Research Article

Received: 16 August 2012, Revised: 29 October 2012, Accepted: 27 November 2012 Published online in Wiley Online Library: 25 January 2013

(wileyonlinelibrary.com) DOI 10.1002/ffj.3143

The influence of thermal reaction and

microbial transformation on the odour
of human urine†
Myriam Troccaz, Yvan Niclass, Pauline Anziani and Christian Starkenmann*
ABSTRACT: The typical stale urine odour is the consequence of thermal or bacterial degradation. Gas chromatography–mass
spectrometry, equipped with a multi-sniffing port for olfactive evaluation (GC-MS-O), was performed to analyse an organic
extract of boiled urine originating from seven male donors. This analysis stressed the importance of guaiacol, 2-methoxy-
4- and -6-methyl phenol, 4-vinyl-guaiacol, indole, skatole and vanillin in urine malodour. Analysis of the headspace of the
highly volatile compounds showed the occurrence of trimethylamine, methyl mercaptan and dimethyl sulfide. However,
odour artefacts such as pyrazines, acetyl thiazole and sugar degradation products were formed during the boiling process
and represented important background noise. In parallel, fermented urine samples were judged by sniffing to be more
characteristic of a urine smell, due to the presence of methional, phenol, p-cresol and a-androstenol. The two urine-isolated
Enterobacteriaceae, Escherichia fergusonii and Morganella morganii (urease positive), were particularly efficient at increasing
urine pH and generating phenol, p-cresol, indole, dimethyl sulfide and trimethylamine after 3 days of incubation of sterile male
urine at 37  C. Streptococcus agalactiae produced a high level of a-androstenol, whereas Micrococcus luteus CIP 103664 and
isolated anaerobic bacteria did not produce any malodours. Analytical comparisons between boiled and fermented aged
urine revealed that incubation of sterile urine with a bacterial mixture of E. fergusonii, Enterococcus faecalis, Citrobacter
koseri, S. agalactiae and M. morganii produced a representative aged urine odour. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: urine odour; Morganella morganii; Streptococcus agalactiae; fermented urine, a-androstenol

Introduction Sato et al.[9] analysed human waste samples collected from a

Urine odour nauseates most people. It may be described as foul,
strong, musty, sweet, or as smelling like sulfur or ammonia.
Although the odour may be caused by diet[1,2] or mild to serious
diseases and disorders,[3,4] in most cases, it is caused by physico-
chemical degradation of urine components or by microbial
fermentation.[5] Many analyses of urine volatiles have been
published in relation to urinary tract infections (UTIs)[6] or
metabolic diseases such as trimethylaminuria.[3]
Urine odour analysis has a long history; in 1914, the presence of
phenol, indole, skatole, benzoic acid, phenylacetic acid, hippuric acid
and ammonia had already been described.[7] However, after
conducting a literature review, we found it difficult to elucidate which
compounds make the smell of stale urine unique. Recently, Banday
et al.[8] analysed urine samples of patients with tuberculosis versus
those of healthy individuals. The headspace of the samples, as ana-
lysed by gas chromatography–mass spectrometry (GC-MS), revealed
the occurrence of o-xylene, isopropyl acetate, 3-pentanol dimethyl
styrene and cymol, but the overall smell was not reported. Storer
et al.[6] investigated the compounds occurring in the headspace
of urine samples inoculated with the UTI-causing microbes
Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphy-
lococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae,
Enterococcus faecalis or Candida albicans. From this work, we might
expect that P. vulgaris, which generates indole, 2-aminoacetophe-
none, trimethylamine and (Me)nSn derivatives, and to a lesser
extent E. coli and E. faecalis, contributes to the characteristic smell
of urine. However, the authors did not report the specific contribu-
200
storage tank in a sewage disposal plant. They provided a list of
volatiles detected in urine that had previously been mixed with
faeces. The concentrations of H2S, methyl mercaptan, ammonia
and trimethylamine were 20–50, 0.5–1, 20–35 and 0.8–1.2 mg/
kg, respectively. Interestingly, the authors speculated that the
offensive urine smell came not from ammonia, but from
trimethylamine, which has an odour detection threshold 1000
times lower than that of ammonia. They also mentioned the rela-
tive occurrence of phenol, skatole (quantified at 0.1–0.5 mg/kg),
p-cresol, 3-ethylphenol and indole. In a study by Sastry et al.,[10]
they underlined the importance of a-androstenol and the role
of bacteria in transforming a-androstenol in androstenone, which
is responsible for stale urine odour, but again, the analytical
evidence was missing.
In 2011, Shirasu and Touhara[11] reviewed the ‘scent of
disease’ from the human body, with one section being dedicated
to urine. They claimed that differences in smell can be detected
* Correspondence to: Christian Starkenmann, Firmenich SA, Corporate
R&D Division, Route des Jeunes 1, CH-1211 Geneva 8, Switzerland. E-mail:
christian.starkenmann@firmenich.com
†
This article is published in Flavour and Fragrance Journal as part of the Special
Issue ‘Human (mal)-odours: chemistry, biochemistry and perception’, edited
by Andreas Natsch (Givaudan Schweiz AG – Bioscience Fragrance Research,
Dübendorf, Switzerland).
Firmenich SA, Corporate R&D Division, Route des Jeunes 1, CH-1211 Geneva

tion of each compound to the urine smell. 8, Switzerland

Flavour Fragr. J. 2013, 28, 200–211 Copyright © 2013 John Wiley & Sons, Ltd.
Bacteria and odorants in human male urine
from ketones, alcohols, furans, pyrroles and sulfides; unfortunately,
only one reference was cited.[12] Ziatkis et al.[12] described a
complex chromatogram from the headspace analysis of stale urine
after the urine was incubated with glucurase, b-D-glucuronidase
and sulfatase. In this paper, no phenolic compounds were men-
tioned. Pause et al.[13] boiled human urine, distilled the volatiles
and presented the distillate to rats. Piperitone, pulegone and
heptanone were detected by MS. The authors noticed that this
extract, without any phenolic compounds, was not recognized
as urine by rats. This raises the question of the occurrence of phe-
nolic compounds in urine. The occurrence of phenolic compounds
after enzymatic hydrolysis was published by Bieniek et al.[14]
Wagenstaller and Buettner [15] analysed the native human urine
of seven female donors and one male donor by two-dimensional
gas chromatography–olfactometry–mass spectrometry. They
discovered 14 odorants, including five lactones, (E)-b-damascenone,
5a-androst-16-en-3-one, two phenols (4-methylphenol and vanillin),
and skatole, but no indole. They also transformed the native urine
with a b-glucuronidase to liberate phenols and found 24 odorants
in total in this extract.
In the literature, the link between bacterial b-glucuronidases
and tryptophanases and the generation of odorant compounds
is widely reported.[16,17] In addition, urease-positive bacteria
such as Staphylococcus intermedius, P. vulgaris, Klebsiella and
Bacteroides ureolyticus are known to metabolize urea to ammo-
nia.[18–20] However, the link to malodour generation in healthy
individuals has not been made, probably because the majority
of authors have historically focused on UTI and its treatment,
and only a limited amount of analytical work has been carried
out on urine volatiles[15] per se.
Urine does not normally contain a significant number of
microbes. However, bacteria sometimes travel to various parts
of the urinary tract;[21–24] since some of these bacteria inhabit
the gastrointestinal tract, they can also travel from the bowel
to the urethra. Other sources of bacterial growth include lack
of personal hygiene, the method of urine collection, environ-
mental exposure and the urine storage technique.[25–27] In this
study, we aimed to isolate and identify the malodour-generating
bacteria present in the urine of healthy male volunteers. We
expected that characterization of the dominant microorganisms
involved in urine fermentation would provide more insight into
the chemical changes that occur in fresh urine in comparison
with sterile and boiled urine.
Experimental
Chemicals and Solvents
Chemical reagents and solvents were purchased from Fluka–Sigma–
Aldrich (Buchs, Switzerland), Novabiochem (Darmstadt, Germany),
Alfa Aesar GMbH & Co. (Karlsruhe, Germany), SDS Carlo Erba Reactifs
(Val-de-Reuil, France) and Acros (Geel, Belgium). More specifically, the
following were from Aldrich: 2,6-dimethylpyrazine W32730-1, CAS 108-
50-9; 2,3-dimethylpyrazine W32710-7, CAS 5910-89-4; 2-acetylthiazole
W33280-1, CAS 24295-03-2; 2-hydroxyacetophenone A44513-4, CAS
582-24-1; indole 13408, CAS 120-72-9; 16-(5a)androsten-3-one, CAS
18339-16-7; 16-(5a)androsten-3a-ol, CAS 1153-51-1. The following
were from Alfa Aesar: Aesar 2-acetylpyrrole A14593, CAS 1072-83-9;
2-acetylthiophene A10062, CAS 88-15-3; 4-ethyl-2-methoxyphenol
A14239, CAS 2785-89-9; 2-aminoacetophenone A10895, CAS 551-93-9; 3-
methylindole l03890, CAS 83-34-1. The following were from Acros: dimethyl
trisulfur 415030050, CAS 3658-80-8; 3-acetylthiophene 188020010, CAS
1468-83-3; 3-mercapto-1-hexanol 347560050, CAS 51755-83-0.
Flavour Fragr. J. 2013, 28, 200–211 Copyright © 2013 John Wiley & Sons, Ltd.
Gas Chromatography–Mass Spectrometry
Compound identifications were performed on a GC 6890 N (Agilent, Palo
Alto, CA, USA) equipped with a fused silica SPB-1 (30 m  0.25 mm i.d.,
0.25 mm film thickness) apolar capillary column (Supelco, Bellefonte, PA,
USA). The initial oven temperature was held at 50  C for 5 min and then
increased by 5  C/min to 250  C, split mode 1/10. The carrier gas was He
(52 kPa). The column was coupled to a MS 5975B Inert XL MSP (Agilent).
The mass spectra in the electron impact mode were measured at 70 eV
in a scan range from 30 to 300 m/z.
Gas Chromatography–Mass Spectrometry–Olfaction
A GC/MS–multi-sniffing system,[28] enabling simultaneous GC/MS and
GC-O analyses, was used to investigate the compounds responsible for
the impact odour of urine. The GC/MS system included a CP-3800 GC
and a 300-MS, both from Varian (Varian–Agilent, Palo Alto, CA, USA).
The extracts were manually injected on an internal TenaxW cold trap
(kept at 30  C). The trap was then rapidly heated to 250  C for 1 min
into a DB-1 ms column (60 m  0.53 mm i.d., 1.5 mm df; J&W Scientific–
Agilent). Helium was used as the carrier gas under an inlet pressure of
12 psi. The column was kept at 50  C for 5 min and then the temperature
was increased by 5  C/min to 260  C (maintained for 8 min). The GC
column outlet was connected to a four-port splitter; one port was
connected to the MS and another to a second four-port splitter, while
the third port was connected to an auxiliary gas outlet. The second
splitter, on the other hand, divided the flow into three deactivated silica
columns (3 m  0.32 mm i.d.), each placed inside a thermostated transfer
line (250  C), ending on a sniffing port. Each sniffing port was equipped
with an electric push button able to generate a 1 V signal when pressed.
Data were collected with Galaxie Station software, version 2.4 (Varian).
Raw GC-O–multi-sniffing data were treated according to the GC–surface
of nasal impact frequency (GC-SNIF) method, with the appropriate
plug-in extension for Galaxie Station software, version 2.4 (Varian).
Three panellists per GC-O session were asked to use free vocabulary
to describe odours perceived at the sniffing port. The olfactive peak
heights were expressed as nasal impact frequency (NIF) units. The
descriptors and NIF units are reported in Table 1 and Figure 1. In the
case of boiled urine, 12 sniffing tests were performed by nine subjects
who had worked on flavour and fragrance projects for many years, and
who are therefore considered trained subjects, and three subjects who
repeated the sniffing (Figure 1).
Extracts were re-injected on the Agilent GC-MS system. MS peaks
were identified by comparing acquired MS data to those contained in
the Firmenich database built on the injection of reference products
and NIST 147 MS libraries and by comparing calculated linear retention
indices (LRIs) to those obtained from our database. The identity of olfacto-
gram peaks, on the other hand, was assigned by LRI comparison, olfactive
description and mass spectra similarity.
Gas Chromatography–Mass Spectrometry (GC-MS Triple Quad)
A GC Agilent 7890A was equipped with a fused silica HP-1 (30 m  0.25
mm i.d., 0.25 mm film thickness) apolar capillary column from Agilent.
The injector was in pulsed splitless mode (30 psi/0.5 min). The initial oven
temperature was held at 150  C for 5 min and then increased by 5  C/min
to 250  C. The carrier gas was He (52 kPa). The column was coupled to a
MS 7000 triple Quad (Agilent). Chemical ionization was carried out using
methane, with the flow at 20 arbitrary units, the source at 250  C and
Quad at 150  C.
Urine Sampling
On the first morning, urine was collected from a minimum of seven
donors out of 10 healthy Caucasian males donors aged between 24
and 60 years (average 45 years) into 250-ml sterile glass containers. Parti-
201
cipants were asked not to donate urine if they experienced penile
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 202

Table 1. Results of gas chromatography–olfactometry analysis

No. LRI Odour descriptor Boiled NIF(%) Aged NIF(%) Identification LRI of reference Method for confirmation
F
1 849 Maggi, stock cube, meaty 83 92 2-Methyl-2-furanthiol 843 Odour, LRI
2 872 Methional — 100 3-(Methylthio)propanal 873 Odour, LRI
3 885 Cereal, roasted coffee 50 — 2-, 5- or 6-Dimethylpyrazine* 884 Odour, LRI, MS
4 894 Popcorn, pyrazine 83 — 2,3-Dimethylpyrazine* 891 Odour, LRI, MS

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5 940 Sewage, truffle, garlic 50 — Dimethyltrisulfure 940 Odour, LRI, MS
6 961 Medicinal — 67 Phenol 965 Odour, LRI, MS
7 1028 Caramel 75F 58 Furaneol 1028 Odour, LRI
8 1046 Liveche, caramel, urine 92 58 3-Acetylthiophene 1041 Odour, LRI, MS
2-Acetylthiophene 1050
9a 1057 Urine, mushroom, barn, phenolic 75F 92 p-Cresol + guaiacol 1056 Odour, LRI, MS
9b 1059
10 1065 Smokey, sotolone, curry, earthy 58F — 2-Ethyl-3,5-dimethylpyrazine 1060 Odour, LRI, MS
11 1080 Floral, honey 25 93 2-Phenyl-1-ethanol 1084 Odour, LRI, MS
12 1081 Urine, coffee, mouldy, smoke, sweat 67 8 2-Acetyl-5- or -6-methylpyrazine — Odour, MS
13 1092 Onion, tropical fruit 67F — 3-Sulfanyl-hexan-1-ol 1096 Odour, LRI
14 1010 Caramel, maltol-like 25 58 Unknown — —
15 1128 Smoke, medicinal 58 — 2-Vinyl phenol 1128 Odour, LRI, MS
16 1134 Spicy, brothy, sweaty 75F — 2-Acetophenol 1133 Odour, LRI, MS
17 1155 Sweaty, amber, barn 25 58 Unknown — —
18a 1159 Meaty, urine, smoke 83 92 2-Methoxy-5- and -4-methylphenol 1161 Odour, LRI, MS
18b 1163
19 1184 Roasted peanut, cereals 58 — 1-(5,6-Dimethyl-2-pyrazinyl)-1-ethanone — Odour, MS
20 1202 Onion, passion fruit 83 33 Unknown — —

Copyright © 2013 John Wiley & Sons, Ltd.

21 1225 Menthol, dill anis 67F — Pipertone 1225 Odour, LRI, MS
22 1237 Leather, medicinal 58 — 3-Propylphenol 1238 Odour, LRI, MS
23 1243 Cumin, barn, phenolic 58 — 4-Ethylguaiacol 1249 Odour, LRI, MS
24 1250 Floral, indol, faeces 92F 100 Indole 1251 Odour, LRI, MS
25 1260 Floral, urine 100 58 Aminoacetophenone 1265 Odour, LRI, MS
26 1288 Eugenol, clove 75 83 4-Vinyl guaiacol 1284 Odour, LRI, MS
27 1301 Goaty, animal 75 83 4-Ethyl octanoic acid 1299 Odour, LRI, MS
28 1347 Urine, burnt, animal 58 — Skatole 1345 Odour, LRI, MS
29 1354 Caramel, vanilla 58F 83 Vanillin 1350 Odour, LRI, MS
30 1379 Coumarine, vanilla 42F 67 Coumarine 1379 Odour, LRI, MS
F = detected in fresh urine, but NIF not reported.
*Regio-isomers not determined.
LRI, linear retention index; MS, mass spectrometry; NIF, nasal impact frequency.
M. Troccaz et al.

Flavour Fragr. J. 2013, 28, 200–211

Bacteria and odorants in human male urine
Figure 1. Gas chromatography–olfactometry using the nasal impact frequency (NIF) of an organic extract of boiled and aged urine. Dotted line marks
NIF > 50%
secretions or bladder, kidney or urinary tract infections. Participants were
also asked not to donate urine if they had used a personal hygiene spray,
cream or powder on the genital/anal area since bathing. No special
recommendations were given regarding nutrition. Specimens were
initially kept at 4  C, and within an hour they were pooled and transported
to the laboratory for analytical or microbiological experiments. One pool
was collected from a minimum of seven subjects and the volumes were
about 1.5–1.8 l/day. When required, pooled urine samples were filter
sterilized on a 0.2 mm filter (Thermo Scientific Nalgene Disposable Filter
Unit, Thermoscientific, Reinach, Switzerland). To control sterility, we
plated urine samples on ChromID CPS and Columbia colistin and nalidixic
acid agar (CNA agar from Biomerieux SA, Switzerland, Geneva).
Extraction of pooled urine samples
The pooled urine samples (~1 litre, volumes specified in Experimental)
were extracted twice with diethyl ether (300 ml and 200 ml, respectively)
containing dodecane (0.6 mg/l) and then washed twice with 100 ml of a
saturated solution of NaHCO3. These water phases were pooled, cooled
and acidified with HCl to pH 1, and then extracted with diethyl ether
to separate the organic acids. The original organic phases were then
washed twice with 100 ml NaOH 2 N. The water phases were cooled in
an ice bath, acidified and re-extracted as described above to obtain a
fraction containing only the phenols. The original organic phases were
finally washed twice with 100 ml H2SO4 2 N. The basic compounds,
obtained after the aqueous phase was basified with NaOH 2 N, were
extracted with diethyl ether as described above. The original organic
phases were dried with Na2SO4, filtered, concentrated and used as a neutral
phase for complementary GC-MS investigations. Five organic extracts were
finally obtained: total (Figure 1), neutral, acid, phenols and base.
Preparation of Boiled Urine Extracts
Freshly collected urine samples from a minimum of seven of the male
donors (1000 ml, pH 5.5) were pooled, filter sterilized and boiled at reflux
for 3 h. A condenser was mounted above the round-bottom flask, and on
top of the condenser, a trap cooled with isopropanol dry ice collected
the very volatile compounds. After 3 h, the trap was disconnected and
solid-phase micro-extraction (SPME) fibres (polyacrylate and DVB/CAR/
PDMS, 1 cm; Supelco) were exposed to the headspace of the trap when
it warmed up. These SPME fibres were desorbed on GC-MS. The urine
was extracted and analysed. Each fraction’s neutral, basic and phenol
acids were injected on GC-MS to confirm the structures.
Preparation of Fermented Aged Urine Extracts
Freshly collected urine samples from a minimum of seven male donors
(1500 ml, pH 5.5) were pooled and stored outside for 5 days in an open
2 litre glass beaker (23–30 August 2011, in Geneva, Switzerland). The
maximum observed temperature was 31  C, the lowest 15  C. The urine
became brownish (pH 7.5, about the same volume). An aliquot (10 ml)
Flavour Fragr. J. 2013, 28, 200–211 Copyright © 2013 John Wiley & Sons, Ltd.
was extracted with ether (3 ml). The test tube was vigorously shaken
and centrifuged at 4  C. The supernatant was dried on anhydrous
sodium sulfate and filtered. The solvent was partially evaporated with a
gentle stream of nitrogen to about 0.1 ml. This was injected on GC-MS-
O (Figure 1, incubated).
GC-MS Analysis of Steroids 44, 45, 50 and 51
Calibrations were established with solutions of steroids 44, 45, 50 and
51 in ethyl acetate. Solution 1: 5a-androst-16-en-3-one (androstenone)
50 (0.950 mg), 5a-androst-4,16-dien-3-one (androstadienone) 51 (0.734 mg)
in 10 ml EtOAc. Solution 2: 5a-androst-16-en-3a-ol (a-androstenol) 44
(1.842 mg) in 10 ml EtOAc. Solution 3: 5a-androst-16-en-3b-ol (b-
androstenol) 45 (1.090 mg) in 10 ml EtOAc. Mother solution: 1 ml each
of solutions 1–3 completed to 5 ml. Solution 1: 44 (19.00 mg/l); 45
(14.68 mg/l); 50 (36.84 mg/l); 51 (21.80 mg/l). Solution 2: 1 ml of solu-
tion 1 diluted to 10 ml in a gauged flask. Solution 3 (200 mg/l), solution
4 (20 mg/l), solution 5 (2 mg/l) and solution 6 (0.2 mg/l) were prepared by
dilution of 1 ml of the mother solution in a 10 ml gauged flask. The
analysis was performed in single reaction mode: for a-androstenol,
the transitions were 257.1 to 147 (collision energy 20 eV, dwell time
60 ms), 257.1 to 95 (25 eV, 60 ms); b-androstenol: 257.1 to 161 (20 eV,
60 ms), 257.1 to 92.8 (25 eV, 60 ms), 257.1 to 80.9 (25 eV, 60 ms); androste-
none: 273.2 to 255.3 (15 eV, 100 ms), 273.2 to 93 (25 eV, 100 ms), 273.1 to
81 (25 eV, 100 ms); androstadienone: 271.1 to 109 (15 eV, 150 ms), 271.1
to 97 (20 eV, 150 ms).
Bacterial Identification
All media and agar plates were purchased from Biomerieux SA
(Switzerland, Geneva) except urea agar plates (Christensen), which were
from Condalab (Buchs, Switzerland). Sterile sodium chloride 0.9% w/v
was used for urine and bacterial dilutions. Bacterial counts were performed
on 40 ml of freshly collected, pooled urine samples after incubation either
at room temperature or at 37  C without agitation for 1, 4 and 5 days.
Within 1 h of collection, each urine sample was inoculated (in triplicate)
into Chromo ID CPS and Columbia CNA agar plates on arrival at the
laboratory. These were incubated aerobically at 37  C for 24 h for
aerobic bacteria counts or anaerobically at 37  C for 72 h for anaerobic
bacteria counts. BioMérieux’s chromID CPS agar allows for the differen-
tiation E. coli, Proteus spp., Providencia, Morganella sp., Enterococci spp.,
Staphylococcus spp., Streptococcus agalactiae and Klebsiella, Enterobacter,
Serratia and Citrobacter (KESC group). Columbia CNA agar was used for
the selective isolation of Gram-positive cocci (Micrococcus, Staphylococcus,
Streptococcus, Enterococcus and yeast). Colimycine (0.010 g/l) and nali-
dixic acid (0.015 g/l) were added to the formula in order to select for
Gram-positive organisms and fungi (by suppressing the growth of
Gram-negative bacteria).
Anaerobic bacterial counts were performed by plating on Schaedler
agar containing 5% sheep blood, with and without the addition of
203
neomycin (0.005 g/l) and vancomycin (0.075 g/l). Plates were incubated
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under anaerobic conditions at 37  C for 72 h. The strict anaerobic
regime was confirmed by sub-culturing on the same medium under
aerobic conditions.
Gram stain, colony morphology and colour studies were performed
on aerobic and anaerobic isolates. Urea indole medium (UI-F; Biomerieux
SA, Switzerland) was used to detect the presence of urease activity.
The detection of catalase was performed with the ID Color Catalase test
(Biomerieux SA) through the release of oxygen from hydrogen peroxide.
The oxidase reagent (Biomerieux SA) was used for the detection of the
bacterial enzyme cytochrome oxidase.
Chromogenic agar plates, ASAP and REBECCA were used to isolate
enterobacteria (Escherichia, Proteus and Enterococcus species). Urea agar
(Christensen) was used as an aid in the differentiation of enteric Gram-
negative bacilli of the genera Escherichia, Klebsiella, Enterobacter, Serratia,
Citrobacter and Proteus.
The final identification of malodour-producing bacteria was confirmed
by API gallery (API 20E and Rapid ID 32 Strept; BioMerieux SA) and
complete 16S rDNA sequencing (Leibniz-Institut DSMZ-Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany).
Urine Incubation with Malodour-generating Bacteria
Malodour-generating bacteria were identified by inoculating 10 ml of
pooled sterile urine with 10 ml of each isolated cell suspension (1  1014
CFU/ml in 0.9% NaCl solution, in the exponential phase) to end up with a
final concentration of 1  1011 CFU/ml. Samples were incubated at 37  C
for 72 h under aerobic conditions (or under anaerobic conditions for
anaerobic bacteria). In cases of urine odour development judged
by smelling the sample, 200 ml of positive isolates were re-inoculated,
individually or in mixed suspensions, into 200 ml of sterile urine for
further analytical experiments (to end up with a final concentration
of 1  1011 CFU/ml per bacterium). Micrococcus luteus CIP 103664 was
used as a reference microorganism. Whenever possible, four repetitions
were carried out for each isolate at different day intervals to guarantee
the reproducibility of results. To analyse volatile compounds, the head-
spaces of sterile and fermented urine were exposed to SPME fibres
for 45 min at the end of the incubation time at 37  C. Samples were then
adjusted to pH 9 if necessary with NaHCO3 and the totality was extracted
with diethyl ether (200 ml) containing 0.1 mg of dodecane [internal
standard, 10 ml (of 10 mg/ml) in 10 ml of toluene]. The organic phase was
dried on Na2SO4, filtered and distilled with a small Vigreux column to
about 1 ml. The response factor was measured by injecting three times
1 ml of a mixture made of 0.1 ml of 10 mg/10 ml of the compounds to be
quantified and 0.1 ml of 10 mg/10 ml of the internal standard The total
ion current peak area of the internal standard and the compounds were
compared, and the ratio was used to estimate the quantity of targeted
compounds in urine. The phenol had the lowest response factor (0.64)
and the a-androstenol the highest (1.86); the rest were between 0.97
and 1.16.
Results
Comparative Aromagram of Boiled Urine and Fermented
Aged Urine
Freshly collected urine samples from seven male donors
were pooled and filter sterilized. This urine had a faint odour
described as ammonia, animal and sweet. The headspace was
trapped on SPME fibres but no volatile compound was detected.
Therefore, organic compounds were extracted with diethyl ether
and analysed by GC-MS-O. The extract odour, evaluated on a
smelling strip by two female subjects and two male subjects,
was described as weak, slightly animalic and phenolic, and the
volatile compounds with an LRI < 800, such as trimethylamine
204
and methyl mercaptan, were lost during the diethyl ether
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M. Troccaz et al.
evaporation. The compounds smelled by GC-MS-O are indicated
in Table 1 (Table 1; see entries marked ‘F’ in ‘Boiled NIF%’ column).
To increase the content of volatiles and to mimic what could
happen when urine dries on surfaces, we boiled the urine at
reflux for 3 h and overnight. After 3 h, the odour profile was
comparable to overnight boiling; therefore, 3 h was chosen for
this study. The highly volatile compounds were extracted by
SPME during the warming of the cold trap mounted on top of
the condenser. Methyl mercaptan, dimethyl sulfur, dimethyl disul-
fur and trimethylamine were detected among other compounds,
such as 2-butanone, pyridine and 2-pentanone. The organic
extract of boiled urine had a urine, antiseptic, animal, barn
and syrup smell; overall, it was considered too sweet to match
the stale urine odour that can be perceived in some public
areas. The reproducibility was evaluated by using five different
pools of boiled male urine. The GC-MS traces were very complex
and quite different, as many compounds such as ibuprofen,
nicotine, caffeine and alkyl isothiocyanate were detected, but
were irrelevant for the aim of this study. However, with GC-
MS-O, we noticed good reproducibility between olfactograms.[28]
In parallel, a pool of freshly collected non-sterile urine was
placed outdoors in an open flask, and the urine microflora spon-
taneously developed for 5 days (aged urine). This experiment
was meant to mimic the conditions encountered when urine is
spilt on surfaces. The odour of this aged urine was described
as aggressive and stale urine. This urine becomes turbid as the
pH increases from 5.5 to 7.5. The compounds extracted by SPME
and positively identified by MS and LRI were methyl mercaptan,
trimethylamine, acetone, dimethyl sulfide, diacetyl, 2-propanone,
benzene, 2-butanone, methyl vinyl ketone, dimethyl disulfide,
pyrrole, 3-hexanone, 4-heptanone and other minor compounds.
The smell was very authentic and less medicinal and syrupy, but
more amine, animalic and stale than boiled urine. This urine was
extracted as described for the boiled urine, and GC-MS-O was
performed (Figure 1, Table 1; see column labelled ‘Aged NIF%’).
When we compared various aromagrams, we observed that
the first odour perceived by GC-O was meaty, stock-cube-like
and typical of 2-methyl-3-furan thiol 1 (Table 1). This compound
has an odour threshold of about 5  10 6 (mg/kg) in water[29]
and is widely used in savoury flavours. No MS fragments for 1
could be detected, but the LRI and odour profile fit perfectly.
Compound 1 was present in fresh, boiled and aged urine. The
odour of methional [3-(methylthio)propanal] 2, the Strecker
aldehyde of methionine, was smelled in aged urine extracts by
all subjects. Compounds 3 and 4 were alkylated pyrazines; the
MS fragments (m/z 108, 81, 42 and m/z 108, 67, 42) confirmed
these occurrences, but 2,5-dimethylpyrazine could not be differ-
entiated from 2,6-dimethylpyrazine. These compounds were
detected only in boiled urine, as they are artefacts formed
during heat treatment. Compound 5 could be a metabolite
derived from food products since it was detected only twice in
our five repetitions. In any case, its occurrence in urine was
reported previously.[15,30] This compound is important for the
sulfury, rotten, cabbage, methyl mercaptan odour of urine, as
are dimethyl sulfide and dimethyl disulfide, which we also
detected by SPME. Phenol 6, with its typical bandage-like, me-
dicinal smell, was present only in aged urine. Compounds 7
and 8 are compounds commonly found in processed foods.
The p-cresol 9a was formed in aged urine, while guaiacol 9b
was more important in boiled urine, as compared to 9a. However,
both compounds are present in both aged and boiled urine.
Because of their close eluting times and closely related odours,
Flavour Fragr. J. 2013, 28, 200–211
Bacteria and odorants in human male urine
it was not possible to differentiate them by GC-O. Compound 10
was a pyrazine similar to 3 and 4. The floral odour of 2-phenyl-1-
ethanol 11 was more prominent in fermented urine. Compound
12 has a MS fragment (m/z 136, 121, 108, 94, 93, 67) that perfectly
matches 2-acetyl-3-methyl pyrazine, but its LRI of 1053 does not
correspond; therefore, the methyl substitution can be attributed
to either position 5 or position 6. 3-Sulfanyl hexan-1-ol 13 has
an odour detection threshold of 12  10 6 mg/l in water[31] with
a characteristic odour profile of tropical fruit, grapefruit and
Sauvignon Blanc wine. The LRI perfectly matches the LRI of the
authentic sample 13. Being naturally present in many food
products, this compound can also result from the detoxification
pathway of hexenal and xenobiotics in general.[32] No MS spec-
trum could help us to elucidate the structure of 14, but its
odour profile was reminiscent of maltol, caramel or methyl
cyclopentenolone, or it could also be an alkylated phenol or
methoxy phenol. No LRI associated with this odour descriptor
could lead us to a specific compound. 2-Vinyl phenol 15 has
the same MS fragmentation pattern as 4-vinyl phenol. 4-Vinyl
phenol was clearly identified by MS in aged and boiled urine
at LRI 1928, but not by GC-O at the relevant LRI. Therefore,
the structure assigned for compound 15 (LRI 1128), which we
could smell only in boiled urine, was confirmed after the synthesis
of the authentic sample. The synthesis of 15 was performed
via the thermal decarboxylation of o-hydroxycinnamic acid.[33]
2-Acetophenol 16 was not smelled in the fermented urine,
but it was detected in both fresh and boiled urine. Compound
17 displayed a MS and LRI corresponding to menthol, but the
odour did not match menthol 43 (Figure 2). Compound 17
co-eluted with menthol, and the structure could not be eluci-
dated. 2-Methoxy-5-methylphenol, LRI 1157, and 2-methoxy-
4-methylphenol, LRI 1166, 18a and 18b were key contributors
to urine smell. The MS of 2-methoxy-6-methylphenol was similar
but its LRI at 1151 disqualified it as a candidate. Compound 19
had a typical smell of acetylated pyrazine, a food flavouring
compound, and it was probably an artefact formed during the
boiling process. For compound 20, no relevant MS spectrum or
fragment could be extracted from the GC-MS background noise,
but it smelled like passion fruit, typical for sulfur compounds
known to sometimes have low odour thresholds. We also had
no obvious candidate in our database that could fit both this
Figure 2. Chemical structures of selected compounds present in urine
Flavour Fragr. J. 2013, 28, 200–211 Copyright © 2013 John Wiley & Sons, Ltd.
odour and the LRI. Compound 21 is piperitone, which was
described in 1973.[12,34] The occurrence of carvone had also been
reported in urine;[35,36] we confirmed its occurrence by MS and
LRI, but it did not show an NIF > 30%. No MS, and therefore no
structure, could be attributed to compound 22, which is most
probably a phenolic compound. A background odour reminiscent
of sweat, cumin and animalic at LRI 1240 could correspond to
3-methyl-3-hydroxy hexanoic acid 31 (Figure 2). This compound
was discovered as an important contributor in human sweat
odour with an odour threshold of 2  10 9 mg/l air.[37] The
LRI and the odour match, but this region of the GC was rich in
compounds, and the elution of acids is not sharp; this may explain
why we could have missed the NIF signal. For this reason, an acid–
base extraction was performed. The extract was methylated with
diazomethane, and by GC-MS, the occurrence of 31 in urine was
confirmed. Compounds 23 to 26 are very typical and well-known
odorants, with a barn, phenolic, floral, faeces and urine odour.
Compound 27 is an acid with a typical goat smell; the reported
odour threshold for (S)-( )-4-ethyloctanoic acid is 6  10 3 mg/
l air.[38] Skatole 28 was next detected and identified only in boiled
urine in all repetitions, but not in aged urine. Vanillin 29 and
coumarin 30 were fully identified in fresh, boiled and fermented
urine. More compounds were smelled after 30 min by only
three panellists, at which time the formal sniffing was stopped
(Figure 1). Raspberry ketone 32 (Figure 2), with an odour thresh-
old of about 1–10  10 3 mg/l,[39] was detected in boiled urine.
Odours reminiscent of woody, musky, coconut fruity and sandal-
wood were also smelled at later retention times. g-Decalactone
at LRI 1426 with the typical MS fragment (m/z 128, 85) was pres-
ent, as well as d-decalactone (m/z 128, 99), which was previously
reported.[15] Two compounds with an LRI > 1380, having a strong,
aged, sweet urine smell, were noticed: indolinone 33 (LRI 1423)
and vanilone 34 (LRI 1440) (Figure 2). Steroids were also smelled,
as described below (Figure 3).
Acid–Base Extractions and Analysis of Acids and Phenols
The acids were analysed after acid–base extractions. Short-chain
fatty acids such as butyric acid, isovaleric acid and isobutyric acid
were 10 times less abundant compared with benzoic acid 35.
The major acids in aged urine were (4-hydroxy-3-methoxyphenyl)
205
wileyonlinelibrary.com/journal/ffj
 M. Troccaz et al.

Figure 3. Chemical structures of relevant steroids analysed in fermented urine. For clarity, the absolute stereochemistry of all centres is not drawn. For
a-androstenol the absolute configuration was (2R,5R,7S,15R)-2,15-dimethyltetracyclo[8.7.0.02,7.011,15]heptadec-13-en-5-ol. This is consistent with all
steroids, unless otherwise specified

acetic acid 36, (4-hydroxyphenyl)acetic acid 37 and 1H-indol-3- Analysis of Steroids

ylacetic acid 38, N-(benzoyl)glycine 39, in agreement with
published data.[40–42] For minor trace compounds such as
3-methyl-3-hydroxy hexanoic acid 31,[43,44] the acid fraction was
methylated and at the expected LRI (1050) the mass spectrum of
this acid was observed in boiled and fermented urine.
The selective extraction of phenols displayed major differences
for phenol 6, which was not detected in boiled urine, but is one of
the major peaks in aged urine. The p-cresol 9a, co-eluting with
guaiacol 9b, was 188 times more abundant in fermented urine.
These values were estimated after we normalized the peak
surface according to the peak surface of the internal standard,
vanillin 29, was present in both extracts (Figure 4). The 4- and
5-vinyl guaiacols 26 and 40 were present in a ratio of 95/5 (peak
area) in the boiled urine and a ratio of 45/55 in the aged urine.
The 3-propyphenol (compound 22) and 4-propylphenol were only
detected on GC-MS (m/z 108, 121) in fermented urine. These differ-
ences in phenolic compounds between boiled and fermented
urine could explain the odour differences.
The major difference between aged and boiled urine was
the occurrence of a-androstenol 44, b-androstenol 45 and b-
androstadienol 46 (androsta-5,16-dien-3b-ol) in aged but not
in boiled urine (Figure 3). In boiled urine, the thermal degradation
of steroid sulfate derivatives led to the formation of 47–49.[45]
Figure 4 focuses on the chromatogram of 44–46, 50 and 51, in
total current ion mode, of fresh sterile urine, fresh sterile urine
stored for 3 days at 37  C (blank), boiled urine and urine fermen-
ted with a blend of selected bacteria (bacteria mix). a-Androstenol
44 and b-androstadienol 45 were detected mainly in urine trea-
ted with the mix of bacteria but not in boiled urine. a-Androstenol
44 (LRI 2176) co-eluted with b-androstadienol 46 (LRI 2176),
but by MS extract ion monitoring mode at m/z 274, 259, 241
and m/z 272, 257, 239 the peak area ratio 44/46 was 9/5 when
the bacteria mix was used. Androsta-4,16-dien-3a-ol (LRI 2149),
androsta-4,16-dien-3b-ol (LRI 2160) and androsta-5,16-dien-3a-ol
(LRI 2154) were also injected on GC-MS and not seen.

Figure 4. The chromatogram corresponds to urine extracted with organic solvent, which was subsequently concentrated (200) and injected on
GC-MS (total ion current). Authentic samples were mixed at the same concentration (0.2 mg/l) and injected. The ratio between 44 and 46 was 9/1,
206

determined in single reaction mode

wileyonlinelibrary.com/journal/ffj Copyright © 2013 John Wiley & Sons, Ltd. Flavour Fragr. J. 2013, 28, 200–211
Bacteria and odorants in human male urine
Using a GC-MS triple quadrupole in single reaction mode,
we detected androstenone 50 and androstadienone 51 only in
fermented urine and in the urine fermented with the bacteria
mix. The sample injected corresponded to urine concentrated
200 times. From the peak surface, in comparison with the
authentic sample injected at 0.2 mg/l, we estimated the concen-
tration of a-androstenol at 0.17 mg/l in this sample, which is
consistent with the results displayed in Table 2. Androstenone
50 and androstadienone 51 are in the range of micrograms
per litre (ppb).
Bacterial Identification in Human Urine
A total of 870 CFU/ml aerobic bacteria and 80 CFU/ml strict
anaerobic bacteria were isolated from freshly collected urine
pools. As expected, this number is inferior to 10 000 CFU/ml, as
is typical for samples from healthy donors.[27] A minor portion
of the aerobic bacteria were identified as aerobic Gram-negative
bacilli (10 CFU/ml), and the remaining portion as aerobic Gram-
positive cocci (860 CFU/ml) isolated on CNA (colistin and nalidixic
acid) agar plates.
The Gram-negative bacilli belonged to the Enterobacteriaceae
family, and included Escherichia, Proteus, Klebsiella, Enterobacter,
Serratia, Citrobacter and Morganella species. Urine-isolated
Gram-positive cocci were catalase negative and identified as
Streptococcus or Enterococcus species.[46] Neither eukaryotic
microorganisms nor Micrococcus spp. were isolated in our urine
samples. Only two genera of anaerobic strains could be isolated
under anaerobic conditions (but not under aerobic incubation)
on Schaedler agar containing sheep blood. They were identified
by 16S rDNA sequencing as Dialister microaerophilus DSM 19965
(100% 16S rRNA gene sequence similarity with bacterial isolate
URI 46) and Actinobaculum schaalii CCUG 27420 (97.7% similarity
with bacterial isolate URI 73).
Upon incubation of urine samples, bacterial counts increased
rapidly for both Gram-positive and Gram-negative species and
reached a maximum concentration of 8.5 log CFU/ml after
1 day of incubation at 37  C and after 4 days of incubation at
room temperature. No Enterobacteriaceae survived after 5 days
at 37  C.
Gram stain, shape, colour identification, urease and oxidase/
catalase tests were performed on 73 urine isolates. After incuba-
tion at 37  C of all 73 isolates in fresh sterile urine samples, 13
were found to generate an intense and repulsive urinary odour.
These strains were further sub-cultured on selective media
[ChromoID CPS, CNA agar, ASAP, REBECCA and urea agar
(for Enterobacteriaceae only)] before identification by API gallery
and 16S DNA sequencing.
Of the 13 malodour-generating isolates, seven different species
were identified, five of which were found to be Gram-negative
and two to be Gram-positive bacteria. Identification of the
Gram-negative isolates by 16S rDNA sequencing revealed the
presence of Escherichia fergusonii ATCC 35469 (99.8% 16S rRNA
gene sequence similarity with URI A sequence; 99.7% 16S rRNA
gene sequence similarity with Escherichia coli ATCC 11775),
Enterobacter aerogenes JCM 1235 (99.9% similarity with URI B),
Citrobacter freundii DSM 30039/C. brackii CD 08058 (99.7% similar-
ity with URI D), Morganella morganii CIP A231 (99.8% similarity
with URI 31, URI 20 and URI 62) and Citrobacter koseri LMG 5519
(99.4% similarity with URI 6). Gram-positive bacteria were identi-
fied as Streptococcus agalactiae JCM 5671 (100% similarities with
Flavour Fragr. J. 2013, 28, 200–211 Copyright © 2013 John Wiley & Sons, Ltd.
URI 3, URI 11 and URI 12) and Enterococcus faecalis V583 (99.9%
similarities with URI 2, URI 27 and URI 30) (Table 2).
Of the isolates, Escherichia sp. and Enterococcus sp. are facultative
anaerobic bacteria and responsible for UTI when present in high
concentrations,[19,23,24] whereas the others are gastrointestinal
commensal microflora. The two Enterobacteriaceae, E. fergusonii
(closely related to E. coli) and E. aerogenes, are part of the faecal
(or thermotolerant) coliforms;[47,48] S. agalactiae (also known
as Group B streptococcus) and E. faecalis are known as faecal
streptococci. They are present in human faeces at densities of
105–107 CFU/g[48] and are known to spread to the vagina.[49]
Isolated Citrobacter species are not urine specific, but can be found
almost everywhere in soil, water and wastewater. M. morganii
(known as Proteus morganii) has been isolated both in the environ-
ment and in the intestinal tract of humans. Morganella was the
only urease-positive bacteria that we isolated.[50,51] However, we
cannot confirm that urease-negative bacteria do not have any
urease activity, but rather only low urease activity in certain
circumstances.[20] The strictly anaerobic bacteria isolated, Dialister
spp. and Actinobaculum spp., did not produce any malodours.
No Bacteroides spp. were isolated from our urine samples and we
did not look specifically for Lactobacillus spp. and Clostridium
spp. Headington and Beyerlein[52] have reported that opportuni-
ties to isolate bacteroides were higher in women than in men. This
may reflect the greater risk for contamination of the distal urethra
in women by genital or faecal flora compared with men.[52–54]
Urine Incubation with Malodour-generating Bacteria
Two hundred millilitres of freshly collected and sterilized urine
was incubated with each previously isolated facultative anaerobic
malodour-generating bacterial isolate, the two strictly anaerobic
isolates (D. microaerophilus and A. schaalii) and the commercial
Micrococcus luteus CIP 103664 species for 3 days at 37  C (Table 2).
Sterile urine samples incubated for 3 days under identical condi-
tions were used as negative controls (blank) in all repetitions.
Because of many variations in DNA sequence within one species,
we decided not to average the analytical results on the same
species, but rather on the same isolates.[4]
At the end of the incubation time, the mixture was extracted
with a solvent containing an internal standard to estimate the
concentration of selected compounds. Standard deviations were
indicated when four repetitions had been carried out with the
same isolate (Table 2). The compounds 6, 9a, 9b, 24, 26, 34,
43 and 44 were selected on the basis of their relevant odour
and the ease of monitoring their chromatographic abundance.
When the urine was incubated with isolated bacteria, it
was possible to observe some trends based on the selected
compounds (Table 2). Phenol 6 and indole 24 were generated
in the presence of C. koseri, E. fergusonii and mainly M. morganii.
The p-cresol 9a was significantly produced by E. fergusonii, and
guaiacol 9b was formed in the presence of C. koseri and E. fergusonii.
Menthol 43 and a-androstenol 44 were observed when urine was
incubated with S. agalactiae and E. fergusonii. Their precursor could
be glucuronide as described previously for 44.[55] 4-Vinylguaiacol
26 was present after incubation with E. faecalis, S. agalactiae,
E. fergusonii and the two anaerobes. Vanilone 34 was always
present in all tested samples. The commercial strain, M. luteus,
did not produce any malodour in our experiments.
The incubation with M. morganii produced the strongest
sulfury, amine type of odour, and the pH rose to 9.0–9.5 due to
207
urease activity.[20] The analysis of highly volatile compounds
wileyonlinelibrary.com/journal/ffj
 208

Table 2. Comparison of quantitative and qualitative analysis of selected volatile compounds using an internal standard obtained with sterile urine incubated with various bacteria
isolates

Species identification Bacterial pHb VOCs in Mean concentration of VOCs in urine samples (mg/l)

wileyonlinelibrary.com/journal/ffj
isolate SPME
Phenol, 6 p-Cresol, 9a Guaiacol, 9b Menthol, 43 Indole, 24 4-Vinylguaiacol, 26 Vanilone, 34 a-Androstenol, 44
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
a
Blank 6.0 0.01 0.01 0.03 0.01 0.00 0.00 0.00 0.01 0.02 0.01 0.10 0.07 0.10 0.06 0.00 0.00
Enterococcus faecalis URI 2 6.0 C2H6S2 0.03 0.01 0.05 0.01 0.00 0.00 0.03 0.05 0.03 0.01 0.18 0.06 0.07 0.02 0.00 0.00
URI 27 6.0 C2H6S2 0.02 — 0.06 — 0.00 — 0.00 — 0.02 — 0.22 — 0.09 — 0.00 —
URI 30 6.0 C2H6S2 0.02 — 0.05 — 0.01 — 0.00 — 0.02 — 0.18 — 0.06 — 0.00 —
Citrobacter koseri URI 6 7.0 C2H6S2 0.60 0.06 0.03 0.01 0.18 0.27 0.04 0.06 3.65 1.75 0.15 0.07 0.12 0.06 0.00 0.00
Streptococcus agalactiae URI 11 6.0 C2H6S2 0.02 0.01 0.06 0.03 0.00 0.00 0.12 0.03 0.02 0.02 0.18 0.10 0.08 0.06 0.15 0.05
URI 3 6.0 C2H6S2 0.02 — 0.02 — 0.00 — 0.06 — 0.01 — 0.14 — 0.06 — 0.09 —
URI 12 6.0 C2H6S2 0.02 — 0.04 — 0.00 — 0.04 — 0.01 — 0.27 — 0.09 — 0.03 —
Morganella morganii URI 62 9.5 (CH3)3 N; 7.55 0.70 0.04 0.06 0.00 0.00 0.01 0.02 4.26 1.26 0.00 0.01 0.01 0.02 0.00 0.00
C2H6S; C2H6S2
URI 20 9.0 (CH3)3 N; 5.18 — 0.00 — 0.00 — 0.00 — 2.91 — 0.03 — 0.03 — 0.00 —
C2H6S; C2H6S2
URI 31 9.0 (CH3)3 N; 5.90 — 0.00 — 0.00 — 0.00 — 4.87 — 0.00 — 0.00 — 0.00 —
C2H6S; C2H6S2
Escherichia fergusonii URI A 7.0 C2H6S2 0.29 0.15 2.09 0.18 0.13 0.04 0.55 0.16 2.01 1.56 0.25 0.23 0.12 0.05 0.08 0.07

Copyright © 2013 John Wiley & Sons, Ltd.

Citrobacter freundii/brackii URI D 6.0 ND 0.12 — 0.04 — 0.00 — 0.00 — 0.02 — 0.02 — 0.03 — 0.00 —
Micrococcus luteus CIP 103664 6.0 C2H6S2 0.02 — 0.01 — 0.00 — 0.00 — 0.01 — 0.00 — 0.00 — 0.00 —
Enterobacter aerogenes URI B 6.0 ND 0.19 — 0.15 — 0.00 — 0.00 — 0.01 — 0.03 — 0.05 — 0.00 —
Dialister microaerophilus URI 46 6.0 ND 0.00 — 0.06 — 0.00 — 0.00 — 0.03 — 0.21 — 0.05 — 0.00 —
Actinobacter schaalii URI 73 6.0 ND 0.02 — 0.03 — 0.00 — 0.00 — 0.02 — 0.32 — 0.19 — 0.00 —
The occurrence of highly volatile compounds was monitored by SPME; only their positive occurrence is mentioned. The pH was measured at the end of incubation time (3 days).
The blank was sterile urine incubated 3 days at 37  C in a closed vessel.
a
Blank: 3-days incubated sterile samples.
b
Final pH after 3-days urine fermentation.
ND, not detected; VOCs, volatile organic compounds; SPME, solid-phase micro-extraction.
M. Troccaz et al.

Flavour Fragr. J. 2013, 28, 200–211

Bacteria and odorants in human male urine

clearly shows the formation of trimethylamine, which is respon-

The compounds were obtained from fresh urine, sterile urine incubated for 3 days at 37  C (= blank), and urine incubated with the mix of six bacteria (URI 2, URI 6, URI 11, URI 62,
a-Androstenol, 44

0.00
0.00
0.00
0.05
sible for the amine smell. The sulfury odour was mainly due

SD

—
to dimethyl disulfide, which was present in most of the
incubated samples.

Mean
0.00
0.00
0.00
0.15
0.07
Urine Fermentation with a Mixture of Bacteria (Bacteria Mix)
Following those results, the five malodour-generating bacteria,

0.01
0.00
0.06
0.04
Vanilone, 34
SD
E. faecalis, C. koseri, S. agalactiae, M. morganii and E. fergusonii,

—
were mixed, added at a concentration of 1  1011 CFU/ml to

Mean concentration of volatile organic compounds in urine samples (mg/l)

sterile urine and incubated to compare these results with those

Mean
0.00
0.19
0.10
0.09
0.07
from the aged urine (Table 3).
All odorants isolated from a single bacterial incubation were
present after incubation with a bacterial mixture. The concentra-

4-Vinylguaiacol, 26

0.00
0.37
0.07
0.04
SD

—
tions of the volatiles were either similar or increased compared
with the aged urine (Figure 5). The pH increased to 9  0.5 after
incubation, but the chemical profile corresponded to what we
would expect from incubations with individual bacteria

Mean
0.01
0.57
0.10
0.09
0.41
(Table 2).
Bacterial counts were carried out on urine incubated with a
bacterial mixture over time. Immediately after inoculation,

0.01
0.08
0.01
0.58
SD
Indole, 24

—
concentrations of E. fergusonii, E. faecalis, C. koseri, S. agalactiae
and M. morganii were confirmed to be 1  1011 CFU/ml of

Mean
urine as defined. After 3 days of incubation at 37  C, no Entero-

0.00
0.37
0.02
4.71
1.95
bacteriacea were detected. Only E. faecalis survived under

NA, not analysed; ND, not detected; SPME, solid-phase micro-extraction; VOCs, volatile organic compounds.
those conditions. Table 3. Qualitative (SPME) and quantitative analysis (organic extraction) of selected volatile compoundsa

0.01
0.00
0.01
0.71
Menthol, 43
SD

—
Discussion

Mean
0.00
0.00
0.00
1.45
0.54
Unpleasant odours in urine basically occur in two ways:
malodorous compounds are already present in freshly collected

0.00
0.03
0.00
0.05
Guaiacol, 9b
SD

—
urine (e.g. diet-dependent odours such as asparagus-like
odours),[1,2,56] or they develop over time, produced by bacterial
Mean

metabolism or thermal reactions.

0.00
0.10
0.00
0.27
0.00
Boiling urine efficiently produced an odour reminiscent
of urine. Many previous studies used thermal treatment to
accelerate odour formation,[12,39,57] or did so in the presence of
0.04
0.01
0.01
0.33
p-Cresol, 9a
SD

—
HCl (which leads to the formation of many chlorinated deriva-
tives).[58] In the present study, some impact odorant compounds,
Mean
0.03
0.02
0.03
3.10
2.19

such as pyrazines 3, 4 and 19, were the result of thermal reactions

(Figure 1). In addition, the loss of key volatiles, such as trimethyla-
0.01
0.01
0.01
0.79

mine and dimethyl (di)sulfur, occurs during boiling. These

Mean SD

—
Phenol, 6

volatiles were detected only in the cold trap, which means that
they were formed and then lost by evaporation. A clear trend
0.01
0.04
0.01
2.90
0.61

was also that 6, 9a, 24, 43 and 44 were not present or less likely
formed in boiled urine compared to aged or fermented urine
C2H6S; C2H6S2

C2H6S; C2H6S2

(Figure 4, Table 3). As a result, the odour profile of boiled urine

VOCs in SPME

was sweet and syrupy and quite different from the odours that
may be encountered in a bathroom or in public toilets.
ND

ND

NA

Aged urine was judged as more representative of a stale

or dirty public toilet smell (Figure 1 and Figure 5, Table 1 and
8.5 (CH3)3 N;

9.0 (CH3)3 N;

URI A) and aged urine (5 days).

Table 3). The most notable differences from boiled urine were
the presence of methional 2, phenol 6, p-cresol 9a, indole 24
and a-androstenol 44 (Table 3). Volatile compounds, such as
5.5

6.0

7.5
pH

trimethylamine, dimethyl (di)sulfur or methyl mercaptan, were

not monitored by SPME and were lost when the volatiles were
Freshly collected

analysed after extraction with an organic solvent, but they could

Urine sample

Bacteria mix

be clearly smelled during the ageing process. We were not able

to confirm the importance of the smell of androstenone 50.[10] Its
concentration was estimated at less than 1 mg/l. The a-androstenol
Boiled
Blank

Aged

44 in urine was in the concentration range of 0.1 mg/l (Table 3),

a

209

which led us to postulate that 44 could be an important compound

Flavour Fragr. J. 2013, 28, 200–211 Copyright © 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj

Figure 5. Comparison of GC-MS traces of a total extract of aged urine (incubated for 5 days outdoors, temperature not controlled) and an extract
of urine incubated with a mixture of six selected bacteria (incubation at 37  C). The blank was a fresh sterile urine sample incubated at 37  C in a
closed flask
for stale urine odour. The relative abundance of 44 compared to 50
after incubation with the bacteria mix is displayed in Figure 4.
The bacteria selected were E. faecalis (URI 2), C. koseri (URI 6), S.
agalactiae (URI 11), M. morganii (URI 62) and E. fergusonii (URI A)
because they generate key impact odorants such as dimethyl
sulfide and trimethylamine: 6, 9a, 24, 44 (Table 2). Proteus
mirabilis, E. faecalis, E. coli and Klebsiella pneumoniae had already
been reported to generate malodours after incubation in
urine, despite the lack of clear analyses of the volatiles.[58] Two
Enterobacteriaceae, E. fergusonii and the urease-positive M.
morganii, were selected for their generation of malodour. The
smell of urine fermented with E. fergusonii was associated
mainly with 6, 9a and 24. The highest pH (9.5) was obtained
with M. morganii and the smell was the result of the formation
of trimethylamine and volatile sulfur compounds. M. morganii
is known for its high urease activity.[19,51] S. agalactiae was particu-
larly interesting for producing a-androstenol 44 and menthol 43.
Previous analytical work confirmed that E. coli (closely related to
E. fergusonii) generates high levels of methanol, formaldehyde,
hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, methyl
mercaptan, trimethylamine and indole.[59,60] Proteus vulgaris
isolated in UTIs (urease positive, tryptophanase positive), but not
in healthy individuals, was also reported to produce high levels
of trimethylamine and indole.[6,61] Citrobacter species were de-
scribed as having differences in their ability to convert tryptophan
to indole 24 through differences in tryptophanase activities.[62]
The incubation of urine with the mix of these five selected
bacteria was representative of the smell of aged urine; Figure 5
and Table 3 display the similarities.
Urine pH is an important parameter, as it may modulate
the smell of urine, bacterial survival and enzyme activities. For
example, a solution of NH4OH at pH 9 is almost odourless,
whereas trimethylamine at 1 mg/l still smells at neutral pH. The
pH of fresh urine is normally between 4.8 and 7.5; however, after
incubation with a mixture of bacteria for 3 days, the pH increased
to 9.0. The increase of pH was attributed to the production of
ammonia.[63] When urine is excreted, the major proportion of
the nitrogen is present as urea (constituting about 2% of human
urine); during bacterial fermentation, this is converted to 2NH+4
and CO23 . Edin-Liljegren et al.[18] have shown that urease activity
was highest at pH 6.4. We confirmed the previous observation
210
that, in pure urine samples, no Enterobacteriaceae survived after
wileyonlinelibrary.com/journal/ffj Copyright © 2013 John Wiley & Sons, Ltd.
M. Troccaz et al.
3 days at 37  C.[60] Faecal streptococci (Enterococci) are consid-
ered more resistant than Enterobacteriaceae at a higher pH and
nitrogen content. Urine composition may certainly influence pH,
bacterial composition and urine odours. Incubation of freshly
collected urine revealed that the maximum concentration of total
bacterial counts was reached after 4 days at room temperature
(20–25  C) and after 1 day at 37  C. Indeed, most of the volatile
compounds were generated during the first hours of incubation.
The selective evaporation of most volatile compounds and the
chemical reactions can influence the change in odour over time.
We did not look at bacteria and odour generated in women,
but we expect a similar odour profile in healthy subjects, except
during menstruation when large changes in substrates, pH and
bacterial ratios may occur. Here, the donors were only men for
simplicity in sampling and to avoid questioning women about
their menstrual cycles.
GC-MS-O analyses also have some limitations; the NIF% gives
a good idea of the impact molecules, but it represents only part
of the information (Figure 1). However, the advantage of this
technique is that the focus is only on smelling compounds and
the GC-O traces are nicely reproducible.
Acknowledgements
We are grateful to Dr Eric Frerot for analytical support with the
MS-triple Quad, Dr Monica Bandera and Dr Laurent Wünsche
for critical discussions and for reviewing the manuscript, and
Dr Isabelle Cayeux for sensory analysis and critical discussions.
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