cations, which leads to the following
two hypotheses for salt stress: 1) Interaction of the positive salt ions with the enzymes
catalysing the ammonia oxidation reaction (ammonia-mono-oxygenase and/or
hydroxylamine oxidoreductase). 2) The positive salt ions may affect the dissociation
constant of ammonia (Clegg and Whitfield 1995) resulting in a reduction of the unionised
form of ammonia (the main substrate of ammonia oxidisers).
Nitrite oxidisers: In contrast with the ammonia oxidisers the inhibitory effects of
various salts on nitrite oxidisers were explained as a function of ionic strength of salt
anions, which leads to the following two hypotheses for salt stress: 1) Interaction of the
negative salt ions with the enzyme catalysing the nitrite oxidation reaction (nitrite
oxidoreductase). 2) Competition of the negative salt ions with NO2−, the main substrate
for nitrite oxidisers.
Besides, a general effect of cations and anions on the membrane processes of
ammonia and nitrite oxidisers, leading to an impaired protonmotive force, can also not be
excluded.
3.5 Conclusions
1- The shock load effects of monvovalent and divalent salt ions on ammonia and nitrite
oxidiser activities can be described as a function of ionic strength of salt cations for
ammonia oxidisers and of ionic strength of salt anions for nitrite oxidisers; formulas
describing this inhibition are proposed;
2- Fluoride and iodide salts have a stronger inhibitory effect (two times higher) on
ammonia oxidisers than other salts tested in this study. Inhibition of fluoride and
iodide on nitrite oxidisers was in the same range as the other salts;
3- SRT has no effect either on the tolerance of ammonia oxidisers for shock loads of salt
or on the type of ammonia oxidisers present. In contrast, it has an effect on the type of
nitrite oxidisers present and consequently on the tolerance of nitrite oxidisers for
shock loads of salt.
4- Nitrobacter is detected in the 30 days SRT reactor is correlated with high nitrite levels
(40 mg NO2–N/L). This result confirms the hypothesis that Nitrobacter can compete
successfully only in environments with relatively high nitrite concentrations.
5- Nitrobacter is more resistant to the shock loads of salt than Nitrospira.
References
4.1 Introduction
Nitrification is the biological oxidation of ammonia to nitrate via nitrite by two groups of
chemolithotrophic bacteria, ammonia oxidisers and nitrite oxidisers; both groups have
low specific growth rates (Bock et al 1991; Prosser 1989). Nitrifying bacteria and the
process of nitrification are sensitive to environmental factors such as temperature,
dissolved oxygen concentration, pH, available substrate, product inhibition and inhibitory
compounds (Antoniou et al 1990; Hellinga et al 1998; Sharma and Ahlert 1977). There is
considerable interest in understanding the ecology of nitrifying bacteria, because
nitrification is the bottleneck for biological nitrogen removal in many wastewater
treatment plants (WWTPs) and the causes for a sub-optimal performance or even absence
of the nitrification process are not always clear. Once nitrifiers have been washed out of a
WWTP, recovery of the nitrification process can take long time due to the slow growth
rates of the nitrifiers. There is an urgent need for interdisciplinary research at the
interface between molecular microbial ecology and process engineering to understand the
links between microbial diversity, process efficiency and process stability (Dabert et al
2002; Nogueira et al 2002; Wagner et al 2002).
Nowadays salt is considered as a common stress factor in WWTPs, especially in the
industrial sector. Industries such as pickling, cheese manufacturing, seafood processing,
tanning, the production of chemicals and pharmaceuticals, oil and gas recovery produce
high inorganic salt concentrations in their wastewater. Other sources of saline wastewater
include infiltration of subsurface water in the coastal areas, landfill leachates and
contaminated ground water. Waste minimisation practices are expected to generate brines
in future via effective water reuse and recycling schemes. Also the use of saline water for
flushing due to the scarcity of fresh water will increase the wastewater salinity that
reaches the treatment plant (Campos et al 2002; Dahl et al 1997; Woolard and Irvine
1995; Yu et al 2002).
Studies on the effect of salt on nitrification are difficult to compare and show
contradictory results (Campos et al 2002; Catalan-Sakairi et al 1996; Dahl et al 1997;
Dincer and Kargi 1999; Hunik et al 1992, 1993; Intrasungkha et al 1999; Panswad and
Anan 1999 a, b; Vredenbregt et al 1997; Yu et al 2002). Reasons for these contradictions
might be: (1) the system configuration and instability in the experimental conditions with
respect to temperature, pH, presence of inhibitory compounds or factors; (2) the way of
salt introduction to the system, as a pulse or by gradual increase; (3) the species involved,
use of pure or mixed cultures and of adapted or non-adapted bacteria.
Nitrification in saline industrial wastewater 70
The work presented here describes the effect of NaCl on the activity, population
structure and floc-forming characteristic of nitrifying sludge, adapted or not adapted to
salt. A systematic approach was followed in which pH and DO were kept constant and
nitrifying sludge was exposed to a gradual increase of NaCl (when full inhibition was
reached, the conditions were brought back to the initial stage).
increasing the salt to the next level. The biomass activity was measured immediately after
increasing the salt level (shock load effect) and again after 2 weeks of adaptation. The
activities of nitrifiers in both reactors R2 (one year at 10 g Cl−/L) and R3 were monitored
and compared four weeks after increasing the salt level to 10 g Cl− /L in R3.
Phase II: The salt concentration was further increased from 10 g Cl−/L NaCl (5 g
−
Cl /L per step) up to almost full inhibition level in R2 and R3. An adaptation period of
two weeks was allowed at each step before increasing the salt to the next level. The
biomass activity was measured immediately after increasing the salt level and again after
two weeks of adaptation. Whenever NO2− concentrations during the cycle became
limiting for optimal activity of nitrite oxidisers, NaNO2 was added manually up to the
concentration needed (25–30 g N–NO2−/L).
Phase III: When almost full inhibition level (95%) was reached, the salt level was
adjusted to its original level (R2:10 g Cl−/L and R3:0 g Cl−/L) by feeding them with their
original media. The activities of nitrifiers were monitored during the recovery period.
4.2.2 Medium
Synthetic medium was prepared with de-mineralised water had the following
composition: (NH4)2CO3 857.95 mg/L as ammonia source, NaH2PO4.H2O 167.5 mg/L,
MgSO4.7H2O 90 mg/L, CaCl2.H2O 14 mg/L, KCl 36 mg/L, yeast extract 1 mg/L, nutrient
solution 0.3 mL/L. The nutrient solution was added to the medium in order to enhance
the microbial growth and contained 1.5g of FeCl3.6H2O, 0.15g of H3BO3, 0.03g of
CuSO4.5H2O, 0.18g of KI, 0.12g of MnCl2.4H2O, 0.06g of Na2MoO4.2H2O, 0.12 g of
ZnSO4.7H2O, 0.15 g of CoCl.6H2O, and 10g of EDTA in one litre of de-mineralised
water.
4.2.3 Analysis
Ammonia and nitrite were measured spectrophotrometrically in accordance with
Standard Methods (APHA, 1998). Nitrate-N was determined using Dionex 4500i series
and Shimadzu C_R5A ion-chromatograph. The mixed liqueur volatile suspended solids
(MLVSS) determination was performed after filtration of a 10 mL sample of mixed
liquor on a Whatman glass micro fibre filter (GC/F) filter. Dry weight was determined
after the filter was dried for 24 h at 105°C and weighted on a microbalance. The ash
content was calculated after incinerating the dried filter in an oven for 1 h at 550°C. The
sludge retention time (SRT) was calculated from the biomass concentration (MLVSS) in
reactors and biomass concentration in the effluent. The floc size in the reactors was
followed using image analysis. The average floc diameter was measured using a
representative sample, in which at least 500 particles were analysed (Tijhuis et al 1994).
4.3 Results
added in two steps). Ammonia oxidisers were more sensitive to short and long term stress
of 10 gCl−/L (36–39% drop in activity) than nitrite oxidisers (5–13% drop in activity).
The effect of 10g Cl−/L on both ammonia and nitrite oxidisers did not show significant
differences between the one-year adapted (R2) and the one-month adapted (R3) sludge.
Table 4.2 Effect of NaCl (10g Cl−/L) on the activity
of ammonia and nitrite oxidisers after shock load
(during the 1st cycle) and in steady state. Both
reactors were operated at 10 gCl−/L and were in
steady state, R2 during one year, R3 one month.
The 10 g of salt/L was added to R2 in one step, to
R3 as in two steps of 5 g Cl−/L salt each. Standard
deviations are indicated between brackets.
Phase II: The effects of the stepwise salt increase in R2 and R3 (5 g Cl−/L) on ammonia
and nitrite oxidisers are shown in Figure 4.3. Increase of salt led to lower activities. Up to
10 g Cl−/L the strong shock load effect was relieved after 2 weeks of adaptation. At 15 g
Cl−/L the inhibition effect took some time to reach its full effect; the long-term effect was
more severe than the short-term. There was no difference in behaviour between the long-
term (one year) and short-term (one month) acclimatised nitrifying sludge. Up to 15 g
Cl−/L the nitrite oxidisers were less affected by the salt, while at higher salt levels the
inhibition was comparable for both ammonia and nitrite oxidisers. More than 95%
reduction in activity was reached for both ammonia and nitrite oxidisers at a salt level of
40 gCl−/L in both reactors (R2 and R3).
Nitrification in saline industrial wastewater 76
state conditions under the new salt levels. Within a week under the same salt stress,
nitrifiers started to adapt to the new saline environment and stabilised their activities
under the new conditions, but never reached the initial activities.
Phase III: Two stages of recovery were observed (Figure 4.4): 1) Throughout the
elimination of salt during the first 1–2 days (9 cycles) the activity for both types of
nitrifiers increased 4 times. 2) During the following period at the initial salt levels (0 in
R3, 10 in R2) the activity increased but slowly. After 2 weeks the specific activities in R2
were 23 mg-N/gVSS.h for both ammonia and nitrite oxidisers, which is 40% of the initial
activity at 10gCl−/L. The specific activity for both ammonia and nitrite oxidisers in R3
reached 32 mg-N/gVSS.h after 2 weeks, which is 33% (ammonia oxidisers) and 53%
(nitrite oxidisers) of the initial activities at 0 g NaCl–Cl−/L.
Phase I:
Four species of ammonia oxidisers were observed in all 3 reactors up to 10 g Cl−/L:
Nitrosomonas oligotropha, Nitrosomonas europaea, Nitrosococcus mobilis and
Nitrosospira sp.. Nitrosomonas oligotropha was the dominant ammonia-oxidiser at 0 g
NaCl–Cl−/L, while N. europaea was the dominant at 10 g Cl−/L salt. In all reactors up to
10 gCl−/L Nitrospira sp. was the dominant nitrite oxidiser and Nitrobacter sp. could only
be detected in small numbers. No difference in the composition of ammonia and nitrite
oxidisers could be observed between R2 (adapted to 10 g Cl−/L for 1 year) and R3
(adapted to 10 g Cl−/L for only 1 month).
Phases II&III:
Above 10 gCl−/L and up to 30 gCl−/L only N. europaea and N. mobilis were found in
both reactors (R2 and R3), while no nitrite oxidisers could be detected. At 40 g Cl−/L
only N. europaea was observed. After 2 months of continuous operation at the initial salt
level only N. europaea and Nitrobacter sp. were detected. This suggests that other
organisms were not able to survive under these elevated salt levels. The quick
reappearance of Nitrobacter sp. after recovery suggests that it was still present even at 40
gCl−/L, but it was below detectable levels (Table 4.3).
Table 4.3 Composition of the nitrifiers during the
different phases of reactor operation under varying
salt concentrations (0–40 g Cl−/L). In phase I R2
and R3 were adapted to 10 g Cl− /L for one year
and one month respectively.
Phases Phase I Phase II Phase III
Back to
−
Salt conc. (gCl L) 0 10 20 30 40 10 0
Reactor R1/R3 R2 R3 R2 R3 R2 R3 R2 R3 R2 R3
Ammonia oxidisers:
Nitrosomonas europaea + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
Nitrosomonas oligotropha +++ ++ ++ − − − − − − − −
Nitrosococcus mobilis ++ + + +++ ++ +++ + − − − −
Nitrosospira sp + ++ ++ − − − − − − − −
Nitrite oxidisers:
Nitrospira sp ++ ++ ++ − − − − − − − −
Long term effects of salt on activity 79
Nitrobacter sp + + + − − − − − − + +
− not detected + present ++ present in relatively high number +++ abundant
4.4 Discussion
The results demonstrated the complexity of the effect of elevated salt levels on the
nitrification process. Nitrification activity, population structures and settling
characteristics are interrelated and are all directly affected by salt. Investigating the
impact of salt on one of these aspects and not considering the others might lead to wrong
conclusions and interpretations. Most of the literature reports emphasise only on the
effect of salinity on nitrifying activity resulting in contradictory interpretation of results.
Similar results were found in a previous enriched culture study by Moussa et al (2003 a,
b) and in a pure culture study by Hunik et al (1993). In contrast with these results, Dincer
and Kargi (1999) and Vredenbregt et al (1997) concluded from the accumulation of
nitrite that nitrite oxidisers were more affected. Probably, nitrite accumulation in their
experiments is not caused by salt stress, but due to oxygen limitation, phosphorous
limitation, and/or the presence of toxicants. Also Campos et al (2002) explained the
accumulation of nitrite in their system under salt stress to oxygen limitation. It is known
from nitrification studies under non-salt stress, that oxygen limitation is crucial and that it
leads to an incomplete nitrification process and to accumulation of nitrite (Garrido et al
1997; Pollice et al 2002; Picioreanu et al 1997). Salt affects directly the maximum
solubility of oxygen and the oxygen transfer rate (van ’t Riet and Tramper 1991), which
can lead to limited oxygen availability.
The inhibition of salt on long-term and short-term adapted nitrifying sludge—
measured as NH4+consumption, NO2− and NO3− formation (this study)—was similar to
the inhibition—assessed as oxygen uptake rate—of non-adapted nitrifying sludge
(Moussa et al 2003 a, b) and of pure cultures of nitrifiers (Hunik et al 1992, 1993) (Figure
4.5). Therefore, short-term activity measurements can be used as a quick tool to assess
the inhibition pattern of salt on nitrifiers.
The constant SRT and biomass levels during the experiments suggest that net yield
remains unchanged at different salt concentrations, which was also found by Dincer and
Kargi (2002) and Yu et al (2002). Elevated salt levels are leading to reduction in specific
activity, but not to changes in biomass content, which suggests that salt causes
inactivation of nitrifiers and/or an increased decay rate. This increased decay rate could
be attributed only to salt, since there are no grazers present under these conditions.
Table 4.4 Reported results on the impact of salt on
the nitrification activity and settling characteristics
in various systems and under different
environmental conditions
due to
initial high
biomass
(20g
VSS/L)
NO2
accumu
lation due
to DO
limitation,
not to
salinity
5–60 NaCl 6,18,30,36 8 25 SW EN no LA nm nm >18 gCl−/L 5
SRTmin is
25 days, at
0gCl−/L 12
days
10–20 NaCl 18,30 8 25 SW EN no LA nm nm NO2− 6
accum
ulation
above 12
gCl−/L
31–55 NaCl 3,6,12,18 nm*5 27–33 SW DA no LA na*5 + MLSS 8,9
decreased
with
increased
NaCl
20–43 NaCl 3,6,12,18 nm 27–33 SW SA to 5g LA na +
Cl−/L
<5 NaCl 3 nm 20–22 SW DA no LS nm nm 7
−
83 NaCl 18–20 8 28–30 SW MS to AQ nm nm NO2 3
sea accumu
water lation due
to
limitation
of trace
elements
and CO2
<5 NaCl 1–4 nm nm DW DA no BA nm nm 1
NaCl 10 7–8 30 IW SA to PF nm nm 10
10g
Cl−/L
0% (com NaCl 20 7–8 30 IW SA to PF nm nm NO2− was
pared to 10g the only
10gCl) Cl−/L product>20
gCl−/L
57% NaCl 34 7–8 30 IW SA to PF nm nm below 20
(compared 10g gCl−/L
to 20gCl) Cl−/L good
fluidizable
particles
Nitrification in saline industrial wastewater 82
are formed
NaCl 10 7– 25–30 IW/SW DA no PA 10 + Shock 4
8.3 load
caused
major
inhibition
33% NaCl 20 7– 25–30 IW/SW DA no PA 10 + Good
(compared 8.3 sludge
to 10gCl) stability
due to
gypsum
precip
itation
*1
DW=Domestic Wastewater; SW=Synthetic Wastewater; IW=Industrial Wastewater from a coal-fired power plant
*2
DA=Domestic Activated sludge performing nitrification; EC=Enriched Culture of nitrifying bacteria;
EN=Nitrosomonas and Nitrobacter in mixed culture SA=Salt Adapted activated sludge performing nitrification;
MS=Marine Sediment
*3
LA=Lab-scale Activated sludge unit; LS=Lab-scale Sequencing batch reactor; AQ=Nitrifiers immobilised in
macro-porous cellulose carrier; PF=pilot-scale Fluid-bed; PA=Pilot-scale Activated sludge unit; BA=Batch Reactor
*4
1=Andreadakis et al 1997; 2=Campos et al 2002; 3=Catalan-Sakairi et al 1996, 1997; 4=Dahl et al 1997; 5=Dincer
and Kargi 1999; 6=Dincer and Kargi 2002; 7=Intrasungkha et al 1999; 8, 9=Panswad and Anan 1999 a, b;
10=Vredenbregt et al 1997; 11=Yu et al 2002
*5
nm=not measured; na=not affected
Long term effects of salt on activity 83
increasing salinity. The increase in salt concentration reduced the electric double layers,
thereby reducing the overall repulsive force between particles. The microbial aggregates
then approached close enough so that increased hydrophobic interactions resulted in
increased aggregation and the formation of larger flocs.
These two mechanisms can be utilised to achieve good floc characteristics. Gradual
increase in salinity will stimulate the selection of dense flocs with minimum washout. On
the other hand, sudden increases in salinity increases water density, causing excessive
wash out of biomass. Our attempt to directly increase salinity from 0 to 20 g Cl−/L
resulted in a severe reduction of biomass due to biomass wash out and biomass flotation.
This observation agrees with the dramatic decrease in biomass content as a result of the
direct increase in salinity from 0 to 18 g Cl−/L as reported by Panswad and Anan (1999).
4.5 Conclusions
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Long term effects of salt on activity 89
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Chapter 5
Modelling Nitrification, Heterotrophic growth
and Predation in Activated Sludge
Submitted as:
Moussa MS, Lubberding HJ, Hooijmans CM, Gijzen HJ, van Loosdrecht MCM
Modelling Nitrification, Heterotrophic growth and Predation in Activated Sludge
Abstract
A mathematical model describing the interaction between nitrifiers,
heterotrophs and predators in wastewater treatment has been developed.
The inclusion of a predation mechanism is a new addition to the existing
activated sludge models. The developed model considered multi-substrate
consumption and multi-species growth, maintenance and decay in a
culture where nitrifiers, heterotrophs and predators (protozoa and
metazoa) are coexisting. Two laboratory-scale Sequenced Batch Reactors
(SBRs) operated at different sludge ages of 30 and 100 days for a period
of four years were used to calibrate and validate the model. Moreover, to
assess the predators activity a simple procedure was developed, based on
measuring the respiration rate with and without the presence of the
predators. The model successfully described the performance of two
SBRs systems. The fraction of active biomass (ammonia oxidisers, nitrite
oxidisers and heterotrophs) predicted by the proposed model was only
33% and 14% at SRT of 30 and 100 days, respectively. The high fraction
of inert biomass predicted by the model was in accordance with the
microscopic investigations of biomass viability in both reactors. The
presented model was used to investigate the effect of increasing sludge
age and the role of predators on the biomass composition of the tested
SBR system.
5
Modelling Nitrification, Heterotrophic growth
and Predation in Activated Sludge
5.1 Introduction
2- Investigate the role of sludge age on the different fractions of biomass (nitrifiers,
heterotrophs, predators) and on the accumulation of inert biomass in the SBR system;
3- Determine the impact of predators on the presence and activity of the nitrifiers and
heterotrophs.
5.2 Theory
period, 4 hours reaction period, 80 minutes for settling, and 30 minutes for effluent
discharge. The characteristics of the operating conditions are summarised in Table 5.1. A
synthetic medium containing mainly ammonia and nutrients to enhance the microbial
growth was used as SBR feeding. 1.5L of medium was fed during the filling period and
the effluent was removed at the end of the settling period. The Sludge Retention Time
(SRT) which was desired, was set by the amount of wasted sludge, which was removed
from the mixed reactor during each cycle and the biomass in the effluent (Figure 5.3).
Aeration was provided during the reaction period with airflow of 120 L/h. The two
reactors were continuously monitored (on-line measuring of DO, pH, addition of NaOH)
and sampled (MLSS, MLVSS, NH4+, NO2−, NO3−) during several cycles. On-line cyclic
measurements of DO and the addition of the amount of base solution consumed and
constant biomass concentration in the reactors confirmed a steady state condition. The
sludge age in each reactor was initially set at 100 days. When steady state was reached,
the second reactor was switched to operate at a lower SRT (30 days).
Table 5.1 Operational conditions of the sequenced
batch rectors (SBR)
N-Load 1200mg-N/L.day pH 7.5
COD Load 60 mg-COD/L.day Temperature 30 °C
HRT 10 h Stirrer speed 650 rpm
(SBR30days) 30 days Aeration 120 L/h
SRT
(SBR100days) 100 days
Nitrification in saline industrial wastewater 100
5.3.2 Media
Synthetic medium prepared with de-mineralised water had the following composition:
(NH4)2CO3 857.95 mg/L as ammonia source, NaH2PO4.H2O 167.5 mg/L, MgSO4.7H2O
90 mg/L, CaCl2.H2O 14 mg/L, KCl 36 mg/L, yeast extract 1 mg/L, nutrient solution 0.3
mL/L. The nutrient solution was added to the medium in order to enhance the microbial
Modelling nitrification, heterotrophic growth 101
growth and was prepared with the following chemicals mixed in one litre of de-
mineralised water: 1.5g of FeCl3.6H2O, 0.15g of H3BO3, 0.03g of CuSO4.5H2O, 0.18g of
KI, 0.12g of MnCl2.4H2O, 0.06 g of Na2MoO4.2H2O, 0.12 g of ZnSO4.7H2O, 0.15 g of
CoCl.6H2O, and 10 g of EDTA. The very low COD in the influent medium (10 mg/L)
was to enhance the growth of nitrifiers over heterotrophs.
the beginning of the next cycle. For the simulation of the solids separation (settling)
another completely mixed compartment (constant volume) was introduced (effluent
compartment) and connected to the main one with an advective link with a purification
recycle to return only the solid content in the outflow back to the SBR compartment. An
additional process for solids removal from the SBR compartment as function of the
required SRT was used to simulate the biomass wasting from the system. Aeration was
simulated by introducing a gas compartment (completely mixed compartment) connected
with the SBR compartment using a diffusive link. A schematic drawing of the SBR
configuration in Aquasim is given in Figure 5.4.
5.4 Results
oxidisers and of heterotrophs was not affected (reversible) in a respirometric activity test
when the salt was washed away (data not shown).
5.5 Discussion
model results are in agreement with experimental results reported by Pollice et al (2002).
They mentioned that the specific ammonia oxidation rate dropped to 14% of its initial
value as the consequence of increasing the SRT from 3 days to 24 days in a nitrifying
SBR system operated at 32°C and pH above 7.2.
These results make the role of SRT in nitrifying SBR systems clear. Increasing the
SRT increases the active biomass fraction and the oxidation rate up to a maximum level
(40 days SRT). Any further increase will not lead to any volumetric improvement. When
increasing the SRT till 40 days (the maximum volumetric oxidation rate), the active
biomass concentration will increase in the SBR, resulting in a faster volumetric substrate
utilisation rate and a longer starvation period per cycle. The active biomass reaches a
saturation level and only the inert biomass increases with increasing SRT. In these model
simulations and experimental tests the cycle length was kept constant. Operating the SBR
system at a shorter cycle length will lead to an increased volumetric oxidation rate.
Therefor, other systems operated at a long SRT (such as membrane bioreactors) need to
be optimised carefully, to avoid accumulation of high amount of inert biomass and high
operational cost without gaining any volumetric improvement.
The proposed model predicted the presence of heterotrophic bacteria in the system as
results of the influent COD (10 mg/L) and generated COD in the decay mechanism. The
contribution of the influent COD and the SRT on the heterotrophic concentration is
illustrated in Figure 5.10. The influent COD is responsible for about 40% of the total
formed heterotrophic biomass. About 60% resulted from the decay mechanism. This can
be calculated by taking the heterotrophic biomass concentration at 10 mg/L and subtract
the biomass concentration at 0 mg/L. Furthermore, the heterotrophic biomass increased
by 11% as a consequence of increasing the SRT from 30 to 100 days. These results show
the significance of a low input of COD on the formed heterotrophic biomass in an
autotrophic system. Such a low value of COD (5–10 mg/L) could be indirectly introduced
to a similar system via aeration (contaminated air with traces of COD) and via media
preparations (water and nutrient compounds can contain traces of organic COD). The
presence of heterotrophs were also detected in non-sterilised chemostat systems used to
cultivate ammonia and nitrite oxidisers, despite the fact that original cultures were free of
heterotrophs and sterilised media were used (Rittman et al 1994). They claim that
ammonia and nitrite oxidisers produce soluble microbial products (SMP) that can support
the detected heterotrophic population. In order to describe this, high yield values for
ammonia and nitrite oxidisers (0.44 and 0.12 mg CODcell mg−1, respectively) were used to
predict proper biomass composition of autotrophs and heterotrophs.
biomass is particulate inert (60 and 80%) and reduction in the amount of predators was
compensated by an increased active biomass fraction. Meanwhile, a dramatic increase in
the active biomass fraction was observed in both reactors when there were no predators
present. An almost double increase of the active biomass fraction was calculated for the
reactor of 100 days SRT, gaining significant higher oxidation rate. In ASM 1 model
(Henze et al 2000) such an effect would have been corrected for by increasing the lysis
rate.
Table 5.3 Model simulation results on the effect of
predators on the active fraction of biomass in the
two reactors operated at 30 and 100 days SRT.
Parameters R30days R100days
unit No predators With predators No predators With predators
MLVSS mg/L 1186 1111 3171 3011
XA mgCOD/L 395 265 442 290
% 23 17 18 7
XN mgCOD/L 166 127 190 153
% 10 8 8 4
XH mgCOD/L 168 129 199 142
% 10 8 8 3
Xpredators mgCOD/L − 97 121
% − 6 3
XI mgCOD/L 955 960 1684 3570
% 57 61 67 83
Active bacteria % 43 33 33 14
Dead fraction % 57 67 67 86
(Inert+predators)
5.6 Conclusions
A rather simple procedure for the determination of the predators activity in suspended
mixed culture has been developed. Salt shock was used as a selective agent acting only
on the protozoa and metazoa and not affecting bacterial activities. The procedure was
successfully tested, verified and applied on a lab-scale nitrifying SBR system. A model
was developed to describe a mixed culture in which nitrifiers, heterotrophs and predators
(protozoa and metazoa) are coexisting.
The developed model proved to be capable of describing the interaction between
nitrifiers, heterotrophs and predators in two laboratory-scale nitrifying (SBRs) systems
operated at different sludge ages. The model was a helpful tool to get insight in the
system and to investigate the impact of increasing sludge age and the role of predators on
the performance of the nitrifying SBR system. The model results showed the need for
careful optimisation of systems operated at long SRT (such as membrane bioreactors), to
avoid accumulation of high amounts of inert biomass and to avoid high operational costs
without gaining any volumetric improvement. The model showed its capacity to elucidate
the biological processes in activated sludge systems by including the effect of the
predators. The practical application of the developed model and assessments of predators
activity needs to be verified under full scale activated sludge plant operation.
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Nitrification in saline industrial wastewater 114
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process: an innovative method for nitrogen removal from ammonia-rich wastewater. Wat. Sci.
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Hellinga C, van Loosdrecht MCM, Heijnen JJ (1999) Model based designed of a novel process for
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Nitrification in saline industrial wastewater 116
APPENDIX 5.1
Process Kinetics and stoichiometry for enriched
culture of nitrifiers in SBR system
Modelling nitrification, heterotrophic growth 117
Appendix 5.2
Stoichiometric and kinetic parameters at 30°C
used to simulate the nitrifying SBR sysstem.
Symbol Expression Unit Definition Reference
SO2 − mg O2/L Concentration of O2
SN2 − mg N/L Concentration of N2
SNH4 – mg N/L Concentration of NH4
SNO2 – mg N/L Concentration of NO2
SNO3 – mg N/L Concentration of NO3
SS – mg COD/L Concentration of organic
substrate
X
NH4 – mg COD/L Concentration of ammonia
oxidisers
XNO2 – mg COD/L Concentration of nitrite
oxidisers
XH – mg COD/L Concentration of
heterotrophic biomass
Xpredators – mg COD/L Concentration of predators
XI – mg COD/L Concentration of participate
inerts
YNH4 0.18 g COD/gNO2–N Yield of ammonia oxidisers Hunik et al (1994)
per NO2−_N
YNO2 0.06 g COD/g NO3–N Yield of nitrite oxidisers Hunik et al (1994)
per NO3−_N
YH 0.63 g COD/g COD Yield of hetrotrophic Henze et al (2000)
biomass on SS ASM2d
Ypred 0.5 g COD/g COD Yield of predators on Curds (1971, 1973)
bacteria
Fxi 0.15 g COD/g COD Fraction of inert COD This study
generated in biomass lysis
iN,XI 0.02 g N/g COD nitrogen content of XI Henze et al (2000)
ASM2d
iN,BM 0.07 g N/g COD nitrogen content of biomass Henze et al (2000)
ASM2d
1.4 day−1 maximum growth rate of Hunik et al (1994)
ammonia oxidisers
Nitrification in saline industrial wastewater 118
1&3 mg O2/L affinity constant for oxygen This study (for SRT
of ammonia oxidisers 30 &100 days)
5 mg NH4−N/L affinity constant for This study
ammonia of ammonia
oxidisers
mNH4 0.35 mg NH4−N/g maintenance coefficient of Tappe et al (1999)
XA–COD.day ammonia oxidisers
bNH4 0.3 day−1 aerobic decay rate of Measured according
ammonia oxidisers to Lesouef et al
(1992)
ηNH4 0.5 – anoxic reduction factor for Siegrist et al (1999)
ammonia oxidisers decay
0.9 day−1 maximum growth rate of (Hunik et al (1994)
nitrite oxidisers
1 mg O2/L affinity constant for oxygen This study
of nitrite oxidisers
2 mg NO2−N/L affinity constant for nitrite This study
of nitrite oxidisers
mNO2 1.15 mg NO2−N/g maintenance coefficient of Tappe et al (1999)
XN–COD.day nitrite oxidisers
bNO2 0.2 day−1 aerobic decay rate of nitrite Measured according
oxidisers to Lesouef et al
(1992)
ηNO2 0.5 – anoxic reduction factor for Siegrist et al (1999)
nitrite oxidisers decay
12 day−1 maximum growth rate of Henze et al (2000)
heterotrophic biomass ASM2d
0.2 mg O2/L affinity constant for oxygen Henze et al (2000)
of heterotrophic biomass ASM3
2 mg COD/L affinity constant for organic Henze et al (2000)
carbon of heterotrophic ASM3
biomass
0.5 mg NO3−N/L affinity constant for NO3 of Henze et al (2000)
heterotrophic biomass ASM3
mH 0.12 mg COD/g XH– maintenance coefficient of Meijer et al (2001)
COD.day heterotrophic biomass
ηH 0.8 – anoxic reduction factor of Henze et al (2000)
heterotrophic growth ASM2d
bH 0.8 day−1 aerobic decay rate of Henze et al (2000)
heterotrophic biomass ASM2d
µpredators 0.2 day−1 predation rate This study
Modelling nitrification, heterotrophic growth 119
6.1 Introduction
and the nitrification activity, not only in full scale WWTPs, but also in pure and enriched
cultures.
The aim of this research was to measure the nitrification activity using activated sludge
from domestic and industrial wastewater treatment plants operated under various salt
levels. The study covers one domestic and three industrial wastewater treatment plants,
all are in the Netherlands. Hoek van Holland is the domestic WWTP (freshwater-no salt),
while Heiploeg (shrimp processing), Ecco (tannery) and Seafarm (production and
processing of sea-fish) represent industries with saline wastewater. Within a period of 3
months, each plant was visited at least two times for measurements and sludge
characteristics. Measurements included: reactor volume, wastewater flow rate, mixed
liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS),
influent and effluent COD, TKN, pH, temperature and salt concentration. Sludge
characteristics include the assessment of the activity of ammonia and nitrite oxidisers and
the population structure.
were used: (ALF968 and BET42a) were used for the alpha and beta subclasses of
Proteobacteria, respectively (Manz et al 1992 1996). The ammonia-oxidising and nitrite-
oxidising bacteria were identified using previously published probes as described by
Nogueira et al (2002). Oligonucleotide probes were purchased as derivatives labelled
with the fluorescent dyes Cy3, Cy5, and 5(6)-carboxyfluorescein-N-hydroxysuccinimide-
ester (FLUOS), respectively (Interactiva, Ulm, Germany). FISH was performed using the
hybridisation and washing buffers as described by Manz et al (1992). The hybridised
samples were analysed with a Zeiss Axioplan2 Imaging microscope.
6.2.3 Analysis
Ammonia, nitrite, total kjeldahl nitrogen (TKN), chemical oxygen demand COD,
Chloride (Cl−), sulphate (SO4−2), pH and conductivity were measured in accordance with
Standard Methods (APHA, 1998). Nitrate-N was determined using Dionex 4500i series
and Shimadzu C_R5A ion-chromatograph. The MLSS and MLVSS determination was
performed after filtration of a 10 mL sample of mixed liquor on a Whatman glass micro
fibre filter (GC/F) filter. Dry weight was determined after the filter was dried for 24 h at
105°C and weighted with a microbalance. The ash content was calculated after
incinerating the dried filter in an oven for 1 h at 550°C.
6.3 Results
The different treatment plants represent a wide range of flow (100–21000 m3/day),
influent COD (300–2175 mg-COD/L), influent TKN (20–557 mg-N/L) and salt level
(0.13–16 gCl−/L) (Table 6.1). All treatment plants perform nitrification as they have high
ammonia removal efficiency (>95%). However, a significant difference among treatment
plants in activity of ammonia and nitrite oxidisers was observed.
Table 6.1 Operating data and measured influent and
effluent parameters of the 4 full-scale WWTP.
Plant Name
Hoek van Heiploeg Ecco Seafarm
Parameter Unit Holland
Type Domestic fish tannery marine
processing Industry aquaculture
Industry Industry
System AS SBR AS BF
3
Volume (m ) 5700 2800 8000
3
Flow (m 21000 440 843 100
day−1)
Temperature (°C) 14 14–18 22
Nitrification activities in full-scale treatment 127
6.4 Discussion
Vredenbregt et al 1997; Panswad and Anan 1999 a, b). However, there is much deviation
under domestic wastewater conditions (Pollice et al 2002, Salem et al 2003) (Figure.2).
(1993)
96 58.5 30 0.1 LSBR EC 10 250 30 Moussa et
al (2003c)
58.5 55.7 30 10 EC 10 250 30
17.7 19.6 30 20 EC 10 250 30
14.7 12.2 30 30 EC 10 250 30
3 1.9 30 40 EC 10 250 30
*1
FA=Full-scale Activated sludge WWTP; PA=Pilot-scale Activated sludge unit; LA=Lab-scale
Activated sludge unit; PF=pilot-scale Fluid-bed; FB=lab-scale Fed-Batch mode; LC= sterile Lab-scale
Chemostat; LSBR=Lab-scale Sequencing batch reactor; PA=Pilot-scale Activated sludge unit
*2
DA=Domestic Activated sludge performing nitrification; SA=Salt Adapted activated sludge
performing nitrification; PCA=Pure Culture of Ammonia oxidisers Nitrosomona europea; PCN =Pure
Culture of Nitrite oxidisers Nitrobacter agilis; MS=Marine Sediment; MC=Nitrosomonas and
Nitrobacter in Mixed Culture; EC=Enriched Culture of nitrifying bacteria
The activated sludge model (ASM1) was used to translate the routine operating data into
parameters to enable the calculation of the fraction of nitrifiers and consequently to
recalculate the actual specific activity of ammonia and nitrite oxidisers (equation 6.1,
6.1).
(6.1)
(6.2)
The ASM1 model was applied to recalculate the actual activity of ammonia oxidisers
both in this study and in other reported studies.
Ammonia oxidisers: The assessment of the fraction of ammonia oxidisers
demonstrates the influence of the above-mentioned operating parameters on the portion
of the nitrifiers to the total biomass. This is in agreement with the results reported by
Rittmann et al (1999) and Wiesmann (1994). Expressing the activity of ammonia
oxidisers in terms of specific activity eliminates the deviation between the specific
activity from different systems and makes the results from pure cultures, enriched
cultures, pilot scale and full scale WWTPs comparable (Figure 6.3). Moreover, the results
of activities of pure and enriched cultures can now be extrapolated to full scale WWTPs.
The activity seems to be dependent on the salt concentration, irrespective of the dominant
species of ammonium oxidisers, which was also demonstrated by Moussa et al (2003c).
Nitrification in saline industrial wastewater 132
Nitrite oxidisers: Despite the fact that nitrite oxidisers were active (oxidation of
nitrite to nitrate), neither of the two probes (NIT3 and Ntspa662) were able to detect
Nitrobacter sp. or Nitrospira sp. The reason could be that the fraction of these nitrite
oxidisers was below the detection limit, since these WWTPs are operated at high sludge
age and/or high organic load. Alternatively, nitrite oxidisers might be present, but are not
detectable with the two available probes.
A number of studies have demonstrated a relationship between the presence of distinct
nitrifiers and specific environmental conditions (Kowalchuk and Stephen 2001; McCagig
et al 1999; Rittmann et al 1999; Stephen et al 1999). However, a relation between salt and
population of nitrifiers cannot be drawn, since within the limited number of WWTPs with
elevated salt levels, two different ammonia-oxidising species became dominant.
Nevertheless, the use of the model to quantify the specific activity and to correlate this to
the presence of specific populations within different systems was promising and could be
applied in future to confirm this relation.
6.5 Conclusions
• The results showed the adverse effect of salt on nitrification in full scale WWTPs. The
domestic WWTP with the lowest salt level (0.13 g Cl−/L) had the highest specific
activity of ammonia and nitrite oxidisers (4.3 and 2.4 mg-N/gVSS.h, respectively).
The lowest specific activities of ammonia and nitrite oxidisers (1.1 and 0.5 mg-N/g
VSS.h) were measured in the WWTP with the highest NaCl concentration (16 g
Cl−/L).
• It is rather complex to compare between the specific nitrification activity of different
types of sludges developed under different operational conditions. This is due to the
variation in active fraction of ammonia and nitrite oxidisers present in the investigated
types of sludge.
• The use of the activated sludge model to calculate the active fraction of nitrifiers and
consequently recalculate the actual specific activity of ammonia and nitrite oxidisers
makes the results from pure cultures, enriched cultures, pilot scale and full scale
WWTPs comparable.
• Model application to quantify the actual specific activity validates the previous results
of salt on nitrification obtained at laboratory-scale. The ammonia and nitrite oxidisers
activity seems to be dependent on the salt concentration, irrespective of the dominant
species of ammonium oxidisers.
• Both Nitrosomonas oligotropha and Nitrosomonas europaea are normally present under
low salt levels, not only in full scale WWTPs but also in laboratory-scale systems. At
elevated salt levels only Nitrosomonas europaea became dominant.
• A clear identification of the nitrite oxidisers present in the investigated WWTPs was not
possible. The reason could be that either the fraction of these nitrite oxidisers was
below the detection limit or they were present, but are not detectable with the two
available probes.
Nitrification in saline industrial wastewater 134
References
Wagner M, Nogueira DR, Juretschko S, Rath G, Koops H-P, Schleifer KH (1998) Combining
fluorescent in situ hybridisation (FISH) with cultivation and mathematical modelling to study
population structure and function of ammonia oxidising bacteria in activated sludge. Water Sci.
Technol. 31:441–449.
Wagner M, Loy A (2002) Bacterial community composition and function in sewage treatment
system. Environ. Biotechnol. 13:218–227.
Wiesmann U (1994) Biological nitrogen removal from wastewater. Advances in biochemical
Engineering/biotechnology 51:113–154.
Chapter 7
Model-based evaluation of the upgrading of a
full-scale industrial wastewater treatment
plant
Previously published as:
Moussa MS, Rojas AR, Hooijmans CM, Gijzen HJ, van Loosdrecht MCM (2004)
Model-based evaluation of nitrogen removal in a tannery wastewater treatment plant.
Accepted for the IWA Conference on wastewater treatment for nutrient removal and
reuse. Bangkok, Thailand (January 26–29, 2004).
Abstract
Computer modelling has been used in the last 15 years as a powerful tool
for understanding the behaviour of activated sludge wastewater treatment
systems. However, computer models are mainly applied for domestic
wastewater treatment plants (WWTP). Application of these types of
models to industrial wastewater treatment plants requires a different
model structure and an accurate estimation of the kinetics and
stoichiometry of the model parameters, which may be different from the
ones used for domestic wastewater. Most of these parameters are strongly
dependent on the wastewater composition. In this study a modified
version of the activated sludge model No. 1 (ASM 1) was used to describe
a tannery WWTP. Several biological tests and complementary physical-
chemical analyses were performed to characterise the wastewater and
sludge composition in the context of activated sludge modelling. The
proposed model was calibrated under steady-state conditions and
validated under dynamic flow conditions. The model was successfully
used to obtain insight in the existing plant performance, possible
extension and options for process optimisation. The model illustrated the
potential capacity of the plant to achieve full denitrification and to handle
a higher hydraulic load. Moreover, the use of a mathematical model as an
effective tool in decision-making was demonstrated.
7
Model-based evaluation of the upgrading of a
full-scale industrial wastewater treatment
plant
7.1 Introduction
The activated sludge system is currently the most widely used biological wastewater
treatment process, treating both domestic and industrial wastewater. The process requires
a high degree of operational control and management. In order to obtain maximum
removal efficiency from the activated sludge plant, the operator must have a full
understanding of this complex process. Much research has been done over the last 15
years to understand the behaviour of activated sludge systems using computer modelling.
A common language for all modellers in this field regarding concepts and nomenclature
is provided by the ASM models developed by the IWA task group (Henze et al 2000).
The ASM models have proved to be a useful tool for the dynamic simulation of activated
sludge systems treating domestic wastewater. However, application of these models to
industrial wastewater treatment plants remains limited. To apply these models to
industrial wastewater treatment plants it could be necessary to extend the ASM models
by additional kinetic reactions (Nowak et al 1995). The use of models especially in the
field of industrial wastewater will support plant operators. The physical, chemical and
biological properties of industrial wastewater and their variation in flow and composition
make the operation more complicated. Moreover, the model could be used as a
quantitative way to predict the effect of different production scenarios on their
wastewater treatment plant (van Zuylen 1993), supporting the operator in decision
making.
The tannery industry is one of the industries generating high amounts of polluted
water while the industry has a low profit margin. Therefore, purification of the generated
wastewater has a high impact on the overall production costs. The work presented here
emphasises on modelling of a tannery activated sludge wastewater treatment plant and its
practical application. The main objectives of this study are:
• To modify activated sludge model ASM1 to satisfactorily describe the COD and N
removal in the tannery wastewater treatment plant;
• To evaluate the plant performance using the modified model;
• To investigate the required modifications for the plant optimisation and future
extensions on plant capacity.
Model-based evaluation of the upgrading 141
7.2.2 Measurements
The staff of WWTP Ecco Tannery Holland B.V. provided the routinely collected
operational data of the bioreactor and its performance over the year 2000. A detailed
sampling and experimental program was conducted in October and November 2000. The
pseudo steady state measurements of the WWTP were performed during two sampling
Model-based evaluation of the upgrading 143
runs (11 October and 24 November 2000). During each run samples were collected from
the influent, return sludge and effluent. The samples were analysed for T (temperature),
pH, Alkalinity, DO (dissolved oxygen), CODtot (total COD), CODS (COD of the micro-
filtrated fraction, 0.45mm pore diameter), NH4–N, NO3–N, TKN (Total Kjeldal
Nitrogen), Ptot (total phosphorus), VSS (Volatile Suspended Solids) and TSS (Total
Suspended Solids). In addition, six different sampling points over the length of the
bioreactor were defined and sampled during the second run (24 November 2000). These
six sampling points were used to describe the hydraulic regime and biological conversion
as function of the reactor length. The average values of the routinely collected data for
the year 2000 and the average measurements of the sampling program are presented in
Table 7.2.
Several biological batch tests were performed at the UNESCO-IHE laboratory to
determine the influent and sludge characteristics (Ekama et al 1986; Orhon et al 1999a,
b). The tests were performed at controlled temperature 20°C, pH of 7.5±0.05 and under
aerobic and anoxic conditions. Nitrification batch tests were performed under aerobic
conditions, in which NaNO2 and NH4 were consequently injected (Moussa et al 2003a).
This test allows measuring the kinetic parameters of nitrite and ammonia oxidisers and
was used for model calibration.
Table 7.2 Measured influent and effluent and the
influent composition required for the model of
WWTP Ecco Tannery B.V. The yearly average
values are printed in Italic, these values were used
to calculate steady-state influent composition.
Measurements Model Influent Composition
Value Value
Description Influent Effluent units Description Symbol Dynamic Steady- units
state
Total COD, 2525 167 gCOD/m3 Soluble
CODTotal (2920) (190) compounds
Soluble 1785 143 gCOD/m3 Dissolved SO2 0.3 0.3 gCOD/m3
COD, CODS oxygen
Total N–Kj 488 7 (11) gN/m3 Readily SS 840 972 gCOD/m3
(515) biodegradable
COD
Soluble N– 454 6 gN/m3 Soluble inert SI 177 205 gCOD/m3
Kj COD
Ammonium, 438 10 gN/m3 Ammonium SNH4 438 448 gN/m3
NH4+
Nitrite, NO2− 0 0 (0.1) gN/m3 Nitrite SNO2 0 0 gN/m3
Nitrate NO3− 0 99 (50) gN/m3 Nitrate SNO3 0 0 gN/m3
Nitrification in saline industrial wastewater 144
each compartment (Appendix 7.1 and 7.2). The main modification incorporated in
ASM1 to simulate the WWTP was that the nitrification was considered as a two-step
process (Nowak et al 1995). The reason for this is that ammonia oxidisers grow faster
than nitrite oxidisers at temperatures above 15–20 °C (Hellinga et al 1999). Moreover,
inhibiting compounds present in industrial wastewater might lead to an adverse effect on
one or both steps of the nitrification process. Thus describing the nitrification in two steps
enables the identification of any inhibition and the detection of partial nitrification (NO2
accumulation).
The readily biodegradable COD (SS) was determined by two different approaches using
an aerobic and anoxic batch test as described by Ekama et al (1986). The soluble inert
COD (SI) was determined according to the approach suggested by Orhon et al (1999a, b).
The Influent XI fraction (XI/CODtotal) ratio was estimated as a result of model calibration
fitting the solid COD balance as proposed by Meijer et al (2001) and consequently the
rest will represent the XS fraction. Average influent measurements and the calculated
model influent compositions are presented in Table 7.2.
After the determination of the main operational parameters and the influent
characterisation, the model of the WWTP was calibrated. A step-wise approach was
applied as proposed by Meijer et al (2001). First the solids were fitted (P, COD and TKN)
on the basis of yearly average measurements. Next the nitrification and denitrification
were calibrated on the basis of yearly average measurements.
gCOD/gMLVSS). When fXSin is used to fit the COD balance, all model uncertainties
related to the production of XI and the influent characterisation of XS and XI are lumped
in the influent fXIin fractionation. As a result of adjusting the fXIin fraction, the model
predicted accurately the soluble COD in the effluent and total P and TKN in the
bioreactor.
Model validation was performed via validating the capacity to predict the measured
concentrations of NH4+, NO2− and NO3− along the bioreactor length using the dynamic
influent data. The recorded average daily influent flow during the period 13–26
November 2000 (Figure 7.3) was used as input flow for the model. The simulated value
of NH4+, NO2− and NO3− of day 24 November (the 12th day of the 14 days dynamic
simulation period) were compared with the values obtained from sampling over the
Nitrification in saline industrial wastewater 148
length of the bioreactor conducted on the same day. The model provides a rather accurate
prediction of the NH4 and NO3 along the bioreactor (Figure 7.4).
This study was conducted in the first year of changing the ownership of the company.
The new owner performed various viability studies on increasing the hide production and
application of different tannery process and operational strategies. The main question,
which arose, was whether the existing WWTP could cope with these future requirements.
Based on the plant performance and effluent quality, an additional volume requirement
(25% of the bioreactor volume) was proposed by the operator. The old equalisation tank
was selected to be the additional volume, and to be operated as an anoxic reactor. This
upgrading aimed to increase the plant capacity and to achieve full denitrification.
Evaluating the upgrading concept for the WWTP was the main focus of our modelling
study. The validation step demonstrated the capability of the proposed model to describe
the WWTP correctly under both steady state and dynamic conditions. Hereafter, the
model was used as a tool to obtain insight in the existing plant performance, possible
extension and ways of process optimisation.
7.7 Discussion
The influent characterisation results of WWTP wastewater do agree with the reported
values of the tannery wastewater (Orhon et al 1999a). The modified ASM1 model
proposed in this paper for COD and N removal described the performance of the WWTP
well with the adjustment of only two parameters An accurate and correct
description of the system configurations, balancing the operational data with the
measurements to accurately calculate the SRT and the use of the stepwise calibration
proposed by Meijer et al (2001) simplified the complexity of the model calibration. The
use of batch tests for model re-calibration was useful because the plant was under loaded
and therefore the effluent concentrations profiles were not sensitive for the rate
parameters. Model validation under dynamic conditions is of great significance in
industrial WWTP because the plant is usually working under highly dynamic conditions
in comparison with domestic WWTP.
The observed reduction in ammonia and nitrite oxidising activity is attributed to the
presence of salt (7.5 g Cl−/L). This decline in the activity for ammonia and nitrite
oxidisers is in agreement with earlier results of salt inhibition effects on nitrifying sludge
(Moussa et al 2003b). The use of a higher decay rate of nitrifiers to mathematically
describe the salt impact simplifies the model description of salt inhibition (only one
parameter to calibrate). Other parameters for calibration could have been chosen but the
decay rate was considered as the most uncertain coefficient in the model.
The potential use of the model was illustrated in the evaluation of the upgrading of the
WWTP. During this evaluation the bioreactor capacity and process configuration were
investigated. The model provided a better and quantitative understanding of the plant
operation and treatment process. The investigation of several modifications to reach
further plant optimisation was very time-effective and cheap (Salem et al 2002). Because
it gives quantifiable results, modelling supported the decisions to be taken with respect to
the plant extension. It was originally proposed to increase the plant volume (25% with the
use of old equalisation tank) to cope with anticipated load expansion. The simulations
clearly indicated that with the present system and future increase in load good effluent
quality can be reached even combined with an increased denitrification.
An additional application of the model is the usage by the plant manger or the plant
staff. These people use the model in a different way, namely, as a tool to quantitatively
predict the effect of certain decisions on the treatment process (Salem et al 2002). This
application of the model is very useful in case of industrial WWTP, like the tannery
studied here. The cost of treating the wastewater generated during the production process
has a crucial impact on the overall production costs. This also increases the awareness of
the impact of each pollutant term in each part of the process and stimulates the practice of
waste minimisation to have an environmentally friendly production.
Model-based evaluation of the upgrading 151
7.8 Conclusions
Activated sludge models commonly applied for domestic wastewater treatment plant
could also be used for industrial WWTPs if the following steps are carefully considered:
1. An accurate description of the system configurations;
2. Balancing the operational data with the measurements to accurately calculate the main
important reactor input parameters (flow rates and SRT);
3. Selection of model process and components which are significant and dynamic in this
system configuration;
4. Complementary analyses to assess the wastewater and the sludge characterisation;
5. Stepwise calibration under steady-sate conditions and finally model validation under
dynamic conditions.
The modified ASM1 model proposed in this paper for COD and N removal proved to be
able to describe the performance of Ecco Tannery Holland B.V. WWTP wastewater
treatment plant.
The model was successfully used to evaluate and optimise the plant performance. In
addition it was demonstrated that the model could be used by the plant manager to
support his decisions quantitatively resulting in saving time and money.
References
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34:846–858.
Ekama GA, Dold PL, Marais GvR (1986) Procedures for determining influent COD fractions and
the maximum specific growth rate of heterotrophs in Activated sludge systems. Wat. Sci. Tech.
18:91–114.
Hellinga C, van Loosdrecht MCM , Heijnen JJ (1999) Model based designed of a novel process for
ammonia removal from concentrated flow. Mathematical and Computer Modelling of Dynamic
Systems 5:351–371.
Henze M, Gujer W, Mino T, van Loosdrecht MCM (2000). Activated sludge models ASM1, ASM2
and ASM3. Scientific and Technical Report, IWA Publishing, London.
Meijer SCF, van Loosdrecht MCM, Heijnen JJ (2001) Metabolic modelling of full-scale biological
nitrogen and phosphorus removing WWTPs. Water Res. 35:2711–2723.
Moussa MS, Lubberding HJ, Hooijmans CM, van Loosdrecht MCM, Gijzen HJ (2003a) Improved
method for determination of ammonia and nitrite oxidation activities in mixed bacterial cultures.
Appl. Microbiol. Biotechnol. 63:217–221.
Moussa MS, Lubberding HJ, Hooijmans CM, van Loosdrecht MCM, Gijzen HJ (2003b). Short
term effects of various salts on ammonia and nitrite oxidisers in Mixed Bacterial Cultures. Appl.
Microbiol. Biotechnol. (Submitted).
Nowak O, Svardal K , Schweighofer P (1995) The dynamic behaviour of nitrifying activated sludge
systems influenced by inhibiting wastewater compounds. Wat. Sci. Tech. 31:115–124.
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models with the help of mass balances. Water Sci. Tech. 39:113–120.
Orhon D, Ates E, Ubay Cokgor E (1999a) Modelling of activated sludge for tannery wastewaters.
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Nitrification in saline industrial wastewater 152
Orhon D, Ubay Cokgor E, Sozen S (1999b). Experimental basis for the hydrolysis of slowly
biodegradable substrate in different wastewaters. Wat. Sci. Tech. 39:87–95.
Reichert P (1998) AQUASIM 2.0 Computer Program for the Identification and Simulations of
Aquatic Systems. Swiss Federal institute For Environmental Science and Technology (EWAG),
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Appendix to Chapter 7
APPENDIX 7.1
Stoichiometry matrix of the activated sludge
model (modified ASM1) applied for the
industrial WWTP Ecco Tannery B.V.
APPENDIX 7.2
Process kinetics of the activated sludge model
(modified ASM1) applied for the industrial
WWTP ECCO Tannery B.V.
Chapter 8
Evaluation and Outlook
8
Evaluation and Outlook
8.1 Introduction
The aim of this research was to achieve a better understanding of nitrification under
saline conditions. The research was carried out in two phases. In the first phase,
laboratory scale activities were conducted to obtain fundamental data to determine the
relationship between salinity and nitrification. In the second phase the results collected
from the laboratory experiments were compared and validated with the results collected
from full-scale treatment plants. Modelling was employed in both phases to provide a
mathematical description for salt inhibition on nitrification and to facilitate the
comparison. The research has lead to an improved understanding of the effect of salinity
on nitrification, while subjects for further research were also identified. The research
findings and challenges are described in more detail below.
In industrial activated sludge wastewater treatment there is always the risk of inhibitory
compounds in the influent due to spilling or other incidents in the industry. An effective
control in order to maximise the use of the plant volume is needed. Therefore an early
detection of inhibitory compounds is essential. The nitrification process is one of the
most sensitive processes within modern WWTPs and any disturbance might lead to
washout of nitrifiers. Moreover, it requires a long time period before these
microorganisms are fully re-established in the plant. Thus, a high priority is always given
to the nitrification parameters when control strategies are applied to the activated sludge
system designed for COD and N removal. One of the achievements of this study was the
development of a simple and reliable method to measure the activity of ammonia and
nitrite oxidisers separately in mixed bacterial cultures. The main advantage of this
method is the possibility to differentiate between the activities of ammonia and nitrite
oxidisers without the use of metabolic inhibitors. The method can be applied to measure
the effects of specific inhibitors on the activity of both groups of nitrifiers. It can,
therefore, also be used as a detection method for early diagnosis of nitrification problems.
Incorporating the method in the existing protocol of the automatic respirometer/on-line is
promising and could be a helpful tool to optimise nitrogen removal processes.
Evaluation and outlook 157
Initially, short-term effects of several salts on nitrification were studied. Different types
of salts appeared to have different inhibition effects on the ammonia and nitrite oxidisers.
Therefore, formulas were developed to unify these differences in inhibition effects on
both ammonia and nitrite oxidisers. These formulas can be used in the design, operation
and control of the WWTPs that operate under salt stress. These findings facilitate the
mathematical description of salt inhibition on nitrifiers from a process-engineering point
of view, although the real inhibition mechanisms are not yet clearly understood.
Investigating the actual inhibition mechanisms in depth (cell membrane, energy
requirement, enzyme inhibition) might not be required for optimal process design.
Nevertheless, it could be an interesting subject for further research, since it might lead to
a more accurate description of inhibition.
Acclimatisation is a term, which is commonly used to describe the recovery of
bacterial cultures when exposed to a certain inhibitor over extended periods of time.
Acclimatised cultures are recommended for seeding a system that will be operated under
similar conditions to shorten the start-up period. Salt acclimatisation was investigated
within the course of this research. The effect of salinity on the activity, the composition
of nitrifying populations and floc characteristics was observed. The main finding was that
acclimatised and non-acclimatised nitrifying sludge were behaving similarly with respect
to activity and population selection in response to different salt concentrations. This
means in practice that sludge from a conventional domestic nitrifying WWTP can be
used as a source for nitrifiers to become adapted to salt stress via gradual adaptation. This
avoids the necessity of seeding a system that is expected to operate under salt stress with
salt acclimatised sludge. The monitoring of the activity and population composition of a
full-scale reactor during the start-up period, after seeding according the proposed
approach could be an interesting subject for further research.
The selection of Nitrosomonas europaea and Nitrobacter sp. as a result of gradual
increase in salinity was shown by the re-growth of only these two species after lowering
the salt concentration to zero values. This result shows the high resistance of these
species to salt stress. Interestingly, the systems were able to fully recover their
nitrification activity with reduced diversity in the population of nitrifiers. These findings
support the hypothesis that the conversion rate depends on environmental conditions and
not on the type of species. In other words, different types of nitrifiers behave kinetically
similar under similar environmental conditions. This hypothesis remains to be verified
and could be an interesting topic for further research.
The current design concepts of activated sludge systems consider bacteria as the sole
active biomass. The activity of all other microbial community members (protozoa,
metazoa, bacteriophages etc.) is hidden in a simple decay process responsible for the
reduction of active biomass. This decay process is the sum of several independent
Nitrification in saline industrial wastewater 158
Activated sludge models have been successfully applied in the last two decades for
domestic wastewater treatment plants. However, their application to industrial WWTPs is
still limited for several reasons. The main reason is the complexity of industrial WWTPs
in terms of dynamic flow, waste composition, etc. Another important reason relates to the
lack of information of model parameters under extreme conditions prevailing in
industries (pH, temperature, toxicant presence). Besides, large variations exist between
similar industries, which make the treatment process specific for each individual plant.
All these factors have limited the applicability of the existing activated sludge model and
the transfer of existing experiences between industries. Therefore, the use of large reactor
volumes, excess aeration end extra chemical dosages is commonly practised in industries
to ensure compliance with effluent quality requirements. Mathematical modelling has
shown to increase the confidence of operators in WWTP design and thereby limits the
use of an unnecessary high safety factor for new or upgraded treatment plants. This will
directly be translated into lower treatment costs.
In this study a modified version of the activated sludge model No. 1 (ASM 1) was
applied under static and dynamic conditions. Firstly, the steady state model was used to
calculate the actual specific activity of ammonia and nitrite oxidisers of different WWTPs
investigated in the course of this research. The routine operating data were used to assess
the fraction of nitrifiers in the sludge. The results from different types of nitrifying
populations (pure cultures, enriched cultures) and different scale reactors (lab scale, pilot
scale, full scale WWTP) were compared. The steady state model confirmed the behaviour
of nitrifiers under salt stress and validated the results obtained from laboratory scale (this
study) to be interpreted on full scale. The use of the model to quantify the specific
activity and to correlate this to the presence of specific populations within different
systems was promising and could be applied in future research. Secondly, the dynamic
Evaluation and outlook 159
model was used to describe a full-scale tannery WWTP. The model was used to obtain
insight in the plant performance, possible extension and options of upgrading. The model
illustrated the potential of the plant to have a better effluent quality and to handle a higher
hydraulic load with simple modifications. The model was used as an effective tool to
visualise and quantify the plant conditions. The results demonstrate the application of the
model as a decision support tool especially in cases when industrial treatment plants are
exposed to substantial fluctuations in production.
Based on the experience gained in the course of this research it is concluded that more
efforts should be made to motivate designers and operators of industrial WWTPs to
become familiar with activated sludge models. A good approach would be to develop a
standard influent characterisation procedure for industrial wastewater similar to the one
successfully developed and applied for domestic wastewater. Moreover, the model
parameters need to be extended to satisfy the operational conditions in industries (e.g.
high temperature and long SRT). Disturbance factors such as pH, salinity, etc could be
incorporated in the model. All this in addition to producing simple and reliable detection
sensors could promote the application of activated sludge modelling in the industrial
field.
In conclusion, the research has lead to an improved understanding of the effect of
salinity on nitrification. The results obtained within the course of this research can be
used to improve the sustainability of the existing WWTPs operated under salt stress. The
findings also form a guideline for more economical and sustainable design and start up of
WWTPs dealing with salt in future.
Samenvatting
Thanks to Allah the exalted, the most merciful, for giving me the strength and persistence
to keep going with this research even during the most difficult moments. May Allah
accept this work and count it as a good deed. My deep thanks to my country Egypt where
I grew up and had the first lessons and experiences in my life, I hope I can pay it back
some day.
I would like to express my thanks to my promoters: Prof. Huub Gijzen who stimulated
and supported the formulation of my PhD joint project. Huub, you had an important role
in guiding and challenging not only during the research but also after the completion;
Prof. Mark van Loosdrecht for his ideas in the earlier stage of the research, valuable
feedback, enthusiasm and friendship. Mark, you were always inspiring, educating and
available when I needed you.
I am also grateful to my supervisors: Tineke Hooijmans who supported me especially
during the difficult initial period of this study and for her efforts in establishing the
project; Henk Lubberding who guided me during the research, inspired my
microbiological experience and spent lots of time helping in finalizing this work.
I would like to acknowledge the MSc students, Samir Ibrahim, Deepthi Sumanasekera,
Alejandro Rivera Rojas, Orleans Garcia, Aboubakar Gomina, Jochem Smit, Yan Song,
Said Rehan, Akram Botorous and Hala Elsadig who were involved in this study for their
valuable contribution.
I would like to express my appreciations for the endless help and support of the
laboratory staff at UNESCO-IHE: Fred Kruis, Frank Wiegman, Kees Bik, Peter
Heerings,. Special thanks to the staff of Kluyver Laboratory of TU Delft: Sjaak Lispet,
Stef van Hateren and Udo van Dongen for their continuous help during this study.
Great thanks to my colleagues at IHE Saleh and Saber for their continuous
encouragement and inspiring discussions on our related research topics. Special thanks to
my friend M.Fiala for all what he did and what he is still doing for me.
These acknowledgments would not be complete without expressing my gratefulness to
GOSD for expanding my background as a structural engineer with environmental
experience and also my appreciation to UNESCO-IHE for providing the best working
environment.
I remain very grateful and gratified to my family, especially my parents, my wife, my
brothers and sister and the two young researchers Adham and Yusuf for their support,
patience, understanding and prayers throughout the period of this work.
Curriculum Vitae
Moustafa Samir Moussa was born in Cairo, Egypt on August 11th, 1965. He graduated in
1987 as a Civil Engineer from the faculty of Engineering, University of Ain Shams,
Cairo. He was awarded his BSc with general grade “very good” and “distinction” for his
awarding project.
After finishing his military service in 1989 he started his professional career in GOSD,
General Organization for Sanitary and Drainage, greater Cairo. Here he was responsible
for the sanitary, hydraulic and structural design of Shoubra El Khimma wastewater
treatment project. In addition he was appointed as instructor for different training courses
in wastewater collection and treatment for the new work orientation training programs.
In October 1995, he studied at the International Institute for Infrastructural, Hydraulic
and Environmental Engineering (IHE) in Delft (now called UNESCO-IHE Institute for
Water Education). In September 1996 he obtained a post-graduate diploma in Sanitary
Engineering and was awarded a scholarship from Shell to continue his MSc research. In
1997 he obtained his MSc degree and continued his research through an additional fund
from Shell. This research formed the starting point of his PhD research, a joint project of
UNESCO-IHE, TU Delft, Shell Global Solutions B.V., Ecco Tannery Holland B.V.,
Heiploeg B.V. and BTS Senter (an agency of the Dutch Ministry of Economic Affairs).
Since Januari 2004 the author works as a researcher/lecturer at UNESCO-IHE.