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Journal
of
General Microbiology
(1989),
135,
1547-1558.
Printed in Great Britain
1547
Nocuvdiu
pirzerzsis
sp.
nov., an Actinomycete Found in Activated Sludge
Foams in Australia
By LINDA L. BLACKALL,'*?
J.
H. PARLETT,3 A. C. HAYWARD,l
D. E. MINNIKIN,3 P.
F.
GREENFIELD2
AND
ANNE
E.
HARBERS2
University
of
Queensland, St. Lucia
4067,
Queensland, Australia
3Department
of
Organic Chemistry, The University, Newcastle upon Tyne
NEl
7RU,
UK
'
Department
of
Microbiology, and Department
of
Chemical Engineering,
(Received
8
February 1989; accepted
15
February 1989)
A new nocardioform actinomycete was isolated by filament micromanipulation during the
course of a study into foaming in activated sludge plants in Australia. It constitutes the second
most prevalent foaming organism in Australia after
Nocardiu amarae.
These two foaming
organisms can be differentiated morphologically, biochemically and chemotaxonomically. The
microscopic appearance of the filaments of the new taxon resembles a pine tree. The filaments
are Gram-positive, non-acid-fast, non-motile, non-sheathed and about 0.5-1
-0
pm in diameter.
On a complex medium, the colonies are orange, opaque, macroscopically dry and friable,
microscopically moist and shiny, with a pasty texture and an entire edge. The strains are positive
for catalase, oxidase and urease and are oxidative in their metabolism of glucose. No strain
could degrade hypoxanthine, xanthine, tyrosine, casein, gelatin or aesculin and none could grow
with lysozyme. The strains contain peptidoglycan type Aly, cell wall type
IV,
whole cell sugar
pattern type A, phospholipid type PII, menaquinones o-cyclo-MK-8
(H4),
fatty acids
comprising straight chain saturated and unsaturated acids and tuberculostearic acid, and
mycolic acids with 58-64 carbons containing substantial amounts of unsaturated chains in the
2-position. The name
Nocardiapinensis
has been chosen for the new taxon because of the pine
tree like appearance of the organism on microscopy. The type strain, UQM3063, is deposited at
the University of Queensland, Department of Microbiology Culture Collection.
INTRODUCTION
The formation of stable, dark, viscous foams or scums on the surfaces of the aeration tank
section of activated sludge processes was first reported in 1969 (Anonymous, 1969). The stable
foam is grey to brown-cream in colour, quite heavy in consistency, up to 30cm deep, and
watersprays applied to the surface have little effect upon its collapse (Lechevalier, 1975; Pipes,
1978; Dhaliwal, 1979). The build up of scum in an activated sludge plant does not appear to
accompany any specific plant design with the exception of the aeration system (Hartley, 1982);
however, operational mode (Ferguson, 1980; Pipes, 1978; Hartley, 1982), influent type
(Lechevalier, 1975; Pipes, 1978; Dhaliwal, 1979; Nelson, 1979; Tricker
&
Thorpe, 1979), and
specific weather patterns (Lechevalier, 1975) have been cited as precipitating factors.
Operational problems have been attributed to the scum, and its added financial cost to the plant
is as a result of extra personnel required for scum control and clean-up.
When the scum is examined microscopically, many Gram-positive, branching filamentous
bacteria are commonly observed (Lechevalier, 1975). Selective accumulation of the actino-
mycetes in the foam over that in the mixed liquor is common. The predominant organism
associated with foams was isolated and identified as
Nocardiu
umarue
by Lechevalier
&
f'
Present address: School
of
Microbiology, University
of
New South Wales,
PO
Box
1,
Kensington 2033,
Abbreviations:
YG, yeast glucose agar; TYG, tryptone yeast extract agar.
Australia.
0001-5399
0
1989 SGM
1548
L. L. BLACKALL AND OTHERS
Lechevalier,
(1
974). This
is
still regarded as the dominant foaming organism; however,
Rhodococcus rhodochrous
(Lemmer
&
Kroppenstedt, 1984) and
'Microthrix paruicella'
(Blackbeard
et
al.,
1986; Hart, 1985) are also implicated.
Foaming in activated sludge plants was investigated in Queensland, Australia, and during the
course
of
the study, a unique actinomycete was isolated. It differed from all the previously
recognized foaming organisms and was therefore fully characterized. This paper describes the
new actinomycete for which the name
Nocardia pinensis
sp. nov.
is
proposed.
METHODS
Isolation and identification.
The strains used in the study are listed in Table 1. All fresh isolates came from
Queensland, Australia. Isolation of the actinomycetes was by micromanipulation using the Skerman
micromanipulator (Skerman, 1968). Bacterial mycelium was manipulated to either yeast glucose agar (YG)
containing (per litre), 10 g yeast extract, 10 g glucose and 15 g agar,
or
to tryptone yeast extract agar (TYG)
containing (per litre),
3
g tryptone,
5
g yeast extract,
5
g glucose and 15 g agar. The pH of all media was adjusted to
7.0 unless otherwise stated and sterilization was at 121 "C for 15 min.
Extraction of cellular DNA was unsuccessful. A summary of the methods used and the possible reasons for the
failure to extract DNA are presented in the Discussion.
Znoculumpreparation.
For
all tests, unless otherwise stated, inoculum was grown on solid TYG for 7 d (reference
strains)
or 21 d
(Nocardiapinensis)
at 28 "C in the dark. Biomass from the plates was vortexed in a bottle containing
5-10 ml sterile distilled water and
a
few 0.25 mm diameter glass beads and the resulting suspension used as
inoculum.
Microcolony and cell morphology,
(a)
Light microscopy.
Inoculum for cultures came from
7
or
21 d old TYG plate
cultures and suspensions were prepared such that when the cells were inoculated onto the TYG media, well
separated cells were visible. Microcolonies of cells were photographed with Kodak Plus
X
pan ASA 125 black and
white film using an Olympus BHS microscope with a BH2-PC phase contrast turret condenser and a PM-1OAD
photomicrographic system.
(b)
Electron microscopy.
Cells
of
N. pinensis
were taken from the surface of TYG plates, negatively stained with
1
%
(w/v) phosphotungstic acid (pH
6.5)
for
20
s
and examined by transmission electron microscopy using an
Hitachi H-800 electron microscope.
Staining reactions.
The Gram reaction (Skerman, 1967
;
Cruickshank
et
al.,
1975), presence of sudanophilic
inclusions (Burdon, 1946), presence of polyphosphate inclusions (Cruickshank
et al.,
1975) and determination of
acid fastness (Cruickshank
et
al.,
1975) were done with all strains with biomass from TYG plates.
Biochemical tests.
The test media were incubated at 28 "C and, unless otherwise stated, read at 14 and 21 d for the
N. pinensis
strains and at 7 and 14 d
for
the remainder
of
strains.
Acid production from carbohydrates.
Tryptone yeast extract medium [0.1% (w/v) tryptone;
0.3%
(w/v) yeast
extract; 1.5% (w/v) agar] with
5
ml 10% (w/v) bromothymol blue
1-l
was the basal medium for these tests. Carbon
sources were at
0.5%
(w/v). The medium was dispensed into tubes and set as slopes. Semi-solid media with glucose
were also used and were stab inoculated. All tests were read at 28 d.
Degradation
of
macromolecules.
Concentrations of the macromolecules were as in Gordon (1967) and the basal
medium was TYG. Inoculum was spotted onto the solid media.
For
aesculin hydrolysis, the method of Sneath
(1956) was slightly modified such that the medium included tryptone and yeast extract in place
of
peptone.
Growth
in
the presence
of
inhibitors.
Mitomycin C
(5
pg ml-'
;
Sigma), isonicotinic acid hydrazide (isoniazid
200
pg ml-*
;
Sigma), rifampicin
(20
pg ml-1
;
Sigma) and 5-fluorouracil (20 pg ml-l; Sigma) were incorporated
into TY G. All antibiotics except isoniazid were filter-sterilized prior to addition to the cooled, sterile basal
medium. Isoniazid was added to the basal medium prior to sterilization. The inoculum was spotted on to the solid
media.
Tween hydrolysis.
TYG containing 0.01
%
(w/v) CaCl, . 2H20 was the basal medium for this test, which was
based on the method of Sierra (1957). Tweens were incorporated at 1% (wlv) and spot inoculation was used.
Detection
of
urease.
TYG (1 litre) with 1.2 ml of 10% (w/v) phenol red and 10 ml of 20% (w/v) filter sterilized
urea was the medium for this test, which was based on the method
of
Christensen (1946).
Nitrite forrnationfiorn nitrate.
The method of Skerman (1967) was used for all strains and liquid TYG was the
basal medium.
Lysozyme resistance.
The method
of
Gordon
&
Barnett (1977) was used for this test and growth was determined
after 7 and 21 d. Ability to grow in glycerol broth without lysozyme was used as a control.
Catalaseand oxidase test.
The catalase test was carried out by adding a drop of 10 vol. H202 onto the growth on a
TYG plate. Instant emission of gas bubbles was positive. The oxidase test was determined by smearing biomass
from a TYG plate onto filter paper impregnated with a drop of the oxidase reagent from a Marion oxidase
dropper. A blue black colouration to the reagent in 10-20
s
was positive.
New Nocardia
sp.
from activated sludge foams
Table
1.
Strains used
in
this study
The first 12 strains are the new actinomycete whilst the remainder are used as reference strains.
Laboratory
Name no.
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia pinensis
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Rhodococcus equi
Rhodococcus equi
Rhodococcus rhodochrous
Rhodococcus rhodochrous
'Gordona aurantiaca'
UQM3063
NM41
NM55b
UQM3064
NMlOl
NM102
NM109
NM110b
NM118
NM167b
NM168b
NM170
UQM2058
UQM28 10
NM26
NM86
N M206
NM296
UQV 1092
UQV 1002
UQM2807
UQM2808
UQM2809
1549
Reference/isolation site*
Bellbowrie STP
Bellbowrie STP
Bundamba STP
Bundamba STP
Burleigh Park STP
Burleigh Park STP
Yandina STP
Bundamba STP
Hayman Island STP
Merrimac STP
Loganholme STP
Nudgee STP
Lechevalier
&
Lechevalier (1974); sewage
plant; type strain
Lemmer
&
Kroppenstedt (1984); sewage plant
Tingalpa STP
Ferny Hills STP
Brackenridge STP
Townsville Golf Course STP
Equine lung
Porcine lymph node
Lemmer
&
Kroppenstedt (1984); sewage plant
Lemmer
&
Kroppenstedt (1984); sewage plant
Lemmer
&
Kroppenstedt (1984); sewage plant
Sourcet
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
ATCC 27808
DSM 43602
Fresh isolate
Fresh isolate
Fresh isolate
Fresh isolate
UQV
UQV
DSM 43583
DSM 43601
DSM 43599
*
STP, sewage treatment plant.
t ATCC, American Type Culture Collection, Rockville, Maryland, USA
;
DSM, Deutsche Sammlung von
Mikroorganismen, Gottingen, FRG
;
UQM, Culture Collection, Department
of
Microbiology, University of
Queensland, Brisbane, Australia; UQV, Culture Collection, Department
of
Veterinary Pathology and Public
Health, University of Queensland, Brisbane, Australia.
Cell wall components. (a) Amino acids and carbohydrates.
The methods followed were those of Harper
&
Davis
(1979) for amino acids and of Staneck
&
Roberts (1974) for carbohydrates. For both, TLC on cellulose-coated
aluminium sheets (20
x
20 cm) (Merck no. 5552) was used and 5-10 pl volumes
of
hydrolysates were applied to the
sheets.
(b) Fatty acids, mycolic acids, polar lipids and menaquinones.
The
N. pinensis
strains were grown for 21 d on
sterile, 0.45 pm pore size, cellulose acetate membrane filters on the surface of TYG plates. The membranes were
lifted from the plates and the cells scraped into sterile 5 ml McCartney bottles and lyophilized without
cryoprotection. Lipids were extracted according to Minnikin
et al.
(1984).
For polar lipids, two dimensional TLC on aluminium backed silica gel sheets was used (Merck no. 5554). The
first direction solvent was chloroform/methanol/water (60
:
30
:
6, v/v) and the second chloroform/acetic
acid/methanol/water (40
:
25
:
3
:
6, v/v). Different spray systems were used for the different polar lipids (Dobson
et
al.,
1985).
Menaquinones were isolated by two dimensional TLC (Nahaie
et al.,
1984) and identified by mass spectrometry
(Collins
et al.,
1977).
Mycolic acid analyses followed the methods of Minnikin
et al.
(1975, 1980) and mass spectrometry of the
tert-
butyldimethylsilyl ethers of the isolated methyl mycolates provided ranges of molecular masses and sizes of the
chains in the 2-position (Minnikin
et al.,
1982).
Whole cell acid methanolysis and isolation by TLC of the fatty acid methyl esters (FAMEs) was carried out by
the method of Minnikin
et a/.
(1980). The FAMEs were identified using a Shimadzu GC Mini 2 flame ionization
chromatograph fitted with both a 25 m OV-1 fused silica bonded column (Alltech) and a 50 m Silar 1OC fused
silica non-bonded column (Alltech)
;
the injected sample was split between both columns. The operating
temperature was programmed from 100 to 200 "C at 5 "C min-1 and peaks quantified using a Shimadzu CE 1B
integrator.
APf
ZYM
determinations.
The API ZYM system was used according to the manufacturer's
(API)
specifications.
Nineteen enzymes were tested per gallery; galleries were incubated for 6 h at 28 "C in the dark prior to detection.
1550
L.
L.
BLACKALL AND
OTHERS
Suspensions in sterile distilled water (described above) were used as inocula.
N.
pinensis
strains were cultured on
TYG media whereas the reference strains were cultured on YG media.
Determination
of
pigment spectra.
N.
pinensis
biomass from 21-d-old TYG plate cultures was harvested into
chloroform and ethanol and vigorously shaken to extract the orange pigment.
A
few drops of 20% (w/v)
KOH
were
added to the ethanol extract to determine the presence of flexirubin-type pigments (Reichenbach
et
al.,
1981). The
chloroform extract was scanned from
200
nm to
800
nm using a Beckman
DU-8
spectrophotometer.
Growth temperature determinations.
Slopes of TYG were inoculated with a drop of bacterial suspension and
placed into a variable temperature incubator. The temperatures selected for the incubator ranged from
11
"C
to
36
"C.
Tubes were examined every
3
d for 21 d.
Determination
of
growth on diflerent media and under diflerent atmospheres.
An
extensive range of complex and
defined media were evaluated for the ability to support the growth of
N.
pinensis.
Incubation
of
all media was at
28
"C
in the dark and growth assessment was made after 21 d. Cultures on TYG were incubated in the light, dark,
anaerobically, microaerophilically, in a candle jar and in an atmosphere containing
5%
(v/v)
C02.
RESULTS
Microcolony and cell morphology
Results from the
N.
amarae strains isolated from the activated sludge plants agreed with the
description given by Lechevalier
&
Lechevalier (1974). Filament fragments of
N.
pinensis in
activated sludge foams and mixed liquors resembled pine trees and as such could be
distinguished from
N.
amarae (Fig.
1).
N.
pinensis strains took 10-21 d to produce colonies that
were about 1-2 mm in diameter on TYG
;
however, the growth rate was variable for no apparent
reason. On some occasions the strains produced 2 mm colonies in 9-10 d whilst on others, the
same strains took 21 d for colonies of the same size to develop. The
N.
pinensis strains on TYG
were orange, opaque, macroscopically dry and friable, microscopically moist and shiny, not
adherent to the agar, difficult to emulsify or subculture, circular with an entire edge. The
majority
of
the colonies were approximately
1
mm in diameter; however, large colonies
approximately 3-4 mm in diameter also occurred. The strains of
N.
pinensis grew in liquid TYG
medium as macroscopically visible, orange coloured colonies in the slightly turbid liquid.
The mycelium of
N.
pinensis did not fragment in undisturbed cultures and secondary
branching was rare to absent. Aerial mycelium was not visible to the naked eye; however, short
branched and unbranched aerial hyphae could be seen microscopically. No specialized
morphological structures have been noted (Figs. 2 and 3).
Examination of microcolonies showed the presence of phase bright spherical regions in the
mycelium
of
N.
pinensis in terminal and intercalary positions. The Gram-positive, septate
mycelia of
N.
pinensis tapered after branch points.
Staining reactions and biochemical tests
The biochemical test results and the staining reactions for the
N.
pinensis strains and for the
reference strains are shown in Table 2.
Cell wall components
All
N.
pinensis strains contained arabinose, galactose, glucose and ribose as the cell wall
carbohydrates and meso-diaminopimelic acid (DAP), alanine and glutamic acid as the cell wall
amino acids. The fatty acids present in the strains are shown in Table 3 and the mycolic acids are
shown in Table
4.
All the mass spectra of the mycolic acid derivatives contained peaks at
m/z
38 1, 355 and 353,
in the approximate ratios 1.0, 0.5 and 0-25, respectively. These fragments are due to cleavage
between carbons 3 and 4, followed by loss of 58 mass units (2-methylpropane), corresponding to
hexadecenyl, tetradecanyl and tetradecenyl chains in the 2-position.
If
the mycolic acid methyl
esters were analysed by pyrolysis gas chromatography (Lechevalier
&
Lechevalier, 1974;
Goodfellow et al., 1982), they would release major amounts of methyl octadecenoate and lesser
proportions of methyl hexadecanoate and hexadecenoate.
Strains UQM 3064, NM1 lob, NM167b and NM41 were found to contain lipoquinones whose
mass spectra had intense peaks at
m/z
187 and 225 consistent with a normal 2-methyl-1,4-
New Nocardia
sp.
from
activated sludge foams
1551
Fig.
1.
Mycelial fragments
of
N.
pinensis
(large arrow) and
N.
amarae
(small arrow) in
a
scum sample
from Bundamba Sewage Treatment Plant. Bar,
20
pm.
Fig.
2.
Microcolonies
of
N.
pinensis
NM41 growing on
TYG
at
28
"C.
(a)
24
h
incubation.
(b)
3
d
incubation. Bars,
10
pm.
Fig.
3.
Microcolony
of
N.
amarae
isolate NM86 grown on
YG
at
28
"C
for
24
h.
Bar,
10
pm.
1552
L. L. BLACKALL AND
OTHERS
Table 2.
Results
for
the biochemical and staining tests
jor
the strains used in this study
None of the strains grew in the presence of rifampicin (20pgml-'), mitomycin-C
(5
pgml-I),
5-fluorouracil (20 pg ml-l) or lysozyme (50 pg
ml-l );
degraded hypoxanthine, xanthine, tyrosine,
casein or gelatin; produced acid from glucose (anaerobic), maltose
or
mannitol
;
were acid-fast. All
strains produced acid from glycerol; contained polyphosphate and sudanophilic inclusions; grew in
glycerol broth; were oxidase and catalase positive; were Gram-positive.
Test
N.
amarae*
N.
pinensist
Growth in the presence of:
Lipolysis of:
Isoniazid (200 pg ml-l)
+
Tween 20
+
Tween 40
+
Tween 60
+
Tween
80
+
Glucose (aerobic)
-
Mannose
-
Salicin
+
Acid production
from
:
Aesculin hydrolysis
Nitrate
3
Nitrite
+
+
*
N.
amarae
strains were UQM2058, UQM2810, NM26 and NM86.
t
N.
pinensis
strains were all those listed in Table
1
except that NM55b was omitted
from
the acid production
from
glycerol, maltose, mannitol, mannose and salicin tests.
Table 3.
Percentage fatty acid composition
of
strains
of
N.
pinensis
Fatty acid type*
Strain 16:l 16
:O
17:l 17
:O
18:l 18
:O
TBS
UQM3063
NM41
UQ
M 3064
UQM3064
NMlOl
NM109
NMllOb
NMll8
NM167b
NM168b
NM170
15-72
18-34
20.29
16.30
15.23
15.07
17.59
17.69
17.38
10.70
29-80
28-85
27-43
26.78
26.23
29.16
19-92
30.30
24-90
2 1.42
40-67
2.43
2.04
0.88
3-16
5.33
2-34
2.59
1.37
3.30
4-37
3.56
1.53
0-99
1.87
6.43
2.29
2.10
1
*46
1.68
5.72
15-19
27-03
20.44
28.84
29.19
27-4 1
26.03
32-36
26.43
22-20
20.6
1
6.47
8.98
3.19
3-88
5.00
6.65
6.27
6-97
4-90
4.2
1
7-88
13.92
12.47
25.60
15-57
13.16
12.71
18.83
12.9
1
17.85
19-12
14-20
23.74
-,
None detected.
*
16
:
0,17
:
0
and 18
:
0
denote hexadecanoic, heptadecanoic and octadecanoic acids; 16
:
1,17
:
1
and 18
:
1 denote
hexadecenoic, heptadecenoic and octadecenoic acids
;
TBS denotes
1
0-methyloctadecanoic acid (tuberculostearic
acid).
naphthoquinone ring system (Collins
et al.,
1977). Molecular ions at
m/z
720 corresponding to a
C,
H,,02 tetrahydrogenated menaquinone with eight isoprene units were observed (Howarth
et al.,
1986). The mass spectra also contained significant fragments at
m/z
584 representing loss
of a terminal cyclized 2-isoprene moiety. These results are consistent with good representatives
of
Nocnrdia
(Collins
et al.,
1977).
Strains UQM3064, NM167b, NM1 lob, NM41, NM109 and NM118 possessedphosphatidyl-
et hanolamine,
p
hosp hat id ylinosi tol,
p
hosp hatidy linosi to1 mannosides and dip hosp hat i dylgly-
cerol as the polar lipids. These strains are therefore representative
of
phospholipid group
PI1
(Goodfellow
&
Lechevalier, 1986). Strains not listed in the tables or the text were not analysed.
The lipid chemotaxonomy indicates membership of the genus
Nocardia
(Goodfellow
&
Lechevalier, 1986).
New Nocardia sp.
from
activated sludge foams
1553
Table 4.
Number of carbons and double bonds in the mycolic acids from the mass spectra of the
mycolates of
N.
pinensis strains
The main component is denoted by
+ +
+
,
any component greater than
50%
of the main peak by
+
+
and all other significant components by
+.
Carbons*
. . .
58 59 60 61 62 63 64
DBt
...
2345345
4
545 4 5 6456 5 6
*A*A-AA
Strain
UQM3064
+++
+
+++
+++
+
+ +
++
++
UQM3064
+++
+++++++
+
+++
++++
+
NM167b
++++++++++++
+
+++
+++++
+
NMl10b
++++
+
+++
+++
+
+
+
+
++
++
NM41
+++
++
+
+
+
++
+++
+
++
+
NM109
++
++
+
+
+
++
+++
+
++
+
*
Number
of
carbons in the parent mycolic acid.
-f
Number
of
double bonds in the mycolic acid.
Table
5.
API
ZYM
assay results for actinomycetes
API
enzyme number?
Strain*
2 3
4
5
6
7
8 9 10
11
12 13 14 15 16 17 18 19
20
Nocardia pinensis
-++-+-
*--
++---
+--+-
Nocardia amarae
+++++-+--++---++---
Rhodococcus rhodochrous
+ +
f
+
+
f.
-
-
+ +
-
-
-
+
+
-
- -
'Gordona aurantiaca'
+
+
+ +
+
- -
-
-
++---
++-+-
Corynebacterium equi
+
+
+
-
+
+
+
-
-
+
+
-
-
-
+
+
-
-
-
*
The strains of
N. pinensis
used were all those listed in Table
1
;
strains
UQM3063
and
NM 170
were duplicated
and gave identical results. For
N.
amarae,
the strains were
UQM2810, NM26, NM86, NM206, NM296;
for
R. rhodochrous,
UQM2807, UQM2808;
and for
C.
equi,
UQV 1002
and
UQV1092.
-f
Enzymes were:
1,
control;
2,
phosphatase alkaline;
3,
esterase (C4);
4,
esterase lipase
(C8);
5,
lipase
(C14); 6,
leucine arylamidase;
7,
valine arylamidase;
8,
cystine arylamidase;
9,
trypsin;
10,
chymotrypsin;
11,
phosphatase
acid;
12,
phosphoamidase;
13,
a-galactosidase
;
14,
P-galactosidase;
15,
P-glucuronidase,
16,
a-glucosidase;
17,
P-glucosidase
;
18,
N-acetyl-P-glucosaminidase
;
19,
a-mannosidase
;
20,
a-fucosidase.
API
ZYM
The results for the API
ZYM
assays are shown in Table
5.
The
N.
pinensis
strains gave
uniform results for the enzymes and were positive for esterase, esterase lipase, leucine
arylamidase, acid phosphatase, phosphoamidase, a-glucosidase and a-mannosidase. Major
variations in the results between
N. pinensis
and
N. amarae
were in the enzymes P-glucosidase
and a-mannosidase. The
N. amarae
strains gave strong positive results for P-glucosidase but
were negative for a-mannosidase. Although both groups were positive for acid phosphatase and
phosphoamidase, the
N. amarae
strains were consistently strongly positive whilst the
N. pinensis
strains were weakly positive. The results for the reference
Corynebacterium equi
strains agreed
with published results (Mutimer
&
Woolcock, 1982).
Pigment spectra, growth temperature determinations and ability to grow
on
diferent media
The orange pigments extracted from
N. pinensis
absorbed maximally at 470 nm. They were
not flexirubin-type pigments according to the reaction with 20% (w/v)
KOH.
It was concluded
that the pigment of
N. pinensis
was carotenoid in nature because of its absorption spectrum
(Stanier
et al.,
1977).
The minimum growth temperature for
N. pinensis
was
15
"C,
the maximum was 31
"C
with
optimum growth between
18
and
25
"C.
Strains of
N.
pinensis
could not grow on Sabouraud's dextrose agar, malt agar plus
0.5%
glucose, corn meal agar plus
0.5%
glucose, corn meal agar plus 1
%
Tween
80
'Microthrix'
1554
L. L. BLACKALL AND
OTHERS
medium (Slijkhuis, 1983). Scant growth was recorded for
N. pinensis
on medium I of van Veen
et
al.
(1982). These strains grew on oatmeal agar, tyrosine agar, peptone yeast extract iron agar,
blood agar, chocolate agar, inorganic salts starch agar, GSP medium (Trick
&
Lingens, 1984),
surfactant medium (Zajic
et
al.,
1977) and medium M69 (Scheff
et al.,
1984). Good growth was
recorded for
N.
pinensis
on TYG, isosensitest agar, medium 2 of Gledhill
&
Casida (1969),
trypticase soy agar, yeast malt extract agar, PPLO agar and glycerol asparagine agar base plus
1
%
glycerol. Isosensitest agar was an Oxoid product whilst the remaining media listed above
without references were Difco Bacto products.
There was no difference in the amount of growth or the pigment of the cultures on TYG
incubated in the light or dark and strains grew as well in the air as in the
C02
amended
atmosphere. The cultures failed to grow anaerobically, microaerophilically or in the candle jar,
DISCUSSION
N. pinensis
is morphologically, biochemically, and in chemotaxonomic properties, different
from
N.
amarae,
but it is found in predominance in foams on the surface of aeration tanks at
activated sludge plants, a feature which it has in common with
N. amarae.
The microcolonies of
N. pinensis
and
N. amarae
showed differences in mycelial morphology (Figs 2 and
3).
Filaments
of
N.
pinensis
contained vesicles; this is not uncommon in actinomycetes and is thought to be a
characteristic cellular reaction to age or toxicity of the medium constituents (Goodfellow
&
Cross, 1984). TYG, being a complex medium, may contain toxic constituents which could vary
between medium batches thus affecting the growth rate of
N. pinensis.
Media that supported the growth of many nocardiae and those used in the identification of
these organisms were generally unsuitable as either growth media or as identification media for
N. pinensis.
TYG, the first medium found to support the growth of
N. pinensis,
was used as the
basal medium for many of the biochemical tests. Its possible toxic properties may explain some
of the negative results obtained for the biochemical tests.
N. pinensis
grew best on a medium
containing glycerol as carbon source and asparagine as nitrogen source and this feature is in
common with the mycobacteria (Ratledge, 1982); however, the reason for this preference is
unknown. It is postulated that glycerol uptake is passive and not carrier mediated.
Using the conventional identification methods of Gordon (1967),
N. amarae
produces acid
from glucose (aerobic), glycerol, maltose, mannitol, mannose and salicin (Goodfellow
et al.,
1982); however, these are not the results obtained when the methods described in the present
paper are used (Table 2). The basal medium for the acid production from carbohydrates tests
was modified to facilitate the growth of
N. pinensis.
The results obtained using these latter
methods show differences between
N. amarae
and
N. pinensis
(Table
2)
and that the metabolism
of
N.
pinensis
is oxidative. The inclusion of complex ingredients in the test medium (tryptone and
yeast extract) could be contributing to the different results with
N.
amarae.
A
defined basal
medium that supports the growth of
N.
pinensis
is required to determine acid production from
carbohydrates.
Attempts to extract the cellular DNA from
N. pinensis
failed as the actinomycetes could not be
lysed. Classical actinomycete lysis methods (Mordarski
et
al.,
1976; Marshall
et al.,
1981) were
unsuccessful. Modification to these protocols included extension of the time, and manipulation
of the temperatures of exposure, to various key lysis ingredients. The cells of
N.
amarae
were
successfully lysed. Lysis of many different bacteria can be difficult, particularly within the
actinomycetes.
N. pinensis
appears to be especially difficult
;
however, new methods and
enzymes for cell lysis are becoming available all the time and hence these need to be evaluated
with this organism. The extreme resistance to lysis highlights another difference between
N. pinensis
and other nocardiae.
Both
N. amarae
and
N. pinensis
are slightly different to most other nocardiae in that their
mycolic acids have major amounts of mono-unsaturation in the 2-branch. The type strains of
N. carnea
and
N.
vaccinii
share this property (Lechevalier
&
Lechevalier, 1974; Goodfellow
&
Lechevalier, 1986). Furthermore, the mycolic acids of
N. pinensis
are slightly longer (58-64
carbons) than those reported for the genus
Nocardia
(44-60 carbons
;
Goodfellow
&
Lechevalier,
1986).
New Nocardia sp. from activated sludge foams
1555
Recent studies (Howarth
et al.,
1986; Collins
et al.,
1987) have shown that the menaquinones
from strains of
Nocardia
have an unusual structure with cyclization of the two terminal isoprene
units. These menaquinones,
11,
III-tetrahydro-o-(2,6,6-trimethylcyclohex-2-enylmethyl)-mena-
quinone-6, abbreviated to o-cyclo-MK-S(H,), contain eight isoprene units, two hydrogenated
(H4) and the terminal
(0)
pair cyclized. The characteristic mass spectral peak at
m/z
584, found
for the menaquinones from
N. pinensis
and other members of the genus
Nocardia
(Collins
et al.,
1977), is explained by the loss of the w-cyclized pair of isoprene units (Howarth
et al.,
1986).
Although Goodfellow
&
Cross (1 984) and Goodfellow
&
Lechevalier (1986) give differing
descriptions for the term ‘nocardioform’, the description of
N.
pinensis
fits both. This new
organism contains mycolic acids and therefore must belong in one of the following taxa:
Mycobacterium, Nocardia, R hodococcus, Corynebacterium, Caseobacter
or the
‘aurantiaca’
taxon.
A combination of the following chemotaxonomic and morphological features was used to align
the new strains with
Nocardia
rather than the other mycolic acid containing taxa
:
size of mycolic
acids, presence of tuberculostearic acid,
phosphatidylethanolamine,
the very characteristic
menaquinones
(cu-cyclo-MK-S(H,)),
presence of aerial mycelium and relatively slow rate of
mycelial fragmentation. The reasons for its inclusion in
Nocardia
rather than the close relative
Rhodococcus
are as follows.
The mycolic acids of
N. pinensis
are slightly longer than those of ‘true’ nocardiae and the
presence of major amounts of unsaturation in the 2-branch is not ‘characteristic’ for either
Nocardia
or
Rhodococcus.
However, of the nine species of
Nocardia
listed in Bergey’s Manual of
Systematic Bacteriology (Goodfellow
&
Lechevalier, 1986), three have this attribute whilst this
is not reported in
Rhodococcus.
Furthermore, although it is true that susceptibility to lysozyme
(50
pg ml-’), 5-fluorouracil (20 pg ml-l) and mitomycin-C
(5
pg ml-l) are features shared by
some species of
Rhodococcus
but not by some species
of
Nocardia
(Tsukamura, 1981
a, b;
Gordon
&
Barnett, 1977), they are characters of
N. amarae.
Isoniazid resistance (20
pg
ml-l) is a
property shared by some 34 rhodococci and 10 nocardiae (Orlean
et al.,
1978); furthermore, this
is a property of
N. amarae.
None of the
N. pinensis
strains showed isoniazid resistance.
Rifampicin resistance (20 pg ml-l) was reported for
225
of 230 ‘true’ nocardiae (Gordon
&
Barnett, 1977). This feature is not characteristic for either
N. amarae
or
N.
pinensis.
There have been problems in accepting
N. amarae
as a ‘true’
Nocardia
(Goodfellow
et al.,
1982; Goodfellow
&
Cross, 1984; Goodfellow
&
Lechevalier, 1986) and the antimicrobial
sensitivity patterns reported for
N.
amarae
in this paper add to that dilemma as they do not fit
those obtained for ‘true’ nocardiae.
The biochemical, morphological and chemotaxonomic data obtained for the
N.
pinensis
strains do not unequivocally align them with
Nocardia
or
Rhodococcus.
However, because there
are obvious parallels and similarities between our novel strains and
N. amarae
and because there
are more chemotaxonomic and morphological attributes shared by the new strains and
Nocardia
(vide supra)
than are common to them and
Rhodococcus,
we have aligned the strains with the
genus
Nocardia.
Atypical nocardial features are the unusual mycolic acids, the antimicrobial
sensitivity patterns, the inability to be lysed by conventional actinomycete lysis methods and the
inability to be identified by the conventional methods.
The separation between
N. amarae
and
N. pinensis
is easily facilitated by API ZYM profile
comparison, nutritional requirements, menaquinone type, size of mycolic acids and microscopic
and macroscopic appearance
of
the filaments in the sewage plant and on laboratory media.
N. pinensis
can be separated from
N. vaccinii
and
N. carnea
on the basis of nutritional
requirements, length of mycolic acids, lysozyme sensitivity and urease production.
N.
pinensis
cannot be identified using the classical media and methods for biochemical
characterization of
Nocardia
(Gordon
et a/.,
1974, 1978; Lechevalier
&
Lechevalier, 1974),
indicating that it is
a
new taxon. Therefore, on the basis of chemical and biological evidence, the
organism that was isolated by micromanipulation from the foams and mixed liquors of activated
sludge sewage treatment plants in Australia is described as a new species of
Nocardia.
Description of Nocardia pinensis
sp. nov.
The name
Nocardia pinensis
sp. nov. (M.L. n.
Pinus
genus of pine trees; M.L adj.
pinensis
pertaining to pines and specifically pine tree like in microscopic morphology) is proposed. The
1556
L.
L.
BLACKALL AND OTHERS
type strain is UQM3063 deposited in the University of Queensland, Department of
Microbiology Culture Collection.
In an activated sludge plant where the organism may proliferate as an extensive surface scum
or foam, the microscopic appearance of the filaments resembles a pine tree. The filaments are
Gram-positive, non-acid-fast, non-motile, non-sheathed and about 0.5-1
-0
pm in width. In a
Gram stained preparation using acetone as decolourizer, the filaments appear blue as compared
to
N.
amarae,
which is dark blue or black. Cells from the activated sludge plant or from artificial
culture media contain intracellular sudanophilic and polyphosphate inclusions. On TYG solid
medium the strains may take 10-21 d to produce colonies that are about 1-2 mm in diameter,
with the growth rate being variable. The colonies are orange, opaque, macroscopically dry and
friable, microscopically moist and shiny, with a pasty texture, not adherent to the agar, difficult
to emulsify or subculture, circular with an entire edge. When picked from the agar surface, the
whole colony can be removed intact. The majority of the colonies are about 1 mm in diameter;
however, some colonies that are 3-4 mm in diameter occur. Good growth occurs on a defined
medium containing glycerol as a carbon source and asparagine as a nitrogen source. In TYG
liquid medium the organisms grow as macroscopically visible colonies in a slightly turbid liquid.
The strains are positive for catalase, oxidase and urease, and their metabolism is oxidative.
Maximum and minimum growth temperatures are 31 "C and 15 "C, respectively, whilst the
optimum lies between 18 and
25
"C. Strains do not degrade hypoxanthine, xanthine, tyrosine,
casein, gelatin or aesculin, reduce nitrate or hydrolyse the Tweens 20,40,60 or
80
when the basal
medium is TYG. Strains do not grow in the presence of rifampicin
(20
pg ml-l), mitomycin-C
(5
pg
ml-l), 5-fluorouracil(20 pg ml-l) or isoniazid (200 pg ml-l) with TYG as basal medium;
or in the presence of lysozyme
(50
pg
ml-l) using glycerol broth as basal medium.
No
growth
occurs anaerobically, microaerophilically or in a candle jar; and growth is not enhanced in an
atmosphere of
5%
C02.
Based upon chemotaxonomy, the strains contain peptidoglycan type
Aly, cell wall type IV, whole cell sugar pattern type
A,
phospholipid type
PII,
menaquinones
o-cyclo-MK-8(H4), fatty acids comprising straight chain saturated and unsaturated acids and
tuberculostearic acid, and mycolic acids with 58-64 carbons containing substantial amounts of
unsaturated chains in the 2-position. Using the
API
ZYM testing protocol the strains contain
esterase (C4), esterase lipase (C8), leucine arylamidase, phosphatase acid, phosphoamidase,
a-glucosidase and a-mannosidase.
We wish to thank
P.
Kelly and
S.
H.
Addison for mass spectroscopy and Ms
J.
Westcott for operation of the
electron microscope.
REFERENCES
ANONYMOUS (1969). Milwaukee mystery
:
unusual
operating problem develops.
Water and Sewage
Works
116,
213.
BLACKBEARD,
J.
R.,
EKAMA, G. A.
&
MARAIS,
G.
vR.
(1986). A survey
of
filamentous bulking and foaming
in activated sludge plants in South Africa.
Water
Pollution Control
85,
90- 100.
BURDON,
K.
L. (1946). Fatty material in bacteria and
fungi revealed by staining dried, fixed slide prepara-
tions.
Journal
of
Bacteriology
52,
665-678.
CHRISTENSEN, W.
B.
(1946). Urea decomposition as a
means of differentiating
Proteus
and paracolon
cultures from each other and from
Salmonella
and
Shigella
types.
Journal
of
Bacteriology
52,
461-466.
COLLINS, M.
D.,
PIROUZ,
T.,
GOODFELLOW, M.
&
MINNIKIN,
D.
E.
(1977). Distribution of menaquin-
ones in actinomycetes and corynebacteria.
Journal
of
General Microbiology
100,
221-230.
COLLINS, M.
D.,
HOWARTH,
0.
W., GRUND,
E.
&
KROPPENSTEDT,
R.
M. (1987). Isolation and struc-
tural determination of new members of the vitamin
K series
in
Nocardia brasiliensis. FEMS Micro-
biology Letters
41,
35-39.
CRUICKSHANK,
R.,
DUGUID,
J.
P., MARMION,
B.
P.
&
SWAIN,
R.
H.
A. (1975). Staining methods. In
Medical Microbiology,
vol. 11, pp. 31-57. New York:
Churchill Livingstone.
DHALIWAL, B.
S.
(1979).
Nocardia amarae
and acti-
vated sludge foaming.
Journal Water Pollution
Control Federation
51,
344-350.
DOBSON, G., MINNIKIN,
D. E.,
MINNIKIN,
S.
M.,
PARLETT,
J.
H.
&GOODFELLOW, M. (1985). Systema-
tic analysis of complex mycobacterial lipids. In
Chemical Methods in Bacterial Systematics,
pp. 237-
265. Edited by M. Goodfellow
&
D. E. Minnikin.
London
:
Academic Press.
FERGUSON, E.
H.
(1980). Actinomycetes
of
sewage
treatment plants. Paper presented to the Queensland
Branch of the Australian Water and Wastewater
Association, 7 pp.
GLEDHILL, W.
E.
&
CASIDA, L. E. (1969). Predominant
catalase-negative soil bacteria. 111.
Agromyces,
gen.
n., microorganisms intermediary to
Actinomyces
and
Nocardia. Applied Microbiology
18,
340-349.
GOODFELLOW, M.
&
CROSS,
T.
(1984). Classification.
In
The Biology
of
the Actinomycetes,
pp. 7-164.
New Nocardia
sp.
from
activated sludge foams
1557
Edited by M. Goodfellow, M. Mordarski
&
S.
T.
Williams. London
:
Academic Press.
GOODFELLOW, M.
&
LECHEVALIER, M. P. (1986). The
gems
Nocardia
Trevisan 1889, 9AL. In
Bergey’s
Manual of Systematic Bacteriology
vol. 2, pp. 1459-
1471. Edited by P. H. A. Sneath. Baltimore:
Williams
&
Wilkins.
GOODFELLOW, M., MINNIKIN,
D.
E.,
TODD, C.,
ALDERSON, G., MINNIKIN,
S.
M.
&
COLLINS,
M.
D.
(1982). Numerical and chemical classification of
Nocardia amarae. Journal of General Microbiology
GORDON, R. E. (1967). The taxonomy of bacteria. In
The Ecology of Soil Bacteria,
pp. 293-321. Edited by
T. Gray, R. G. Parkinson
&
B. Parkinson. Liver-
pool
:
Liverpool University Press.
GORDON, R.
E.
&
BARNETT, D. A. (1977). Resistance to
rifampin and lysozyme of strains of some species of
Mycobacterium
and
Nocardia
as a taxonomic tool.
International Journal of Systematic Bacteriology
27,
GORDON, R. E., BARNETT, D. A., HANDERHAN,
J.
E.
&
PANG, C. H.-N. (1974).
Nocardia coeliaca, Nocardia
autotrophica
and the nocardin strain.
International
Journal of Systematic Bacteriology
24,
54-63.
GORDON, R. E., MISHRA,
S.
K.
&
BARNETT, D. A.
(1978). Some bits and pieces of the genus
Nocardia:
N. carnea,
N.
uaccinii, N. transvalensis, N. orientalis
and
N.
aerocolonigenes. Journal of General Micro-
biology
109,
69-78.
HARPER,
J. J.
&
DAVIS, G. H. G. (1979). Two-
dimensional thin-layer chromatography for amino
acid analysis of bacterial cell walls.
International
Journal
of
Systematic Bacteriology
29,
56-58.
HART, M. A. (1985). Scum formation in a nutrient
removing activated sludge plant.
Water South Africa
HARTLEY, K.
J.
(1982).
Nocardia
in activated sludge plants.
Paper presented to 1982 Queensland Local Authority
Engineers Conference Proceedings, pp. 109-1 32.
HOWARTH,
0.
W., GRUND, E., KROPPENSTEDT, R. M.
&
COLLINS,
M.
D.
(1986). Structural determination
of a new naturally occurring cyclic vitamin
K.
Biochemical and Biophysical Research Communica-
tions
140,
916-923.
LECHEVALIER,
H.
A. (1975). Actinomycetes of sewage
treatment plants.
US
Dept. of Commerce National
Technical Information Service Report no. PB
245 9 14.
LECHEVALIER, M. P.
&
LECHEVALIER, H.
A.
(1974).
Nocardia amarae
sp. nov., an actinomycete common
in foaming activated sludge.
International Journal of
Systematic Bacteriology
24,
278-288.
LEMMER, H.
&
KROPPENSTEDT, R. M. (1984). Chemo-
taxonomy and physiology of some actinomycetes
isolated from scumming activated sludge.
Systematic
and Applied Microbiology
5,
124-1 35.
MARSHALL, R. B., WILTON, B. E.
&
ROBINSON, A.
J.
(1
981). Identification of leptospira serovars by
restriction endonuclease analysis.
Journal
of
Medical
Microbiology
14,
163-1 66.
MINNIKIN, D.
E.,
ALSHAMAONY, L.
&
GOODFELLOW,
M.
(1975). Differentiation of
Mycobacterium, Nocar-
dia
and related taxa by thin-layer chromatographic
analysis of whole-organism methanolysates.
Journal
128,
1283-1297.
1
76-
1
78.
11,
171-178.
MINNIKIN, D.
E.,
HUTCHINSON, L. G., CALDICOTT,
A. B.
&
GOODFELLOW, M. (1980). Thin-layer
chromatography of methanolysates of mycolic acid-
containing bacteria.
Journal
of
Chromatography
188,
MINNIKIN, D.
E.,
MINNIKIN,
S.
M.
&
GOODFELLOW,
M. (1982). The oxygenated mycolic acids of
Myco-
bacterium fortuitum, M. farcinogenes
and
M. senega-
lense. Biochimica et biophysica acta
712,
6 16-620.
MINNIKIN, D. E., O’DONNELL, A. G., GOODFELLOW,
M.,
ALDERSON, G., ATHALYE, M., SCHAAL, A.
&
PARLETT,
J.
H. (1984). An integrated procedure for
the extraction of bacterial isoprenoid quinones and
polar lipids.
Journal of Microbiological Methods
2,
MORDARSKI, M., SZYBA,
K.,
PULVERER, G.
&
GOOD-
FELLOW, M. (1976). Deoxyribonucleic acid reasso-
ciation in the classification of the
‘rhodochrous’
complex and allied taxa.
Journal of General Micro-
biology
94,
235-245.
MUTIMER,
M.
D.
&
WOOLCOCK,
J.
B. (1982). API
ZYM
for identification of
Corynebacteriurn equi. Zentral-
blatt
fur
Bakteriologie und Hygiene, Abteilung
I,
Originale
C
3,
410-415.
NAHAIE, M. R., GOODFELLOW, M., MINNIKIN, D.
E.
&
HAJEK, V. (1984). Polar lipid and isoprenoid quinone
composition in the classification of
Staphylococcus.
Journal of General Microbiology
130,
2427-2437.
NELSON,
J.
K.
(1979). Start-up and operation of
Denver’s pure oxygen-activated sludge plant.
Jour-
nal Water Pollution Control Federation
51,
907-
917.
ORLEAN, P. A. B., GOODFELLOW, M.
&
MINNIKIN, D.
E.
(1978). Isoniazid susceptibility as a criterion for
the differentiation of mycobacterial species from
other mycolic acid containing taxa.
International
Journal of Systematic Bacteriology
28,
194-1 96.
PIPES, W.
0.
(1978). Actinomycete scum production in
activated sludge processes.
Journal Water Pollution
Control Federation 50,
628-634.
RATLEDGE, C. (1982). Nutrition, growth and metabo-
lism. In
The Biology of the Mycobacteria,
vol. 1, pp.
185-271. Edited by
C.
Ratledge
&
J.
Stanford.
London
:
Academic Press.
REICHENBACH, H., KOHL, W.
&
ACHENBACH, H. (1981).
The flexirubin-type pigments, chemosystematically
useful compounds. In
The Flavobacterium-Cyto-
phaga Group
(GBF Monograph Series no.
5),
pp.
101-108. Edited by H. Reichenbach
&
0.
B. Weeks.
Weinheim
:
Verlag Chemie.
SCHEFF, G., SALCHER,
0.
&
LINGENS,
F.
(1984).
TrichococcusJlocculiformis
gen. nov. sp. nov.
A
new
gram-positive filamentous bacterium isolated from
bulking sludge.
Applied Microbiology and Biotechno-
SIERRA, G. (1957).
A
simple method for the detection
of
lipolytic activity of micro-organisms and some
observations on the influence of the contact between
cells and fatty substrates.
Antonie van Leeuwenhoek
SKERMAN, V. B. D. (1967).
A Guide to the Identification
of Genera
of
Bacteria,
2nd edn. Baltimore
:
Williams
&
Wilkins.
SKERMAN, V. B. D. (1968).
A
new type of micromani-
pulator and microforge.
Journal of General
Micro-
22 1-233.
233-241.
logy
19,
114-1 19.
23,
15-22.
of General Microbiology
88,
200-204.
biology
54,
287-297.
1558
L.
L.
BLACKALL
AND
OTHERS
SLIJKHUIS, H.
(1983).
Microthrix parvicella,
a filamen-
tous bacterium isolated from activated sludge
:
cultivation in a chemically defined medium.
Applied
and Environmental Microbiology
46,
832-839.
SNEATH,
P.
H. A.
(1956).
Cultural and biochemical
characteristics of the genus
Chromobacterium. Jour-
nal
of’
General Microbiology
15,
70-98.
STANECK,
J.
L.
&
ROBERTS, G. D.
(1974).
Simplified
approach to the identification of aerobic actinomy-
cetes by thin-layer chromatography.
Applied Micro-
biology
28,
226-23
1.
STANIER, R.
Y.,
ADELBERG,
E.
A.
&
INGRAHAM,
J.
L.
(1
977).
General Microbiology,
4th edn. London
:
Macmillan.
TRICK,
I.
&
LINGENS,
F.
(1984).
Characterization of
Herpetosiphon
spec.-a gliding filamentous bacte-
rium from bulking sludge.
Applied Microbiology and
Biotechnology
19,
191-198.
TRICKER, T.
D.
A.
&
THORPE, A.
H.
(1979).
The
commissioning and operation of Belper Sewage
Treatment Works.
Water Pollution Control
78,
5
15-
523.
TSUKAMURA, M.
(1981
a).
Differentiation between the
genera
Mycobacterium, Rhodococcus
and
Nocardia
by
susceptibility to 5-fluorouracil.
Journal
of
General
Microbiology
125,
205-208.
TSUKAMURA, M.
(1981
b).
Test for susceptibility to
mitomycin C as aids for differentiating the genus
Rhodococcus
from the genus
Nocardia,
and for
differentiating
Mycobacterium fortuitum
and
Myco-
bacterium chelonei
from other rapidly growing myco-
bacteria.
Microbiology
&
Immunology
25,
1197-1 199.
VAN
VEEN, W. L., KRUL,
J.
M.
&
BULDER,
C.
J.
E.
A.
(1 982).
Some growth parameters of
Haliscomeno-
bacter hydrossis
(syn.
Streptothrix hyalina)
-
a
bacterium occurring in bulking activated sludge.
Water Research
16,
531-534.
ZAJIC,
J.
E.,
GUIGNARD,
H.
&
GERSON,
D.
F.
(1977).
Emulsifying surface active agents from
Corynebac-
terium hydrocarboclasters. Biotechnology and Bioen-
gineering
19,
1285-1301.
