Evaluation of nitrogen removal in a small-scale system for

biological treatment of tannery wastewater

Sofia Andersson*, Fantahun Misganaw**, Seyoum Leta** and Gunnel Dalhammar*

*
Department of Environmental Microbiology, Royal Institute of Technology, AlbaNova University Centre
S-106 91 Stockholm, Sweden. (E-mail: sofia@biotech.kth.se; gunnel@biotech.kth.se )
**
Department of Biology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.
(E-mail: letaseyoum@yahoo.com; fantahun21@yahoo.com )

Abstract
The diversity and abundance of nitifying bacteria in a biological pilot scale system treatning tannery
wastewater was investigated using fluorescent in situ hybridisation (FISH) in combination with process
monitoring and nitrification activity meassurements. Also, the posibility to enhance the denitrification
prosses’ resistance to shock-loads in incoming tannery wastewater was investigated. Agar-embedded
denitrification bacteria was bioaugmented into an anoxic lab-scale reactor and the system was monitored by
analysing process parameters and using FISH-technique. The aim of the two studies was to gain knowledge
about biological processes when exposed to high-loaded, tannery wastewater. The nitrification in the pilot
system performed well. Approximatly 0.14% of the total suspended solids in the sludge was comprised of
nitrifying bacteria. Nitrosococcus togehter with Nitrosomonas was the most abundant genus of ammonia
oxidisers while bacteria within the phylum Nitrospira were found responsible for nitrite oxidisation.
Bioaugmentation with agar-embedded denitrifying bacteria was not sucsessful. Neither good retention of
bacteria in the system nor enhanced denitrification was achieved.

Keywords
Bioaugmentation; denitrification; nitrification; nitrogen removal; tannery wastewater treatment; fluorescent
in situ hybridisation,

INTRODUCTION
One of the most important industries in Ethiopia is the tanning industry. Leather is the second
largest export merchandise after coffee (EPA, 2002). A problem with the leather industry is that the
tannery wastewater composition is complex and toxic. Typical combined tannery effluent is
characterized by a high content of organic matter, salts (chromium, sulphides and chlorides) and
suspended and dissolved solids. In addition, it is strongly alkaline (UNEP 1991). Despite these toxic
properties of the wastewater, only two out of 20 tanneries in Ethiopia have some kind of treatment
process linked to their production (EPA, 2003).

In most tanning industries the wastewater streams from soaking, pickling and tanning processes are
separated and treated individually by chemical treatment, electrochemical oxidation, various
membrane techniques or biological treatment. One of the currently most interesting and developed
techniques is the membrane technique. Using pressure-driven membranes in tannery wastewater
treatment conveys a reduction of the environmental impact and a decrease in disposal costs, by
obtaining uncontaminated sludge, a recovery of by-products and in some cases savings of direct and
indirect energy (Cassano et al. 2000). However, the membrane technique, as well as
electrochemical oxidation, is a technologically advanced technique that requires both knowledge
and sophisticated equipment. Therefore it is presently not suitable to use in developing countries.
Biological treatment, on the other hand, is an internationally widespread method of treating tannery
effluents but, due to its’ high-load and toxic composition, it is difficult to obtain satisfactory results.
The difficulty with biological tannery wastewater treatment is discussed in a UNEP report (1991)
and especially biological nitrogen removal is pointed out as problematic. This attitude, however,
could be changed if the techniques are further developed and better adapted to tannery effluents.

In this paper the results from two related studies with the aim to gain more knowledge about
tannery wastewater treatment are presented. First, a study on a biological pilot plant in Ethiopia,
treating tannery effluents, was performed. Fluorescent in situ hybridisation (FISH) technique was
utilized to investigate the population of nitrifying bacteria in aerobic sludge. Since the nitrification
process performed well but the denitrification process was functioning inadequately another study
was performed. This time a lab-scale system was used to investigate whether bioaugmentation with
immobilised denitrifying bacteria could enhance the performance and make the system resistant to
concentration shock-loads in the inflow.

METHODS

Pilot plant setup

The pilot plant was constructed on the premises of the Department of Biology, Addis Ababa
University campus. Figure 1 shows the schematic setup the predenitrification system, comprising of
a 100 L anoxic tank, followed by a 200 L aerated activated sludge tank and a 50 L funnel for
sedimentation as described by Leta et al. (2004). The initial seed for the anoxic and aerated tanks
was collected from the Kaliti municipal wastewater treatment pond system. The inlet feed consisted
of combined (soaking, pickling and tanning process) raw tannery wastewater obtained from Mojo
tannery, located 70 km south of Addis Ababa (Table 1). The inflow and the mixed liquor and
suspended solids (MLSS) recycle flow were controlled by peristaltic pumps. All other flows were
due to gravity. The inflow was adjusted to give a load of 0.5-1.5 kg COD/m3 per day which was
equivalent to a flow of 10-14.4 L/d, and the MLSS recycle flow was set to 1.5 times the inflow.
Approximately 40 % of the settled sludge, which corresponded to around 24 g dry weight sludge
per day, was returned manually twice a week from the sedimentation funnel to the anoxic tank. The
dissolved oxygen (DO) concentration was kept between 2 and 5 mg O2/L in the aerated tank and
below 0.3 mg O2/L in the anoxic tank. The water temperature was 19-24°C. The hydraulic retention
time in the system was approximately 24 days.

Lab scale system setup

Two identical lab-scale systems were utilized, consisting of a 1.5 L anoxic tank with a magnetic
stirrer and a 0.66 L sedimentation funnel (Figure 2). Initial sludge inoculum was collected at
Överjärva treatment plant in Sweden (Norström, 2004) and the systems were fed by sterile synthetic
tannery wastewater (STWW) with a composition that resembled the Mojo tannery wastewater used
in the pilot plant (Table 1). The inflow was controlled by a peristaltic pump and set to give a feed of
0.8 L/d. All other flows were due to gravity. Sludge was returned manually every day. Water
temperature was kept constant at 20 °C and DO was below 0.3 mg O2/L.
Synthetic tannery wastewater Mojo WW
Ingredients Analyze, n=5 Analyze, n=10
Table 1. The composition of the
CrKSO4 150 COD 7373±123 13056±1775
synthetic tannery wastewater used in the
lab-scale system (the chemical Na2S×XH2O 1500 S2- 215±4 474±76
ingredients and the analysis results) is Bushnell 60 SO4 157±19 508±37
displayed in the table below as well as NH4Cl 200 Tot-Cr 35±3 32.4±5.7
the composition of raw tannery NaNO2 50 Tot-N 253±24 1198±248
wastewater from Mojo used in the pilot KNO3 140 Tot-P 26±2.7 1.5±0.4
system. Concentrations are given in mg/L NaAc 2500 pH 10±0.1 11.2±0.8
except for pH. CrSO4 100
Peptone 4500
 Pilot system Lab-scale system

STIRRING AIR STIRRING

MLSS
RECYCLING

IN
OUT
IN OUT

SLUDGE
SLUDGE
SLUDGE RECYCLE

Figure 1. A schematic picture of the pilot plant setup Figure 2. The setup and flows of the
displaying the anoxic tank, the aerated tank and the lab-scale system. The dashed line
final settling tank as well as the different flows. symbolizes manual transfer.

Nitrifying bacteria in pilot system

The pilot plant was run for 3.5 month, including start up. During this period the system was
stabilised and the running parameters were optimized. Samples were taken twice a week throughout
the experimental period and were analyzed for chemical oxygen demand (COD), biological oxygen
demand (BOD) total phosphorus (Tot-P), total nitrogen (Tot-N), ammonia (NH4-N), nitrate (NO3-
N), nitrite (NO2-N), sulphate (SO42), sulphide (S2-), total chromium (Tot-Cr), temperature, pH and
dissolved oxygen (DO), in order to supervise the system performance. To control the sludge
properties MLSS from the anoxic and aerated tank was analysed one to three times a week for
settleable solids (SS), total suspended solids (TSS), total volatile suspended solids (TVSS), total
solids (TS) and denitrification/nitrification properties. Samples of anoxic and aerated sludge for
FISH analyze were taken continuously two to four times a week. The occurrence and abundance of
different nitrifying bacteria was monitored using FISH during the start up and the stable phase.

Bioaugmentation with agar-embedded denitrifying bacteria

The bioaugmentation experiment was conducted in the lab-scale system. The two anoxic reactors
was first run until the processes were stabilised, then a series of five measurements was carried out
where samples were taken from the inlet and outflow of the system. The samples were analysed for
COD, Tot-N, Tot-P, NH4, NO3, NO2, SO4, S2-, pH, temperature and DO. In addition MLSS from
the anoxic reactors were sampled and fixated for FISH. At three occasions prior to bioaugmentation
the denitrification activity of the sludge in the two anoxic reactors was measured. Subsequently the
two systems were inoculated with agar-embedded denitrifying bacteria. One reactor was seeded
with Brachymonas denitrificans CCUG 45880T and the other with Comamonas denitrificans ATCC
700936T. B. denitrificans was chosen because of its’ documented denitrification properties and
tolerance against chromium (III) and sulphide (Leta et al. 2004). C. denitrificans was chosen
because of its’ documented denitrification properties and abundant appearance in anoxic zones of
various biological treatment plants (Gumaelius et al. 2001). Overnight culture of the two bacterial
species were mixed with agar and cast to form 1% agar beads of varying size in the range of 0.5-1
cm3 and with a bacteria concentration of around 1×109 cells per bead for B. denitrificans and 1×108
cells per bead for C. denitrificans. The beads were made larger than the outlet tube of the anoxic
reactor in order to avoid a washout. Approximately 500 beads, which occupied a volume of 400
mL, were added. After inoculation the system was run for three weeks. During this period a series
of five analyses of physical parameters were performed in the same way as before inoculation.
Similarly, samples for FISH were fixated and denitrification activity was measured.
Fluorescent in Situ Hybridization
The diversity of nitrifying bacteria in the pilot system and the abundance of B. denitrificans and C.
denitrificans in the lab-scale reactors were visualized using FISH technique. The sampled MLSS
was immediately fixated for two hours in 4% (w/v) paraformaldehyde in 10 mM sodium phosphate
buffer solution (pH 7.2) for 2 h at 4°C according to protocol described by Amann (1995). After
washing, the fixated samples were stored at -20°C in 50% (v/v) ethanol-PBS solution.
Hybridization followed Manz´s protocol (1992) and was performed on gelatine-coated slides at
46°C for 60 minutes in a hybridization solution with formamide concentration as recommended
individually for each probe in their respective references. The probes that was utilised are displayed
in Table 2.

Analyses of Physical Parameters

COD, Tot-P, Tot-N, NH4, NO3, NO2, SO42- and S2- were analyzed using Hach reagent kits (Hach
Water Analysis Handbook, methods number 8000, 8190, 10071, 8038, 8171, 8153, 8051 and 8131)
and a Hach DR/2010 spectrophotometer. BOD was analysed according to standard method number
5210 B. Tot-Cr was analysed as described in standard methods number 3030 and 3500-Cr B
(APHA 1995) using a Buck Scientific 210 VGP spectrometer. Temperature was measured with a
mercury thermometer, pH was measured with a GOnDO electronics pH-metre and DO was
analyzed using a Jenway 970 portable oxygen-meter. SS, TSS, TVSS and TS analyses were
performed according to standard methods, number 2540 F, 2540 D, 2540 E and 2540 B (APHA,
1995). Nitrification and denitrification activity was measured by sampling 100 mL MLSS from the
aerated and anoxic tank and adding standard solution to obtain an initial concentration of 500 mg
NH4-N/L and 100 mg NO2-N/L, and then measuring the NH4-N and NO2-N concentration over
time. TSS was also measured in the samples to provide weight correlation.

RESULTS AND DISCUSSION

Pilot system
The startup of the pilot system was slow and almost two months passed before the process could be
considered stable. The average removal of COD and Tot-N in the stable system was 78% and 54%.
Compared to removal efficiencys of standard municipal biological treatment plants, where 80-95%
and 70% removal can be achieved, the performance is not remarkably good. However, considering
the wastewater characteristics, it is not expected. Chromium removal was unexpectedly high, 99%,
but since Tot-Cr was only meassured in the inflow and outflow, the fate of the chromium is not
known. It is most probable that it presipitated within the system. Sulphide removal also reached
99%. Sulphate removal was lower, approximatley 23%, which was expected since the sulphide
oxidates to sulphate in the presence of dissolved oxygen. Combined tannery effluents are typically
high in fats, proteins and other organic materials. This conveys a degradation of these components
in the first step of the treatment process, in this case the anoxic tank. This was shown by a 176%
increase of NH4-N in the anoxic step. The reduction of NH4-N over the aerated tank, however, was
39% which indicates a functioning nitrification process. The aerobic sludgeage was approximatley
20 days in the stable process and since a sludgeage of 5 days is sufficient for full nitrification at
20°C (Henze et al. 1995) this also indicates a functioning nitrification process. The nitrification rate
of the aerobic sludge reached an average of 57 mg NH4-N/g TSS per hour. Pure culture of nitrifying
bacteria removes approximatly 40 000 mg NH4-N/g TSS per hour (Grunditz, 1999) which means
that nitrifying bactertia made up, by a rough estimete, 0.14% of the aerated sludge TSS. The sludge
showed good settling properties with an average sludge volumed index, SVI, of 44 ml/g TSS. The
high nitrification rate in the aerated sludge combined with the high sludgeage in the pilot system
and the high incoming concentrations of nitrogen from the tannery wastewater should provide good
growth conditions for nitrifying bacteria.
Table 2. The different probes used in this study, their names, specificity, the phylum and class they belong to as well as the reference for each
probe is listed in the table below. The results from FISH performed on sludge from the pilot system at start-up and when ran under stable
conditions are also shown in the table. The occurrence is rated on a scale ranging from (-) when no signal could be detected to (+++++) when the
probe-specific target bacteria appeared in large numbers.
*Domain specific
Name Specificity Reference Phylum Class Occurrence in pilot
Start-up Stable
General
ALF968 Alphaproteobacteria, except of Rickettsiales Neef A. (1997) Proteobacteria Alpha ++++ ++++
BET42a Betaproteobacteria Manz et al. (1992) Proteobacteria Beta +++ +++++
EUB 338 most Bacteria Amann et al. (1990) Bacteria* -
EUB 338 II Planctomycetales Daims et al. (1999) Planctomycetes -
EUB III Verrucomicrobiales Daims et al. (1999) Verrucomicrobia -
EUBmix 1:1:1 mixture of EUB 338, EUB 338 II and EUB III +++++ +++++
GAM42a Gammaproteobacteria Manz et al. (1992) Proteobacteria Gamma + ++
Denitrifying bacteria
COM1424 Comamonas spp. Amann et al. (1996) Proteobacteria Beta - -
DEN Comamonas denitrificans Hemström (2000) Proteobacteria Beta - -
OTU6-178 Brachymonas denitrificans, activated sludge clone A6b Juretschko (2002) Proteobacteria Beta + +++
Nitrite oxidising bacteria
NIT3 Nitrobacter spp. Wagner et al (1996) Proteobacteria Alpha - +
NSR447 Nitrospira spp. Schramm et al. (1998) Nitrospira Nitrospira - +
Ntspa662 genus Nitrospira Daims et al. (2001) Nitrospira Nitrospira - -
Ntspa712 phylum Nitrospira Daims et al. (2001) Nitrospira - + ++++
Ammonia oxidising bacteria
Cluster Nitrosomonas oligotropha lineage (Cluster 6a) Adamczyk et al. (2003) Proteobacteria Beta ++ ++
NmV Nitrosococcus mobilis ("Nitrosomonas") lineage Juretschko et al. (1998) Proteobacteria Gamma ++ ++++
Nse1472 Nitrosomonas europea, N. halophila, N. eutropha, Juretschko et al. (1998) Proteobacteria Beta - -
Kraftisried-Isolat Nm103
Nsm156 Nitrosomonas spp., Nitrosococcus mobilis Mobarry et al. (1996) Proteobacteria Beta, Gamma ++++ ++++
Nso1225 Betaproteobacterial ammonia-oxidizing bacteria Mobarry et al. (1996) Proteobacteria Beta - ++
Nsv443 Nitrosospira spp. Mobarry et al. 1996) Proteobacteria Beta + +
Nitrification population. The EUBmix probe gave a high response throughout the experiment,
showing that the sludge contained large amounts of bacteria at all times. The occurance of
proteobacteria was investigated using three different probes targeting the alpha, beta and gamma
class. The abundance of delta and epsilonproteobacteria was not investigated in this study since
theese groups are relatively small. Amongst the proteobacteria the alphaproteobacteria was the
dominating class both at startup and when the system had stabilised. The occurence of
betaproteobacteria was sparse at startup but very abundant at stabilised conditions. The
gammaproteobacteria, on the other hand, could not be detected at startup and only a moderate
number was observed in the stabilised system. This is somehow contradictory compared with
results from a study made by Norström (2005) on sludge from an aerated system treating domestic
wastewater, where betaproteobacteria was the dominating class and alphaproteobacteria only
ocurred sporadically in moderate numbers. Furthermore, the majority of the target organisms of the
probes that gave a high response belonged to the beta or gammaproteobacteria and not to the
alphaproteobacteria class. The denitifying bacteria C. denitrificans was not observed at any time,
while the other investigated denitrifying species B. Denitrifican, could not initially be detected, but
was subsequently discoverd to be an abundant species in the stabilised system. The bacteria mainly
responsible for ammonia-oxidising, out of the ones tested, seemed to be bacteria belonging to the
Nitrosomonas spp. Together with Nitrosococcus mobilis. The probes targeting these groups formed
small clusters that produced strong signals. The nitrite oxidation was performed by bacteria in the
phylum Nitrospira but none of the Nitrospira spp. genus or species-specific probes gave any
significant response. This might indicate that bacteria belonging to the phylum Nitrospira that are
still unknown were present in the system or that the NIT3-probe, specific to Nitrobacter spp., was
not properly hybridised. The abundant occurance of nitrifying bacteria in the aerated sludge
reflected the favourable growth conditions, that were mentioned above, provided in the system. The
results from the FISH analysis as well as a list with information about the probes and their target
organisms are provided in Table 2.

Lab-scale system
The attempt to create a stable and shock-load resistant predenitrification systrem by
bioaugmentation with immobilised denitrifying bacteria was not succesful. The denitrification rates
of both of the inoculated reactors were lower after than before inoculation and no significant
enhancement could be seen in the process performance.

Reactor 1. The physical appearance of the agar beads remained the same throughout the
expeiments. FISH results showed no presence of B. denitrificans in the reactor prior to inoculation.
However, two days after inoculation a large number of B. denitrificans could be detected in the
sludge and only a few days later the bacteria could be seen in the outflow from the system.
Furthermore, three weeks after inoculation no B. denitrificans was detected in the sludge. This
indicates that the method of immobilisation was not suitable and could not retain the bacteria in the
system.

Reactor 2. Shortly after inoculation a large number of C. denitrificans was observed in the sludge.
This number increased even more during the following days simultaneously as the agar beads
dissolved. After five days no remainders of the beads could be found in the reactor but the number
of bacteria stayed high for two more days and then started to decline until ten days after inoculation
when no more bacteria could be detected. The disolvance of the agar beads was probably caused by
the high sulphide content in the feed but since the two systems were fed by the same synthetic
tannery wastewater with equal flow rates another explanation must be found. However, no further
investigations in this matter were performed.
CONCLUSIONS
The most commonly occuring nitrifying bacteria in biologic tannery wastewater treatment sludge
(out of the different specificitys tested in this study) was found to be Nitrosococcus mobilis or
species belonging to the genus Nitrosomnas (ammonia oxidizers) and bacteria within the phylum
Nitrospira (nitrite oxidizers). Large numbers of nitrifyers in clusters was observed at steady-state,
eventhough very few were seen during startup. B. denitrificans was found to be an abundant
denitrifying species in the anoxic sludge. Bacteria within the phylum proteobacteria appeard
abundantly and the dominating classes were alpha and beta.

The approach to use agar as an immobilisation matrix for bioaugmentation of denitrifying bacteria
in biological predenitrification systems treating tannery effluents was not sucsessful. The high-
strength wastewater dissolved the agar beads and in neither a good retention of bioaumented
bacteria in the system, nor an enhancement in denitrification activity was achieved. The aim to
improve the shock-resistance of such system was not fulfilled.

ACKNOWLEDGEMENT
The authors thank Civilingenjörsförbundet and Miljöfonden for partial funding of this study. The Swedish
international development cooperation agency, Sida, through the East African Regional Programme and
Research Network for Biotechnology, Biosafety and Biotechnology Policy Development, BIOAERN, also
supported this work.

REFERENCES
Adamczyk J., Hesselsoe M., Iversen N., Horn M., Lehner A., Nielsen P. H., Schloter M., Roslev P. and
Wagner M. (2003).The isotope array, a new tool that employs substrate-mediated labelling of rRNA for
determination of microbial community structure and function. Appl. Environ. Microbiol. 69, 6875-6887.

Amann R. (1995). In situ identification of micro-organisms by whole cell hybridisation with rRNA-targeted
nucleic acid probes. In: Molecular Ecology Manual, Kluwer Academic Publishers, Dordrecht, pp1-15.

Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R. and Stahl D. A. (1990). Combination of
16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.
Applied Environmental Microbiology, 56, 1919-1925.

Amann R., Ludwig W., Schulze R., Spring S., Moore E. and Schleifer K.-H. (1996). rRNA-targeted
oligonucleotide probes for the identification of genuine and former pseudomonads. Systematic and Applied
Microbiology, 19, 501-509.

Cassano A., Molinari R., Romano M., Drioli E. (2000). Treatment of aqueous effluents of the leather
industry by membrane processes: A review. Journal of Membrane Science, 181, 111-126

Daims H., Brühl A.., Amann R., Schleifer K.-H. and Wagner M. (1999). The domain-specific probe EUB338
is insufficient for the detection of all Bacteria: Development and evaluation of a more comprehensive probe
set. Systematic and Applied Microbiology, 22, 434-444.

Daims H., Nielsen J. L., Nielsen P. H., Schleifer K. H. and Wagner M. (2001). In situ characterization of
Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67,
5273-5284.

Ethiopian Privatization Agency, EPA (2002). The leather industry: Ethiopian Privatization Agency webpage.
Updated July 15, 2002. http://www.telecom.net.et/~epa/Sectors/leather.html
Environmental Protection Authority, EPA (2003). State of the environment report for Ethiopia.
Environmental Protection Authority, Addis Ababa.

Grunditz C. (1999). Bioasseys for the determination of nitrification inhibition. PhD thesis, Department of
Biotechnology, Royal Institute of Technology, Stockholm

Gumaelius L., Magnusson G., Pettersson B. and Dalhammar G. (2001). Comamonas denitrificans sp. nov.,
an efficient denitrifying bacteria isolated from activated sludge. International Journal of Systematic and
Evolutionary Microbiology, 51, 999-1006

Hemström T. (2000). In situ identification of Comamonas denitrificans by whole cell hybridization with
rRNA-targeted probes, Department of Biotechnology, Royal Institute of Technology, Sweden

Henze M., Harremoës P., la Cour Jansen J. and Arvin E. (1995). Wastewater Treatment: Biological and
Chemical Processes. Springer, Heidelberg.

Juretschko S., Loy A., Lehner A. and Wagner M. (2002). The microbial community composition of a
nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle
rRNA approach. Syst. Appl. Microbiol. 25, 84-99.

Leta S. (2004). Developing and Optimizing Process for Biological Nitrogen Removal from Tannery
Wastewaters in Ethiopia. PhD thesis, Department of Biotechnology, Royal Institute of Technology

Manz W., Amann R., Ludwig W., Wagner M. and Schleifer K.-H. (1992). Phylogenetic
oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst. Appl.
Microbiol.15, 593 - 600.

Mobarry B. K., Wagner M., Urbain V., Rittmann B. E. and Stahl D. A. (1996). Phylogenetic probes for
analyzing abundance and spatial organization of nitrifying bacteria. Appl. Environ. Microbiol. 62, 2156-
2162.

Neef A. (1997). Anwendung der in situ Einzelzell-Identifizierung von Bakterien zur Populations-analyse in
komplexen mikrobiellen Biozönosen. PhD thesis, Technische Universität München.

Norström A. (2004). Treatment of domestic wastewater using microbiological processes and hydroponics in
Sweden. PhD thesis, Department of Biotechnology, Royal Institute of Technology

Pommerening-Röser A., Rath G. and Koops H.-P. (1996). Phylogenetic diversity within the genus
Nitrosomonas. Syst. Appl. Microbiol. 19: 344-351.

Schramm A., De Beer D., Wagner M. and Amann R. (1998). Identification and activities in situ of
Nitrosospira and Nitrospira spp. as dominant populations in a nitrifying fluidized bed reactor. Appl. Environ.
Microbiol. 64: 3480-3485.

Standard Methods for the Examination of Water and Wastewater (1995). 19th edn, American Public Health
Association/American Water Works Association/Water Environment Federation, Washington DC, USA

United Nations Environment Program, UNEP (1991). Tanning and Environment - A Technical Guide to
Reducing the Environmental Impact of Tannery Operations. In: UN publication series III, UNEP, Paris.

Wagner M., Rath G., Koops H.P., Flood J. and Amann R. (1996). In situ analysis of nitrifying bacteria in
sewage treatment plants. Wat Sci Techn 34: 237-244.