NZ239297A - Gram negative alkaliphilic bacterial cultures and their use in producing alkali tolerant enzymes - Google Patents
Gram negative alkaliphilic bacterial cultures and their use in producing alkali tolerant enzymesInfo
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- NZ239297A NZ239297A NZ23929791A NZ23929791A NZ239297A NZ 239297 A NZ239297 A NZ 239297A NZ 23929791 A NZ23929791 A NZ 23929791A NZ 23929791 A NZ23929791 A NZ 23929791A NZ 239297 A NZ239297 A NZ 239297A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Description
Henry Hughes Ltd
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N.Z. No.
NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION
GRAM-NEGATIVE ALKALIPHILIC MICROORGANISMS
We, GIST-BROCADES N.V., a Dutch Body Corporation of The Netherlands, of Wateringseweg 1, P O Box 1, 2600 MA Delft, The Netherlands, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: -
- I - (Followed by 1A)
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Gram-Negative Alkaliphilic Microorganisms
The present invention is in the field of microbiology and more particularly in the field of alkaliphilic microorganisms.
Alkaliphiles are defined as organisms which exhibit optimum growth in an alkaline pH environment, particularly in excess of plH 8, and generally in the range between pH 9 and 10. Alkaliphiles may also be found living in environments 15 having a pH as high as 12. Obligate alkaliphiles are incapable of growth at neutral pH.
Alkaliphiles may be found in such everyday environments as garden soil, presumably due to transient alkaline conditions caused by biological activity such as 20 ammonification, sulphate reduction or photosynthesis. A much richer source of a greater variety of alkaliphilic organisms may be found in naturally occurring, stable alkaline environments such as soda lakes.
organisms in general is provided in Grant, W.D., Mwatha, W.E. and Jones, B.E. ((1990) FEMS Microbiology Reviews, 75, 255-270) , the text of which is hereby incorporated by reference. Lists of alkaline soda lakes may be found in the publications of Grant, W.D. and Tindall, B.J. in Microbes in Extreme 30 Environments. (eds. R.A. Herbert and G.A. Codd); Academic Press, London, (1986), pp. 22-54; and Tindall, B.J. in Halophilic Bacteria, Volume 1, (ed. F. Rodriguez-Valera) ; CRC Press Inc., Boca Raton, FL, (1988), pp. 31-70, both texts are also hereby incorporated by reference.
Alkaliphiles, the majority of which are Bacillus species, have been isolated from non-saline environments and are discussed by Horikoshi, K. and Akiba, T. in Alkalophilic
Background of the Invention
A more detailed study of soda lakes and alkaliphilic
23
Microorganisms (Springer-Verlag, Berlin, Heidelberg, N.Y., (1982)). However, alkaliphilic organisms from saline and alkaline environments such as lakes are not discussed therein. Strictly anaerobic bacteria from alkaline, 5 hypersaline, environments have been recently described by Shiba, H. in Suoerbugs (eds. K. Horikoshi and W.D. Grant) ; Japan Scientific Societies Press, Tokyo and Springer-Verlag, Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and by Nakatsugawa. N., ibid, pp. 212-220.
Soda lakes, which may be found in various locations around the world, are caused by a combination of geological, geographical and climatic conditions. They are characterized by the presence of' large' amounts of sodium carbonate (or complexes thereof) formed by evaporative concentration, as 15 well as by the corresponding lack of Ca2+ and Mgz+ which would remove carbonate ions as insoluble salts. Other salts such as NaCl may also concentrate resulting in environments which are both alkaline and saline.
Despite this apparently harsh environment, soda lakes 20 are nevertheless home to a large population of prokaryotes, a few types of which may dominate as permanent or seasonal blooms. The organisms range from alkaliphilic cyanobacteria to haloalkaliphilic archaeobacteria. Moreover, it is not unusual to find common types of alkaliphilic organisms 25 inhabiting soda lakes in various widely dispersed locations throughout the world such as in the East African Rift Valley, in the western U.S., Tibet, China and Hungary. For example, natronobacteria have been isolated and identified in soda lakes located in China (Wang, D. and Tang, Q., 30 "Natronobacterium from Soda Lakes of China" in Recent Advances in Microbial Ecology (Proceedings of the 5th International Symposium on Microbial Ecology, eds. T. Hattori et al.); Japan Scientific Societies Press, Tokyo, (1989), pp. 68-72) and in the western U.S. (Morth, S. and Tindall, B.J. (1985) System. Appl. Microbiol., 6, 247-250). Natronobacteria nave also been found in soda lakes located in Tibet (W.D.
nV
23 92 9 7
Grant, unpublished observations) and India (Upasani, V. and 0 Desai, S. (1990) Arch. Microbiol., 154. pp. 589-593).
Alkaliphiles have already made an impact in the application of biotechnology for the manufacture of consumer 5 products. Alkali-tolerant enzymes produced by alkaliphilic microorganisms have already found use in industrial processes and have considerable economic potential. For example, these enzymes are currently used in detergent compositions and in leather tanning, and are foreseen to find applications in the 10 food, waste treatment and textile industries. Additionally, alkaliphiles and their enzymes are potentially useful for biotransformations, especially in the synthesis of- pure enantiomers.
Summary of the Invention
The present invention provides pure cultures of novel aerobic, Gram-negative alkaliphilic bacteria. These bacteria have been isolated from samples of soil, water, sediment and a number of other sources, all of which were obtained from in 20 and around alkaline soda lakes. These alkaliphiles have been analyzed according to the principles of numerical taxonomy with respect to each other and also to a variety of known bacteria in order to confirm their novelty. In addition, these bacterial taxa are further circumscribed by an analysis 25 of various chemotaxonomic characteristics.
The present invention also provides data as to the composition of the environments from which the samples containing the microorganisms were obtained, as well as the media required for their efficient isolation and culturing 30 such that one of ordinary skill may easily locate such an environment and be able to isolate the organisms of the present invention by following the procedures described herein.
It is also an object of the present invention to provide 35 microorganisms which produce useful alkali-tolerant enzymes, r as well as methods for obtaining substantially pure
V i
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^preparations of these enzymes. These enzymes are capable of
27 AUGI99I
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?79297
performing their functions at high pH which makes them uniquely suited for applications requiring such extreme conditions. For example, alkali-tolerant enzymes may be employed in detergent compositions, in leather tanning and in 5 the food, waste treatment and textile industries, as well as for biotransformations such as the production of pure enantiomers.
The genes encoding these alkali-tolerant enzymes may be isolated, cloned and brought to expression in compatible 10 expression hosts to provide a source of larger volumes of enzyme products which may be, if desired, more easily purified and used in various industrial* applications, should the wild-type ■ strain fail to produce sufficient amounts of the desired enzyme, or does not ferment well.
Brief Description of the Figures
1. Simplified dendrogram showing clusters (phenons) obtained with the SG coefficient and Unweighted Average Linkage procedure.
2. Simplified dendrogram showing clusters (phenons) obtained with the Sj coefficient and Unweighted Average Linkage procedure.
3. Simplified dendrogram obtained with the Sc coefficient and Unweighted Average Linkage procedure using the derived minimum discriminatory tests.
Detailed Description of the Invention Sampling
Several hundreds of strains of bacteria have been isolated from samples of soil, water, sediment and a number of other sources in and around alkaline lakes. These samples were obtained as part of an investigation over a period of three years. The isolated bacteria are non-phototrophic ^ eubacteria. Up until now, such bacteria have not been well cha^racteri z ed.
27 AUG 199)
Figure 20 Figure Figure
The samples were collected in sterile plastic bags. Sampling was conducted at lakes Elmenteita, Nakuru, Bogoria, Crater (Sonachi), Little Naivasha (Oloidien), Magadi, and Little Magadi (Nasikie Engida), all of which are located in 5 Kenya, East Africa. Alkaline soda lakes having similar environments may also be found in Tibet, China, Hungary and the western U.S.. At each sampling site, physical parameters such as pH, conductivity and temperature were measured as well as the physical appearance of the site and the sample. 10 Some of the samples were treated locally within 36 hours of collection of the sample but the majority were examined off-site, several weeks after collection.
Table 1 lists various strains which have been isolated. The strains are listed according to the location from which 15 the sample was taken, the physical appearance of the sample itself and a reference to Table 2 which provides the chemical analysis of the lake water samples.
Table 3 provides a list of the isolated strains arranged according to the results of the numerical taxonomic analysis. 20 Furthermore, Table 3 provides physical properties of the sample, in particular the temperature, conductivity and alkaline pH, as well as the numerous isolation media required for obtaining pure cultures of the new bacteria. These media are letter coded with reference to Appendix A.
Tables 1, 2 and 3 provide data from which the environment of the sampling locations may be characterized. The chemical and physical analysis of the samples confirm the presence of alkaline pH, as well as the presence of unusually high levels of
Na2C03,
coupled with low levels of Ca2+ and
Mg'
.2+
No chemical analysis is available for mud samples. Furthermore, no chemical analysis is available for a few samples (see Table 1) . However, other samples taken at the same location have been analyzed and are described in Tables 1-3. It is known that the basic environments of soda lakes are stable with respect to their pH and ionic composition, oreover, the microbial populations found at these sites
27 AUG1991
remain largely stable. Thus, it is to be expected that despite the lack of a chemical analysis of certain samples, the environment from which the bacteria were obtained may nonetheless be determined from the data presented in Tables 1-3.
The fresh soda-lake water samples were plated out on an alkaline nutrient medium (Medium A) soon after collection. Microscopic inspection showed an unexpectedly high diversity of bacterial types. Considering the extremely alkaline nature of the environment, viable counts showed unexpectedly high numbers of organotrophic bacteria,, in the range of 105 - 106 colony forming units per ml. The samples were stored either cooled or at ambient temperatures. After a few weeks' storage, the total numbers of bacteria in the sample rose, whereas the diversity of types decreased.
Table 1
Alkaliphilic Strains Arranged According to Their Place of Origin
ANALYSIS
STRAINS
SAMPLE LOCATION
SAMPLE APPEARANCE
(Table 2)
1E.1, 4E.1, 35E.2, 37E.2,
2E.1, 5E. 1, 36E. 38E.
2, 2
Lake Elmenteita (east bay)
Mud from dried up lake bed
N.
R.
wE.5, WE12
WE11,
Lake Elmenteita (east bay)
Sediment and water, littoral zone.
N.
T.
39E.3, 41E.3, 44E.3, 56E.4
40E. 42E. 53E.
3, 3 <
4,
Lake Elmenteita ' (east bay)
. Mud and water, littoral zone. Spirulina scum.
1
45E.3, 57E.4
47E.
3,
Lake Elmenteita swamp, southeast arm
Brown water and sediment
2
48E.3,
58E.
4
tt
Grey mud
2
59E.4
Lake Elmenteita, (north-west bay)
Water and sandy sediment, littoral zone
3
16N.1, 17N. 18N.1, 19N. 2ON.1, 26N. 28N.1, WN1, WN2, wNkl, wNk2.
1/ 1, 1,
Lake Nakuru, north beach between Hippo Point and Njoro Point.
Mud and water, littoral zone.
N.
T.
49N.3, 6IN. 4
50N.
3,
it
Water column, littoral zone.
4
51N.3,
52N.
3
ii
Lake sediment,
4
littoral zone.
9 A
J* * »;
1 (\ 9 U
'*S
jt T- ') ^ «• >
-~y
Table 1
Alkaliphilic Strains (continued)
STRAINS
SAMPLE LOCATION
SAMPLE APPEARANCE
ANALYSIS (Table 2)
63N.4
Lake Nakuru; water hole, SW salt flats.
Mud and water
N.T.
6B.1, 7B.1, 8B.1, 9B.1, 10B.1, 24B.1, 25B.1, wBl, WB2, WB4, WB5 wBn4
Lake Bogoria, northern mud flats.
Mud and water, littoral zone.
N.T.
64B.4
Dried crust of soda mud.
N.R.
65B.4
Mud at water line wBs4
Lake Bogoria, south-west shore.
Mud and water, littoral zone.
N.T.
lie.1, 12C.1, Crater Lake, 29C.1, North point
73aC.4, 73bC.4, "
74C.4
Mud and water, littoral zone.
N.T.
75C.4
Soda-mud, shore line.
N.R.
77LN.4, 78LN.4
Little Lake Naivasha, south shore.
Water column and sediment
7
21M.1, 22M.1,
Lake Magadi,
Mud and water
N.T.
27M.1
causeway upper
western arm.
92LM.4, 94LM.4
Little Lake Magadi, northwest springs.
Spring water and sediment
8
1.
- 9 -Table 2
Chemical Analysis of Kenyan Soda Lake Waters
ANALYSIS
Na+
K*
Ca2+
Mg2+
Si02
i ro o A
CI"
so*-
co32"
TON*
T A
(mM)
(mM)
(mM)
(mM)
(mM)
(mM)
(mM)
(mM)
(mM)
(mM)
1
196
3.58
0.07
b.l.d.
2.91
0.03
65.1
2.0
68.0
0.8
119
2
140
3.32
0.48
0.13
1.85
0.02
46.8
1.7
32.0
1.2
86
3
167
3.32
0.06
b.l.d.
3.10
0. 03
51.8
1.7
64.0
2.2
103
4
326
.63
0.15
b.l.d.
3.25
0.15
57.5
0.5
198.3
1.9
259
735
.50
0.21
0.01
2.23
0.09
100.9
1.0
476.7
0.9
612
6
140
8.95
0. 06
0.01
2.13
0.04
12.4
0.8
90.0
1.1
133
7
8.7
1.79
0.28
0.65
1.02
0. 003
4.8
0.5
<10.0
<0. 07
18
8
483
4.35
0.03
0.03
0.64
0.08
157.8
1.7
166.0
1.2
105
b.l.d. = below the limits of detection * TON = Total Organic Nitrogen
§ TA = Total Alkalinity in milliequivalents/liter
a
CO
-J
Table 3
Origin of the Strains Arranged bv Cluster
SAMPLE
Temp. Conductivity ISOLATION
USTER
STRAIN
LOCATION
pH
"C
mS/cm
MEDIUM
1
1E.1CT
Elmenteita
9.5
n.t.
A
1
2E.1
Elmenteita
9.5
n.t.
A
1
WB2
Bogoria n. t.
n.t.
n.t.
A
1
WB5
Bogoria n. t.
n.t.
n.t.
A
1
wBs4
Bogoria
.5
n.t.
19
A
1
10B.1
Bogoria
.5
36
45
• A
1
2 ON. 1
Nakuru
.5
36
-40
A
1
27M.1
Magadi
11. 0
36
100
A.
1
Comamonas terrigena (NCIMB 8193)
-
1
wNk2
Nakuru
.5
n.t.
19
A
1
Pseudomonas putida (NCIMB 9494)
-
2
39E.3
Elmenteita
-10.5
23
13.9
M
2
4 IE. 3
Elmenteita
-10.5
23
11.3
N
2
45E. 3CT
Elmenteita
27
11.3
P
2
47E.3
Elmenteita
27
11.3
0
2
IN. 3
Nakuru
-10.5
29
40.1
P
2
52N.3
Nakuru
-10.5
29
40.1
P
2
42E.3
Elmenteita
-10.5
23
13.9
N
2
5ON. 3
Nakuru
-10.5
29
40.1
N
2
Pseudomonas stutzeri1 (NCIMB 11358)
-
-
wN2
Nakuru n. t.
n.t.
n.t.
A
-
Pseudomonas beijerinckiiT
(NCIMB
9041)
—
-
4E.1
Elmenteita
9.5
n.t.
A
-
5E.1
Elmenteita
9.5
n.t.
A
3
6B.1
Bogoria
.5
36
45
A
3
7B.1
Bogoria
.5
36
45
A
3
8B.1
Bogoria
.5
36
45
A
3
38E.2
Elmenteita n.t.
n.t.
n.t.
B
3
56E.4
Elmenteita
-10.5
23
13.9
C
3
25B.1
Bogoria
.5
36
45
A
3
26N.1
Nakuru
.5
36
-45
A
3
11C.1
Crater
9.0
A
3
wBl
Bogoria n.t.
n.t.
n.t.
A
3
12C.1
Crater
9.0
A
3
28N. 1CT
Nakuru
.5
36
-40
A
3
6 IN. 4
Nakuru
-10.5
29
40.1
E
3
36E.3
Elmenteita n.t.
n.t.
n.t.
K
3
40E.3
Elmenteita
-10.5
23
13.9
M
3
65B.4
Bogoria n.t.
n.t.
41.9
C
3
94LM.4
Little Magadi 9-9.5
81
.0
L
3
19N.1
Nakuru
.5
36
-40
A
3
24B.1
Bogoria
.5
36
45
A
3
21M.1
Magadi
11.0
36
100
A
3
29C.1
Crater
9.0
A
3
35E.2
Elmenteita n.t.
n.t.
n.t.
I
37E.2
Elmenteita n.t.
n.t.
n.t.
J
48E.3
Elmenteita
.0
27
11.3
P
\\ 27 AUG 1991*
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Table 3 (continued)
Origin of the Strains Arranged bv Cluster* (continued^
SAMPLE
Temp. Conductivity ISOLATION
rSTER
STRAIN
LOCATION
PH
•c mS/cm
MEDIUM
3
78LN.4
Little
Naivasha
8.5-9
1.2
G
3
73aC.4
Crater n.t.
n.t.
.2
D
3
75C.4
Crater n.t.
n.t.
n.t.
H
3
73bC.4
Crater n.t.
n.t.
.2
D
3
74C.4
Crater n.t.
n.t.
.2
H
3
77LN.4
Little
Naivasha
8.5-9
1.2
F
3
wNl
. Nakuru n.t.
n.t.
n.t.
A
3
49N.3
Nakuru
-10.5
29
40.1 '
Q
3
44E.3
Elmenteita
.0
27
13.9
0
3
58E.4
Elmenteita
.0
27
11.3
G
3
57E.4
Elmenteita
.0
. 27
11.3
C
4
WE5
Elmenteita n.t.
n.t.
n.t.
A
4
WB4CT
Bogoria n.t.
n.t.
n.t.
A
4
wNkl
Nakuru
.5
n.t.
19
A
4
wEll
Elmenteita
.4
n.t.
13
A
4
WE12
Elmenteita
.4
n.t.
13
A
9B.1
Bogoria
.5
36
45
A
16N.1
Nakuru
.5
36
-40
A
17N.1CT
Nakuru
.5
36
-40
A
22M.1
Magadi
11.0
36
100
A
6
18N.1
Nakuru
.5
36
-40
A
6
59E.4
Elmenteita
.0
31-33
12.7
G
6
64B. 4CT
Bogoria n.t.
n.t.
n.t.
E
6
63N.4
Nakuru
9.0
n.t.
n.t.
C
6
53E.4
Elmenteita
-10.5
23
13.9
G
-
92LM.4
Little Magadi 9-9.5
81
.0
L
-
wBn5
Bogoria
.5
n.t.
19
A
Clusters of microorganisms are obtained by analysis according to the principles of numerical taxonomy using the Sg/UPGMA method (see discussion below and Figure 1).
n.t. = not tested
The letter codes given for the Isolation Media refer to Appendix A.
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\ 2 7 AUG 199!
Treatment of the Samples: Enrichment and Isolation of
Alkaliphilic Bacteria A wide diversity of enrichment and isolation methods were applied. Some of the methods were specifically designed 5 for the enrichment and isolation of alkaliphilic bacteria which exhibit specific types of enzyme activity at an alkaline pH. Other techniques of a more general nature were applied for the isolation of diverse sorts of alkaliphilic bacteria. In some cases, the specific conditions prevailing 10 in the lakes (Table 2) were taken into account when experiments were performed for the isolation of bacteria.
The different nutrient media employed for the isolation of the new alkaliphilic bacteria are designated Medium A -Medium Q. The composition of the various media employed is 15 shown in Appendix A.
For the isolation of non-specific alkaliphilic organotrophic bacteria, soda-lake water samples or dilutions thereof were streaked out on an alkaline nutrient agar, pH 10 - pH 10.5 (Medium A). Samples of a more solid consistency, 20 mud, sediment, etc. were first suspended in an alkaline nutrient broth (Medium A) before spreading on an alkaline nutrient agar (Medium A) . The bacteria were cultivated in a heated incubator, preferably at 37 "C. In some cases, the samples were suspended in an alkaline nutrient broth 25 (Medium A) and the bacteria cultivated by shaking, preferably at 37 °C for 2-3 days before spreading the broth onto an alkaline nutrient agar (Medium A) for the isolation of bacterial colonies.
For the isolation of alkaliphilic bacteria exhibiting 30 specific types of enzyme activity, samples were spread onto alkaline nutrient agar containing specific substrates such as lactalbumin or casein or olive oil. In some instances, the bacteria in the sample may be enriched for 1 day or several weeks in a non-specific alkaline nutrient broth such as Medium A before spreading the broth onto an alkaline nutrient jigcfer specific for the detection of bacteria exhibiting enzyme tivity such as lipolytic or proteolytic activity.
z o •? Q 1
. 13 . «. 0 '
Taxonomic Analysis Seventy strains of bacteria isolated from in and around alkaline lakes were assigned to the type of bacteria known as Gram-negative bacteria on the basis of (1) the Dussault 5 modification of the Gram's staining reaction (Dussault, H.P., (1955), J. Bacteriol., 70, 484-485); (2) the KOH sensitivity test (Gregersen, T., (1978), Eur. J. Appl. Microbiol, and Biotech. 5, 123-127; Halebian, S. et al. , (1981), J. Clin. Microbiol., 13., 444-448); (3) the aminopeptidase reaction 10 (Cerny, G., (1976), Eur. J. Appl. Microbiol., 3, 223-225; ibid, (1978), 5," 113-122); and in many cases, confirmation also on the basis of (4) a quinone. analysis (Collins, M.D. & Jones, D., (1981), Microbiol. Rev., 45, 316-354) using the method described by Collins, M.D. in Chemical Methods in 15 Bacterial Svstematics (eds. M. Goodfellow & D. Minnikin) pp. 267-288, Academic Press, London, 1985.
The seventy strains were tested for 104 characters. The results were analyzed using the principles of numerical taxonomy (Sneath, P.H.A. and Sokal, R.R., in Numerical 20 Taxonomy. W.H. Freeman & Co., San Francisco, 1973). The characters tested and how they were tested are compiled in Appendix B. In addition, Appendix C records how each character was coded for taxonomic analysis.
Since there are no well-documented strict or obligate 25 non-phototrophic, alkaliphilic Gram-negative eubacteria known to the inventors, a diverse collection of 20 known Gram-negative bacteria were subjected as controls to the same analysis, using modified pH conditions. These 20 known reference bacteria are recorded in Table 4 from which it will 30 be seen that in most cases the "Type Strain" of the known species has been used.
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Table 4
Gram-Negative Non-Alkaliphilic Reference Strains
(C.t. (P.p. (P.s. (A. t. (V.c. (P.a. (P.v. (M.v. (E.t. (A.h. (A. s. (F.a. (E.c. (E.a. (K.a. (H. a. (C.f. (S.m. (P.b. (H.e.
Comamonas terrigenaT NCIMB 8193
Pseudomonas putida T NCIMB 9494
Pseudomonas stutzeriT NCIMB 11358
"Alcaligenes tolerans" Leicester University strain
Vibrio costicolaT NCIMB 701
Providencia alcalifaciens7 NCTC 10286
Proteus vulgarisMT ATCC 13315
Moellerella wisconsensisT NCTC 12132
Edwardsiella tarda1 NCTC 10396
Aeromonas hydrophilaT NCTC 8049
Aeromonas sp S5 Leicester University strain
Flavobacterium aquatileT NCIMB 8694
Escherichia coliT NCTC 9001
Enterobacter aerogenes1 NCTC 10006
Klebsiella pneumonia ATCC 15380 ("K.aerogenes")
Hafnia alveiT ATCC 13337
Citrobacter freundiiT NCTC 9750
Serratia marcescensT NCTC 10211
Pseudomonas beijerinckiiT NCIMB 9041
Halomonas elongataT ATCC 33173
* abbreviation used in Figure 1 and Figure 2 Tdenotes "Type Strain"
NT
denotes "Neotype Strain"
Analysis of Test Data The Estimation of Taxonomic Resemblance
The phenetic data, consisting of 104 unit characters were scored as indicated in Appendix C, and set out in the 5 form of an "n x t" matrix, whose t columns represent the t bacterial strains to be grouped on the basis of resemblances, and whose n rows are the unit characters. Taxonomic resemblance of the bacterial strains was estimated by means of a similarity coefficient (Sneath, P.H.A. and Sokal, R.R., 10 Numerical Taxonomy. supra, pp. 114-187). Although many different coefficients have .been' .used . for biological classification, only a few • have found regular use in bacteriology. We have chosen to apply two association coefficients (Sneath, P.H.A. and Sokal, R.R., ibid, p. 129 et 15 seq.). namely, the Gower and Jaccard coefficients. These have been frequently applied to the analysis of bacteriological data and have a wide acceptance by those skilled in the art since they have been shown to result in robust classifications.
The coded data were analyzed using the TAXPAK program package (Sackin, M.J., "Programmes for classification and identification". In Methods in Microbiology. Volume JL9 (eds. R.R. Colwell and R. Grigorova), pp. 459-494, Academic Press, London, (1987)) run on a DEC VAX computer at the University 25 of Leicester, U.K.
A similarity matrix was constructed for all pairs of strains using the Gower Coefficient (SG) with the option of permitting negative matches (Sneath, P.H.A. and Sokal, R.R., supra, pp. 135-13 6) using the RTBNSIM program in TAXPAK. As 30 the primary instrument of analysis and the one upon which most of the arguments presented herein are based, the Gower Coefficient was chosen over other coefficients for generating similarity matrices because it is applicable to all types of characters or data, namely, two-state, multistate (ordered 35 and qualitative), and quantitative.
Cluster analysis of the similarity matrix was accomplished using the Unweighted Pair Group Method with
Arithmetic Averages (UPGMA) algorithm, also known as the Unweighted Average Linkage procedure, by running the SMATCLST sub-routine in TAXPAK.
The result of the cluster analysis is a dendrogram, a 5 simplified version of which is provided in Figure 1. The dendrogram illustrates the levels of similarity between the bacterial strains. The dendrogram is obtained by using the DENDGR program in TAXPAK.
The phenetic data, omitting multistate characters 10 (characters 1-5, 11 and 12; Appendix C) and thus consisting of 193 unit characters, and scored in binary notation (positive = 1, negative = 0) were re-analyzed using the Jaccard Coefficient (Sj) (Sneath, P.H.A. and Sokal, R.R.-., ibid, p. 131) by running the RTBNSIM program in TAXPAK. A 15 further dendrogram was obtained by using the SMATCLST with UPGMA option and DENDGR sub-routines in TAXPAK. A simplified version of this dendrogram is illustrated in Figure 2. Appendix E gives the percentage positive states of characters in each cluster.
Results of the Cluster Analysis
Sc/UPGMA Method
Figure 1 shows the results of cluster analysis, based on the Gower Coefficient and the UPGMA method, of 70 new, Gram-25 negative, alkaliphilic bacteria isolated from in and around alkaline lakes, together with 20 known Gram-negative bacteria.
Six natural clusters or phenons of alkaliphilic bacteria which include 65 of the 70 alkaliphilic strains are generated 30 at the 73% similarity level. Although the choice of 73% for the level of delineation may seem arbitrary, it is in keeping with current practices in numerical taxonomy (Austin, B. and Priest, F., in Modern Bacterial Taxonomy, p. 37; Van Nostrand Reinhold; Wokingham, U.K., (1986)). Placing the delineation 35 at a lower percentage would combine groups of clearly Unrelated organisms while a higher percentage would produce a
•A multitude of less well-defined clusters. At the 73% level,
o)l i
y e«m
,7^007
o u £. <J *
the individual clusters may represent separate bacterial genera. Furthermore, the significance of clustering at this level is supported by chemotaxonomic data (see below) and the pattern of clusters obtained using the Jaccard Coefficient (Figure 2).
The significance of the clustering at the 73% level is supported by the results of the TESTDEN program. This program tests the significance of all dichotomous pairs of clusters (comprising 4 or more strains) in a UPGMA generated dendrogram with squared Euclidean distances, or their complement, as a measurement. The program assumes that the clusters are hyperspherical. The critical overlap was set at 0.25%. As can be seen from Table 5, the separation of the clusters is highly significant.
Table 5
Significance of the Clusters Generated bv the SC/UPGMA Method Provided bv TESTDEN
CLUSTER separates from CLUSTER
at significance Level
1
2
p = 0.99
1+2
3+4
p = 0.99
1+2+3+4
+6
p < 0.90
1+2+3+4+5+6
controls p = 0.99
6
p = 0.99
A further measure of cluster separation can be estimated from the probability of cluster overlap. This was achieved using the 0VERMAT program in TAXPAK with the critical overlap set out at 2.5%. As can be seen from Table 6, there is a greater than 95% probability of less than 2.5% overlap between the clusters. For many of the cluster combinations the overlap is effectively nil. Only Clusters 3 and 4 have a lower probability of < 2.5% overlap, but these clusters may be clearly distinguished from one another on the basis of chemotaxonomic data (see below).
.TE/vp « o
27 AUG/99/"|
■V
297
Table 6
Percentage Probability that Cluster Overlap is < 2.5' CLUSTER 12 3 4 5 6
1
2
3
4
6
95 99 95 99 >99
95 95 >99 >99
90 >99 99
>99 >99
>99
The controls show that, as expected, the cluster analysis groups the Enterobacteriaceae separately. Additionally, the Aeromonas and Pseudomonas species, included as controls, also group separately. This is entirely consistent with the current taxonomy of these organisms (Bergey's Manual of Systematic Bacteriology. Volume 1, Williams and Wilkins, Baltimore/London, 1984).
Five of the alkaliphilic strains fall outside the major clusters. Two strains, 4E.1 and 5E.1 form a separate but related pair and are obviously associated with the major groups of alkaliphilic bacteria. Strain wN2 is also unclustered but is apparently related to a Pseudomonas species and the major phenons of alkaliphilic bacteria. Strains 92LM.4 and wBn5 do not associate with the major alkaliphilic phenons and probably represent distinct groups of new alkaliphilic bacteria.
Clusters 1 and 2 are the only phenons which show an association with known organisms, i.e. Pseudomonas and Comamonas species. The separation of Pseudomonas putida and Pseudomonas stutzeri into separate taxa is entirely in keeping with the current taxonomic status of these organisms (Palleroni, N.J. et ajL, (1973), Int. J. Systematic Bacteriol., 23., 333-339? Gavini, F. et al, (1989), ibid, 19, 135-144; Bergev's Manual of Systematic Bacteriology, supra).
It was clear from the original dendrogram that Pseudomonas stutzeri is an outlier to Cluster 2 and is not closely related to the other members of the cluster. This is seen when the Euclidean distances of the strains from the entroid of the cluster are computed and used to calculate
7 406/991
C £ » -- r
w
7
the cluster radius (Sneath, P.H.A. and Sokal, R.R., supra, pp. 194 et sea) . The cluster radius is 3.91 (99% confidence level) and the mean distance of the strains from the centroid is 2.84 (standard deviation 0.4 6). Pseudomonas stutzeri at a distance from the centroid of 3.91 is clearly at the very boundary of phenetic hyperspace which defines Cluster 2.
A clear discrimination between Clusters 1 and 2 is possible using the concept of the minimum discriminatory tests (see below).
Each of the alkaliphilic strains in Cluster 2 have been examined by two independent laboratories expert in the identification of bacteria, namely, the German Culture Collection (DSM, Braunschweig, FRG) and the Laboratory for Microbiology at Delft University of Technology, The Netherlands. Neither of these laboratories was able to make a positive identification of the strains, although both agreed there was a resemblance with Pseudomonas either placing them in RNA homology group I (Palleroni, N.J. et al, supra) or more specifically in the Comamonas testosteroni/Pseudomonas alcaliqenes or Pseudomonas pseudoalcaligenes groups (Gavini, F. et al, supra) . However, no Pseudomonas species are known which are able to grow under the same highly alkaline conditions (pH 10) as the new strains described herein. An attempt was made to cultivate Pseudomonas pseudoalcaligenes1 DSM 50188 and Pseudomonas alcaliqenes1 DSM 50342 in an alkaline broth medium (Medium A, Appendix A) , but without these experts together with the here, clearly indicate that these in Clusters 1 and 2 represent new
;/V *
success.
The results of discoveries described alkaliphilic strains species of bacteria.
Clusters 3, 4, 5 and 6 are discrete phenons distinguished from each other on the basis of the minimum discriminatory tests (see below) and chemotaxonomic markers (see below). These phenons show no significant similarity with known groups of bacteria, and thus represent new genera r species.
Whole cell protein patterns generated by PAGE-electrophoresis indicate that a number of strains are likely to be identical. Examples include: 1E.1CT and 2E.1; 6B.1, 7B.1 and 8B.1; 45E.3CT and 47E.3. The dendrogram reveals that 5 these strains are related at an average SG value of 92.3%, indicating a probable test error of 3.8% (Sneath, P.H.A. and Sokal, R.R., supra). Strains 73bc.4 and 74C.4, which appear to be closely related (90% SG), have similar but not identical gel patterns.
Sj/UPGMA Method
The Jaccard coefficient is a useful adjunct to the Gower generated by negative matches or distortions owing to undue 15 weight being put on potentially subjective qualitative data. Consequently, the Jaccard coefficient is useful for confirming the validity of clusters defined initially by the use of the Gower coefficient. The Jaccard coefficient is particularly useful in comparing biochemically unreactive 20 organisms (Austin, B., and Priest, F.G., supra, p. 37).
In the main, all of the clusters generated by the Sq/UPGMA method are recovered in the dendrogram produced by the Sj/UPGMA method (Figure 2) . Although the composition of the clusters is virtually identical in both dendrograms, a 25 few strains have changed position. Non-clustering strains 4E.1 and 5E.1 move into Cluster 1/5, strains 42E.3 and 5ON.3 move from Cluster 2 to Cluster 3/4. The strains wNk2, Pseudomonas stutzeri, Pseudomonas putida. wE5 become non-clustering.
Not surprisingly, the Sj transformation combines (SG)
Clusters 1 and 5. Both of these clusters are characterized as consisting of biochemically fairly unreactive strains. However, Clusters 1 and 5 are clearly distinct. Cluster 1 consists of strains producing cream/beige, circular colonies while the strains of Cluster 5 exclusively produce bright
coefficient as it can be used to detect phenons in the latter oWeilow, irregular colonies.
C\ r) n, n
21
ts.«
Furthermore, the Sj transformation groups most of the strains of Cluster 4 with the strains of Cluster 3. However, it is evident from the chemotaxonomic data (see below), which shows that the strains of Cluster 4 contain Q9 and the 5 strains of Cluster 3 contain mainly Q6, that these clusters should not be combined since they contain distinctly different strains. For these reasons, it is considered that the clustering produced by the SG/UPGMA method is the better representation of the actual taxonomic status of these 10 strains. However, the Sj/UPGMA serves to re-emphasize that with the single exception of a Comamonas species none of the known strains, not even the Pseudomonas control strains, bear significant resemblance to the clusters of the new alkaliphilic bacteria.
Chemotaxonomic Definition of the Clusters Chemotaxonomy is the study of the chemical compositions of organisms in relation to their systematics. The analysis of chromosomal DNA, ribosomal RNA, proteins, cell walls and 20 membranes, for example, can give valuable insights into taxonomic relationships and may be used as a further tool to construct or to verify the taxonomies of microorganisms (Goodfellow, M. and Minnikin, D.E. in Chemical Methods in Bacterial Svstematics. (eds. Goodfellow, M. and Minnikin, 25 D.E.), Academic Press, London and Orlando, FL, (1985), pp. 1-15) . However, it is not always possible to decide a priori which type of chemical information will be most diagnostic for a given classification. The amphipathic polar lipids, the major respiratory quinones, fatty acids located in the 3 0 bacterial membranes and analysis of chromosomal DNA all have taxonomic significance for the classification of various bacteria (Lechevalier, H. and Lechevalier, M.P., in Microbial Lipids. volume 1 (eds. Ratledge, C. and Wilkinson, S.G.) Academic Press, London and San Diego, CA, (1988) , pp. 869-5 902).
2 "z n o 0 ^ i
Polar Lipids
The extraction of polar lipids from bacteria and their analysis by two dimensional thin layer chromatography (2D-TLC) may yield patterns of diagnostic value. Stationary phase 5 cells were extracted in 1:1 (v/v) CHC13:CH30H and examined by 2D-TLC as described by Ross, H.N.H., Grant, W.D. and Harris, J.E., in Chemical Methods in Bacterial Systematics. (eds. Goodfellow, M. and Minnikin, D.E.), Academic Press, London and Orlando, FL. (1985), pp. 289-300. The types of lipids 10 present on the chromatograms were visualized using a variety of differential stains (Ross, H.N.M., et al., supra, p. 291; and Trincone, A., et al., J. Gen. Microbiol., (1990), 136. pp. 2327-2331). The identity of components were confirmed by co-chromatography with known lipids.
The results of this analysis for representative strains of Gram-negative alkaliphiles are set out in Table 7. These show no clear polar lipid pattern which is distinct for any one cluster. All strains contain phosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerol phosphate and 20 phosphatidylethanolamine. In addition, certain strains, particularly in Cluster 3, contain phosphatidylglycerol sulphate (PGS). The distribution of PGS within Cluster 3 coincides broadly with the suspected sub-group structure of the cluster evident from the phenetic and other 25 chemotaxonamic data. PGS is therefore a non-exclusive marker for Cluster 3.
We were surprised to find that a majority of the bacteria contained a glycolipid which on the basis of numerous co-chromatographic analyses appeared common to Gram-3 0 negative bacteria of the present invention. Glycolipids have not previously been demonstrated to be present in alkaliphilic bacteria (Krulwich, T.A., et al, CRC Critical Reviews in Microbiology, (1988), 16, 15-36). Furthermore, as judged by co-chromatography of lipids obtained from several strains, the glycolipid is also found in Gram-positive alkaliphiles isolated from soda lakes. It is possible lerefore, that the chemical structure of the glycolipid may a chemotaxonomic marker for the obligate alkaliphiles.
oil
Table 7
CLUSTER
STRAIN
PG
DPG
PGP
PE
PGS
GL
AL
UPL
1E.1CT
+
+
+
+
2E.1
+
+
+
+
+
1
10B.1
+
+
+
+
3+
20N.1
+
+
+
+
+
27M.1
+
+
+
+
wNk.2
+
+
+
+
+
39E.3
+
+
+
+
+
4IE. 3
+
+
+
+
+
45E.3CT
+
+
+
+
+
2
51N.3
+
■ +
+
+
+
52N.3
+
+
+
+
+
42E.3
+
+■
+
+
+
50N.3
+
+
+
+ .
+
P.s.
+
+
+
-
5E.1
+
+
+
+
+
+
+
6B.1
+
+
+
+
+
7B.1
+
+
+
+
+
8B.1
+
+
+
+
+
25B.1
+
+
+
+
+
26N.1
+
+
+
+
+
12C.1
+
+
+
+
+
+
3
2 8N. 1CT
+
+
+
+
+
36E.2
+
+
+
+
+
+
40E.3
+
+
+
+
+
+
94LM.4
+
+
+
+
+
+
19N.1
+
+
+
+
+
24B.1
+
+
+
+
+
21M.1
+
+
+
+
+
29C.1
+
+
+
+
+
35E.2
+
+
+
+
+
37E.2
+
+
+
+
+
+
48E.3
+
+
+
+
+
+
7 3 aC.4
+
+
+
+
+
+
74C.4
+
+
+
+
+
+
49E.3
+
+
+
+
+
+
44E.3
+
+
+
+
+
+
58E.4
+
+
+
+
+
wE5 + + + + +
wB4CT + + + +
4 WNkl + + + + + +
• ft **-r " -"\ , ■> ■ -'7
i** TJ J
Table 7 (continued)
CLUSTER STRAIN PG DPG PGP PE PGS GL AL UPL
9B. 1 + + + +
16N.1 + + + +
17N. 1CT + + + +
22M.1 + + + +
18N.1 + + + + +
59E.4 + + + +
64B. 4CT + + + +
63N.4 + + + + +
53E.4 + + + 4- +
(PG) phosphatidylglycerol; (DPG) diphosphatidylglycerol ; (PGP) phosphatidylglycerol phosphate; (PE) phosphatidylethanolamine;
(PGS) phosphatidylglycerol sulphate;
(GL) unidentified glycolipid(s) , a-naphthol positive (the number in the column gives the number of positive spots on the TLC plate);
(AL) unidentified amino-lipid (ninhydrin positive) ; (UPL) unidentified phospho-lipid(s).
Isoprenoid Ouinones
The isoprenoid or respiratory quinones are characteristic components of the plasma membrane of aerobic bacteria. There are two types; menaquinones and ubiquinones. The value of isoprenoid quinones as taxonomic criteria lies in the variation in the length of the polyprenyl side-chain and the degree of saturation (Collins, M.D. and Jones, D. (1981), supra).
Freeze dried stationary phase bacterial cells were extracted, using a modified procedure of Collins, M.D. (in Chemical Methods in Bacterial Svstematics. supra, pp. 267-284), in 1:1 (v/v) CHC13:CH30H at 50°C, for 16 hours. The inones were examined by reverse phase thin layer omatography as described by Collins, M.D. (supra).
9
The results of quinone analyses of nearly all the strains of Gram-negative alkaliphiles are illustrated in Table 8. All of the strains tested contained exclusively ubiquinones which confirms their status as Gram-negative bacteria (Collins, M.D. and Jones, D., supra). Table 8 shows quite clearly that the major ubiquinones are Q6 and Q9. It is also evident that the strains containing Q6 are exclusive to Cluster 3 and that this distinguishes Cluster 3 from all the other clusters since they contain strains possessing Q9 as the major ubiquinone.
Table 8
Maior Respiratory Quinones of the Strains
Arranaed per Cluster
CLUSTER 1 CLUSTER
2
CLUSTER
3
STRAIN Q STRAIN
Q
STRAIN
Q
1E.1CT Q9 39E.3
Q9
6B.1
Q6
2E.1 Q9 4IE.3 WB2 Q9 45E.3CT
Q9
7B.1
Q6,
Q9
Q9
8B.1
Q6,
Q9
WB5 Q9 47E.3
Q9,
Q10 38E.2
Q6
WBs4 Q9 52N.3
Q9
56E.4
Q6
10B.1 Q9 42E.3
Q9
25B.1
Q6,
Q9
20N.1 Q9 50N.3
Q9
26N.1
Q6
27M.1 Q9
12C.1 28N. 1CT
Q6
C.t.* [Q8+Q9(t)]
Q6
wNk2 Q9
6 IN. 4
Q6 .
P.p.* [Q9]
36E.2
Q6
40E.3
Q6,
Q9
65B.4
Q6
94LM.4
Q6,
Q9
19N.1
Q6,
Q9
24B.1
Q6,
Q9
21M.1
Q6
29C.1
Q6
35E.2
Q6
37E.2
Q6
48E.3
Q6
78LN.4
Q6
73aC.4
Q6
75C.4
Q6
73bC.4
Q6
74C.4
Q6,
Q9
77LN.4
Q6
49E.3
Q6
58E.4
Q6
57E.4
Q6
I
A ° h * J } '■• /' Vw3 /
i.;' v /
Table 8 (continued)
CLUSTER 4 STRAIN Q
WE5 Q9
WB4CT Q9 WNkl Q9 WE11 Q9,Q10(t) WE12 Q9
CLUSTER 5 STRAIN Q
9B.1 Q9 16N.1 Q9 17N. 1CT Q9, Q10 22M.1 Q9 22M.1 Q9
CLUSTER 6 STRAIN Q
18N.1 Q9
59E.4 Q9
64B. 4CT Q9
63N.4 Q9
53E.4 Q9
NON-CLUSTER STRAIN Q
WN.2 Q9
5E.1 Q8
92LM.4 Q9
wBn5 Q8,Q9(t)
Q = Ubiquinone, the number indicates the number of side-
chain isoprene units.
(t) = trace
* C.t. = Comamonas terriaena1 NCIMB 8193, the quinone result is obtained from J. Tamaoka et al, International Journal of Systematic Bacteriology, 37, 52-59, (1987).
P.p. = Pseudomonas putidaT NCIMB 9494, the quinone result is obtained from M.D. Collins and D. Jones, Microbiological Reviews, 45, 316-354, (1981).
7 rj n <1 c'7
0 & c y '
Fatty Acids
The analysis of fatty acid profiles has had a significant impact on bacterial classification especially in the circumscription of genera and species among Gram-positive 5 bacteria and actinomycetes (Kroppenstedt, R.M., in Chemical Methods in Bacterial Svstematics (eds. M. Goodfellow and D.E. Minnikin), Academic Press; London and Orlando, FL, (1985), pp. 173-199); Lechevalier, H. and Lechevalier, M.P., supra.
Freeze dried stationary phase cells (200-300 mg) were extracted for 16 hours at 75 "C in toluene: methanol :conc. sulphuric acid (2.5 ml:2.5 ml:0.2 ml) and after cooling, the lipids were partitioned into hexane (twice times 1 ml) . Residual acid was removed using NH4HC03. Lipid extracts were 15 concentrated under o2-free N2, dissolved in 300 fil hexane and applied to preparative silica gel plates (Merck F254, Type T). The plates were developed in hexane: diethyl ether 85:15 (v/v) and the fatty acid methyl esters scraped off, extracted with hexane and concentrated under a stream of 02-free N2. 20 The fatty acid methyl esters were dissolved in heptane and analyzed by gas chromatography using a Packard model 439 chromatograph equipped with flame ionization detectors. The samples were divided by a sample splitter and analyzed simultaneously-over two columns, namely, CP-SIL-88 (Chrompack) 25 (length 50 meter1,' internal diameter 0.22 mm) and Ultra-2 (Hewlett/Packard) (length 50 m, internal diameter 0.20 mm). The carrier gas was nitrogen; the injection temperature 120°C; temperature gradient 2.5°C per minute to 240"C and isothermal at 240°C for 3 0 minutes. Fatty acid methyl esters were 30 assigned by reference to known standard mixtures. The identity of some peaks was confirmed by means of gas chromatography-mass spectrometry using a Carlo Erba HRGC 5160 Mega series gas chromatograph equipped with a CP-SIL-88 column (length 50 meter, internal diameter 0.22 mm) with helium as carrier gas 35 and direct injection into the source of a AMD 403 mass spectrometer.
o
/ KMlM
The fatty acid compositions of representative individual Gram-negative bacteria are set out in Table 9. Table 10 shows the unique fatty acid profiles of each of the clusters. Clusters 1, 2, 3 and 4 are fairly typical of the majority of 5 Gram-negative bacteria where the major saturated fatty acid is C16:0 with lesser amounts of C14:0 and C18:0. The major unsaturated fatty acids in these alkaliphilic bacteria are C16:0 and C18:l (11-cis), which is also typical, as is the lack of odd-numbered fatty acids (Wilkinson, S.G., in 10 Microbial Lipids. volume 1 (eds. Ratledge, C. and Wilkinson, S.G.), Academic. Press', London and San Diego, CA, (1988), pp. 299-488). Minor amounts of C17:0 and C19:0 cyclopropane acids are found in some strains of Gram-negative bacteria. The strains of Cluster 3 exhibit fairly simple fatty acid profiles 15 with C16:0 and C18:l contributing 67-88% of the total acids, and C16:l plus C18:0 up to 20% of the remainder. Even so, the fatty acid patterns support the notion that Cluster 3 contains several sub-groups, a conclusion that is also inferred from phenetic (numerical taxonomy) and polar lipid analyses (Table 20 7) .
The strains of Cluster 1 can be distinguished from those of Cluster 2 on the relative abundance of straight chain saturated and unsaturated fatty acids, as well as the percentage amounts of C18:1(11-cis). The alkaliphilic bacteria 25 of Clusters 1 and 2 have more complex fatty acid profiles than those of Cluster 3, with many more minor components. From the numerical taxonomy evidence, the alkaliphilic strains of Clusters 1 and 2 exhibit some resemblance to Pseudomonas species. However, the total lack of any hydroxy-fatty acids 30 which are typical of most Pseudomonas species, further indicating that a close relationship is doubtful.
The strains of Clusters 5 and 6 are remarkable in that besides containing major amounts of C16:0, the other major fatty acids are odd-numbered branched chain acids (40-85%).
lso, these strains lack significant amounts of C18:l or any '^feher unsaturated acids which are present in appreciable affiibunts in the alkaliphilic strains of Clusters 1, 2, 3 and 4,
The presence of large amounts of C15:0 and C17:0 iso and anteiso acids is characteristic of only a very few classes of Gram-negative bacteria, notably species from exotic environments such as Thermus. or poorly defined taxa such as 5 Flavobacterium (Wilkinson, S.G., supra). This result further emphasizes the novelty of the alkaliphilic strains of the present invention. The strains of Clusters 5 and 6 can be distinguished from each other by the proportion of branched-chain fatty acids they contain and more especially by the 10 relative proportions of even- and odd-numbered fatty acids.
Table 9
Fatty Acid Composition* of Gram-Negative Alkaliphiles
40
45
CLUSTER -
1
2
FATTY ACID
IE. 1CT
2E.1
45E. 3CT
50E.3
0
12
0
t t
3.6
4.7
12
1
0.2
0.3
13
0
< 0.1
14
0
0.7
0.7
3.8
3.9
14
0 iso
14
1
2.6
0.9
0
0.3
0.6
0.9
0 iso
0.1
0.2
0 anteiso
< 0.1
0.3
0 cyclo
16
0
29.0
32.0
28.5
34.4
16
0 iso
0.3
0.2
16
1
7.4
9.6
4.1
4.3
17
0
0.5
2.3
0.9
1.1
17
0 iso
0.2
0.6
17
0 anteiso
0. 4a
17
0 cyclo
1.8
17
1
0.3
1.6
17
l br
1.7
18
0
12.0
4.7
17.9
.8
18
0 unknown
0.2
0.4
18
1 9-cis
0.2
t
0.5
0.3
18
1 9-trans
0.5
.0.6
.9
2.9
18
1 11-cis
42.0
44.0 '
23.9
24.1
18
1 unknown
0.2
0.3
18
'2
0.5
1.9
19
0
0.1
19
0 cyclo
1.3
19
1 br
0
0.4
.3
2.7
1
3.6
1.2
22
0
. 3.3
1.5
24
0
0.2
50
+ = % total fatty acids t = trace br = branched
= C17:0 cyclo or C18:0 unknown
*<°i
//<<
^27 AUG 1991
?1Q0
O f
Table 9 (continued)
40
45
CLUSTER -►
3
FATTY ACID
25B.1
28N. 1CT
3 6E.2
24B.1
37E.2
48E.3
0
0.4
12
0
0.5
0.5
0.3
t
12
1
13
0
14
0
4.5
4.4
3.4
2.6
2.9
2.6
14
0 iso
14
1
0.1
0
0.7
0.6
0.9
0.3
0 iso
0 anteiso
0 cyclo
16
0
37.3
24.1
42.0
36.7
33.3
26.0
16
0 iso
16
1
11.6
.0
.0
.8
3.4
8.0
17
0
0.2
1.2
0.3
17
0 iso
17
0 anteiso
17
0 cyclo
1.8
0.3
17
1
0.6
17
1 br
18
0
8.9
0.2
0.9
2.0
0.6
1.1
18
0 unknown
18
1 9-cis
t t
t
0.6
t
18
1 9-trans
2.4
0.2
t
1.0
0.6
0.6
18
1 11-cis
27.5
54.2
43.0
50.6
41.6
57.7
18
1 unknown
18
2
2.7
0.5
t t
19
0
19
0 cyclo
0.4
12.9
2.0
19
1 br
0
2.4
1
22
0
1.4
24
0
50 + = % total fatty acids t = trace br = branched a = C17:0 cyclo or C18:0 unknown
T a*
•n/
.^o
27 AUG 1991'
,,.y
Table 9 (continued)
10
CLUSTER -
4
FATTY ACID
WB4CT
WEll
9B.1
16N.1
17N. 1CT
0
0.5
12
0
0.9
0.2
12
1
0.8
13
0
14
0
3.8
1.3
3.2
1.9
1.2
14
0 iso
0.7
t t
14
1
0.1
0
0.7
0.2
0 iso
8.7
9.3
8.2
0 anteiso
32.3
27.1
.3
0 cyclo
< 0.1
16
0
26.8
26.9
22.5
17.4
12.5
16
0 iso
4.7
.8
6.7
16
1
7.9
2.4
17
0
0.4
0.3
17
0 iso
0.3
3.2
6.0
.1
17
0 anteiso
21.9
24.4
29.8
17
0 cyclo
0.2
17
1
< 0.1
17
1 br
18
0
.2
3.5
2.3
.3
1.2
18
0 unknown
18
1 9-cis t
2.5
*
18
1 9-trans
1.7
0.9
*0.5
1.6
1.0
18
1 11-cis
47.4
48.1
18
1 unknown
18
2
1.0
0.3
19
0
40
19
0 cyclo
1.0
19
1 br
11.2
0
1.3
0.4
1
0.3
22
0
0.8
0.2
45
24
0
* = includes all C18:l isomers so + = % total fatty acids t = trace br = branched a = C17:0 cyclo or C18:0 unknown
Table 9 (continued)
40
45
CLUSTER -
6
non-clustering
FATTY ACID
59E.4
64B. 4CT
5E.1
92LM.4
0
12
0
12
1
13
0
14
0
3.9
6.4
3.8
1.5
14
0 iso
0.7
2.0
14
1
0
t
3.8
t
0 iso
3.9
12.6
44.0
0 anteiso
24.8'
18.1
9.9
0 cyclo
16
0
26.3
40.5
74.7
18.7
16
0 iso
2.4
3.7
2.1
16
1
2.9
17
0
t
2.4
17
0 iso
0.6
1.7
.7
17
0 anteiso
6.1
3.7
6.8
17
0 cyclo
17
1
17
1 br
18
0
16.2
4.8
2.6
1.5
18
0 unknown
.9
18
1 9-cis
18
1 9-trans
*5.7
*0.5
*10.0
18
1 11-cis
18
1 unknown
18
2
2.7
19
0
19
0 cyclo
19
1 br
0
4.6
1
22
0
2.6
24
0
= includes all C18:l isomers 50 + = % total fatty acids t = trace br = branched a = C17:0 cyclo or C18:0 unknown
N
i 27AUG
1991
ss-V
fill
- JO
' XI
>
ffl cz z
o
"fy o
O /)
% —
~JL2
- 35 -Table 10
Fatty Acid Profiles of the Clusters of Gram-Negative Alkaliphiles
br = branched
Cluster
1
2
3A/B
3C
Predominant Fatty
C16:0
C16: 0
C16:0
C16:0
Acids (> 10%)
C18:l 11-cis
C18:0
C16:1
C18:1 11-cis
C18:1 11-cis
C18:1 11-cis
n-saturated
- 40%
60 - 65%
- 55%
~ 40%
n-unsaturated
~ 60%
~ 33%
45 - 70%
~ 60%
iso
< 1%
< 1%
0%
0%
anteiso
0%
< 1%
0%
0%
total branched
< 1%
< 3%
0%
0%
cyclo
0%
< 5%
0%
even carbon no.
> 95%
> 90%
> 99%
> 99%
odd carbon no.
< 5%
< 10%
< 1%
< 1%
additional markers
C17:0 cyclo
C19:0 cyclo
C17:1 br
-/
Cluster .
3D
4
6
Predominant Fatty
C16: o
C16:0
C15:0 anteiso
C15:0 br
Acids (> 10%)
C18:1 11-cis
C18:1 11-cis
C16:0
C16:0
C17:0 anteiso
n-saturated
- 40%
- 40%
- 30%
55 - 60%
n-unsaturated
50 - 65%
50 - 60%
< 2%
< 10%
iso
0%
< 1%
~ 20%
- 20%
anteiso
0%
0%
50 - 65%
- 30%
total branched
0%
1 - 12%
> 70%
~ 40%
cyclo
2 - 15%
< 2%
0%
0%
even carbon no.
> 80%
> 85%
- 35%
55 - 65%
odd carbon no.
< 20%
< 15%
65 - 80%
- 45%
additional markers
C17:0 cyclo
C19:0 cyclo
br = branched C ,
Q, i
ItX j
Nucleic Acids
An essential component of any taxonomic study is an analysis of the genetic material - the nucleic acids. The composition of chromosomal DNA is unaffected by the growth conditions of the organism and an appropriate analysis may confirm or refute the taxonomic position of the organism. Chromosomal DNA may be analyzed by the determination of the base composition (G+C mol%) of individual strains, and the base sequence homologies between pairs of strains by DNA-DNA reassociation (hybridization) (Owen, R.J. and Pitcher, D., in Chemical Methods in Bacterial Svstematics (eds. M. Goodfellow and D.E. Minnikin), Academic Press, London and Orlando; FL (1985), pp. 67-93).
DNA Base Composition
The guanine plus cytosine (G+C mol%) composition is constant for the chromosomal DNA from any given organism. Closely related organisms have similar G+C compositions. However, G+C results must be interpreted within the context 20 of independent taxonomic data since similar G+C mol% of DNA samples from different organisms does not in itself imply biological relatedness.
DNA was extracted from cells grown to exponential phase in Medium A by the chloroform:phenol method and was 25 precipitated with ethanol. Base composition was determined by the thermal denaturation method (Marmur, J. and Doty, P. (1962), J. Mol. Biol., 2, 585-594) on a Phillips model PV8764 spectrophotometer with temperature programming. A second method involved HPLC analysis on a Beckraan system gold using a Beckman ultrasphere ODS column and 0.04 M potassium dihydrogen phosphate plus acetonitrile (9 +1, v/v) as eluent at a flow rate of 1.5 ml/min, after treatment of the DNA with nuclease PI and alkaline phosphatase.
The results of these analyses are set out in Table 11. The G+C mol% values for the alkaliphilic bacteria cover a krange of 30 mol% (37.6 - 67.1 mol%) . However, within the lusters the variation is only 3-7 mol%, which further
21 AUG 1991
confirms that the strains within a cluster are closely related to each other.
Table 11
DNA Base Composition of Gram-Negative
Alkaliphilic Bacteria
Cluster
Strain
G+C HPLC
mol%
tm
1
2E.1 wBs4 2 ON. 1 wNk2
55.2 51.1
51.2 53 .0
2
42E.2
62.7
3
wBl
28N. 1CT
37E.2
wNl
64.1 67.1
63.0 64.8
4
WE5
wB4CT
wNkl wEll
WE12
58.5 65.3
61.0 59.7
58.1
17N. 1CT 22M.1
50.0 43 .8
40
6
64B. 4CT 53E.4
41.0 37.6
45
non wN2 wBn5
64.1 54.6
DNA—DNA Molecular Hybridization The method used was essentially that of Crosa, J.H. et Q^al. (Int. J. Systematic Bacterid., 29, 328-332, 1979). 50 'jfiritium labelled DNA was prepared using a nick-translation 27AUG1991*Cy (Amersham, N5000) according to the manufacturer's
40
45
50
instructions. The reassociation mixtures were incubated at 65°C for 16 hours. The results are set out in Table 12 from which it can be seen that the DNA sequence homology is higher within the clusters that between the clusters.
Table 12
Inter-Cluster and Intra-Cluster DNA-DNA Homology Values for Gram-Negative Alkaliphilic Bacteria
Cluster
1
2
3
4
6
Strain
2E.1
45E. 3CT
28N. 1CT
WE12
17N. 1CT
64B.4cr
1
2E.1 20N.1
100 56
33
2
45E.3CT 42E.3
100 76
3
28N. 1CT 56E.4 21M. 1 37E.2 44E.3
34 37
100 51 53
55
26
4
WE12 WB4CT
41
100 43
17N.1CT 22M.1
45
44
36
24
100 65
44
6
64B.4CT 53E.4
21
31
34
100 60
SS
17
The values give the percent hybridization between the strains (rows) and fT-labelled strains (columns).
SS = salmon sperm DNA.
17 AUG 199!
0 X
40
Determination of Representative Strains The centroid of each individual cluster generated by the Sq/UPGMA method was computed using the RGROUPS program in TAXPAK. The centroid of a cluster of points representing real organisms projected into hyperspace represents a hypothetical average organism. The centroid rarely, if ever represents a real organism. Therefore, the Euclidean distances of each of the members of the cluster from the centroid of the cluster were calculated in order to establish which strain was closest to the hypothetical average organism. The strain closest to the centroid was designated the "centrotype" organism (indicated with the superscript "CT").
The centrotype organism can ' be thought of as the "Type Strain" which most closely represents the essential and discriminating features of each particular cluster. The centrotype strains are recorded in Table 13.
Table 13
Centrotype Strains
Cluster Number
Number of Strains in Cluster
Mean Euclidean Distance of Strains Standard from Deviation Centroid
Centrotype
Strain
Euclidean
Distance from
Centroid
1
11
3.67
0.30
IE. 1
2.88
2
9
3.20
0.52
45E.3
2.30
3
34
3.52
0.32
28N.1
2.90
4
3.97
0.29
WB4
2.87
4
3.25
0.29
17N.1
2.13
6
3.11
0.41
64B.4
1.93
A description of each of the centrotype organisms has been made so as to be able to distinguish these organisms from all other bacteria previously known and described. In addition, the minimum number of discriminating tests to define each cluster has been computed so that it may be clearly seen that the clusters containing these novel bacteria can be easily disti:
■' t>- ^
each other and from all other known bacteria. / <<
\ 27 AUG 1991*)
Description of Centrotype Strains Strain IE. 1CT (Cluster 1)
An aerobic, motile, Gram-negative rod-shaped bacterium, 1-7-3-3 im x 0-5-0-7 /im.
Obligate alkaliphile, grows best between pH 9 and pH 10.
On alkaline-agar, (Medium A) forms smooth, cream colored colonies, initially translucent but becoming opaque after a few days. The colonies are circular, entire and convex, 2-3 mm in diameter.
In alkaline-broth, (Medium A) growth (37 °C) is flocculent with the formation of a sediment and surface pellicle.
Grows well between 20°C and 40°C. Grows slowly at 10°-15°C. No growth at 8°C or 45°C.
KOH test: positive
Aminopeptidase: weak positive
Oxidase: negative
Catalase: pos itive
NaCl tolerance: 0% to < 8%. No growth at 8%
Hydrolysis of Gelatin: positive
Hydrolysis of Starch: positive
Major polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate
phosphatidylethanolamine
Major ubiquinone: Q9
Major fatty acids: C16:0, C18:0, 11-cis C18:l
Chemoorganotroph. Grows oh complex substrates such as yeast extract and peptones. Growth on simple sugars and organic acids
very restricted (e.g., growth only observed on ribose, sucrose and pyruvate).
Strain 45E.3CT (Cluster 2)
An aerobic, Gram-negative, rod-shaped bacterium, 3-4.5 /zm x 35 0.6 im. Motile by a single polar flagellum.
Obligate alkaliphile growing between pH 7.8 and pH 11.2. 0 n alkaline-agar, (Medium A) forms smooth, opaque, cream colored konies, 1-2 mm in diameter. The colonies are circular, convex id entire.
In alkaline-broth, (Medium A) growth (37°C) is slow, slight /ith an even turbidity, surface pellicle and no sediment.
27 AUG199V
Grows well between 20°C and 40°C. Grows slowly at 10°C. No growth at 8°C or 45°C.
KOH test: Aminopeptidase: Oxidase: Catalase:
NaCl tolerance:
Hydrolysis of Gelatin: Hydrolysis of Starch:
Major polar lipid components:
positive positive positive positive
\ to 12%. Growth at 12% is slow No growth at 15% positive positive (weak) phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamine , glycolipid (a-naphthol positive)
Major ubiquinone: . Q9
Major fatty acids: C16:0, C18:0, 11-cis C18:l
Chemoorganotroph. Grows on complex' substrates such as yeast extract and peptones. No growth on simple sugars. Grows on organic acids (e.g., fumarate, succinate, pyruvate, acetate,
lactate) and some fatty acids (e.g., propionate, valerate) and amino acids (e.g., proline, alanine, phenylalanine).
40
Strain 28N.1CT (Cluster 3)
An aerobic, motile, Gram-negative, rod-shaped bacterium,
4-8-5-5 fim x 0-6-0-8 jam. Obligate alkaliphile growing between pH
8•5 and pH 10•7.
On alkaline-agar, (Medium A) forms smooth, circular, opaque colonies with a stringy texture. The colonies have a convex elevation and entire margin. The colony color is initially cream/beige becoming pink after a few days.
In alkaline-broth, (Medium A) growth (37° C) is heavy,
flocculent with a surface pellicle and a sediment.
Grows well between 20°C and 45°C. Grows slowly at 10°C and
159C. No growth at 50°C.
KOH test: positive positive positive
Aminopeptidase:
Oxidase:
Catalase:
NaCl tolerance:
Hydrolysis of Gelatin:
Hydrolysis of Starch:
Major polar lipid components:
positive 0% to 12%. No growth at 15%
negative positive phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate
AUG 19917
Major ubiquinone: Major fatty acids: G+C:
? 3 Q?o t.* tsjj phosphatidylglycerol sulphate phosphatidylethanolamine
Q6
C16:0, C16:l, 11-cis C18:1 64.1 mol% (HPLC)
40
Chemoorganotroph. Grows well on complex substrates such as yeast extract and peptones. Grows on simple sugars, organic acids, fatty acids and amino acids.
Strain wB4CT (cluster 4)
An aerobic, Gram-negative, rod-shaped bacterium, 3-4 /im x
0.6-0.8 im, frequently occurring as pairs of cells.
Alkaliphile, grows well between pH 7.5 and pH 10.9.
On alkaline-agar, (Medium A) forms smooth, beige to brown colonies. The colonies are somewhat variable: 1 to >5 mm in size,
circular to irregular in form, low convex or raised in elevation with an undulate or entire margin.
In alkaline broth, (Medium A) growth (37#C) is flocculent,
sediment forming with a surface pellicle.
Grows best between 15°C and 45°C, no growth at 50°C.
KOH test: positive
Aminopeptidase: positive
Oxidase: very weakly positive, may be seen as negative
Catalase: positive
NaCl tolerance: 0% to >12%, no growth at 15%
Hydrolysis of Gelatin: negative
Hydrolysis of starch: negative
Major polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamine Major ubiquinone: Q9
Major fatty acids: C16:0, 11-cis C18:l
G+C: 65.3 mol% (Th)
Chemoorganotroph. Grows well on complex substrates such as yeast extract. Growth on simple sugars is restricted. Grows on organic acids (e.g., lactate, acetate, fumarate), fatty acids (e.g., propionate, valerate, caprate) and amino acids (e.g., proline, serine, lysine).
Strain 17N.1CT (cluster 5)
//
4'
An aerobic, Gram-negative, long, thin, rod-shaped, bacterium, 5.5 - 10.5 im x 0.6 £im, sometimes forming short chains of cells. With age pleomorphic, peculiar swollen forms predominate.
Obligate alkaliphile, grows best between pH 8 and pH 10.5.
On alkaline-agar, (Medium A) forms smooth, opaque, yellow, colonies, 2-3 mm in diameter. The colonies vary from circular to irregular in form, with a convex to umbonate elevation, and entire, undulate or lobate margin, depending upon age.
In alkaline-broth (Medium A), growth (37 °C) is even and sediment forming with no surface pellicle.
Grows well between 15"C and 37°C. Grows slowly at 10°C and not at all at 8°C. No growth at 40°C or above.
KOH test:
positive
Aminopeptidase:
Oxidase:
Catalase:
NaCl tolerance:
Hydrolysis of Gelatin: Hydrolysis of Starch:
Major polar lipid components:
Major ubiquinone: Major fatty acids: G+C:
weakly positive negative positive
0% to <12%, grows best at 0% NaCl positive weakly positive phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamine glycolipid (a-naphthol positive)
Q9, Q10
C15:0 anteiso, C16:0, C17:0 anteiso
50.0 mol% (HPLC)
Chemoorganotroph. Grows wel-l on complex substrates such as yeast extract. Growth on simple sugars is restricted (e.g., growth observed only on fructose; no growth observed on glucose, ribose, lactose). Grows on organic acids (e.g., fumarate, succinate, pyruvate, 2-ketogluconate) and amino acids.
Strain 64B.4CT (cluster 6)
An aerobic, Gram-negative, rod-shaped bacterium 2.0 - 3.5 /im x 0.8 - 1.0 /im.
40 Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.
On alkaline-agar, (Medium A) forms smooth, opaque colonies, first creamy yellow in color, becoming beige with age. The olonies are about 4 mm in diameter, circular becoming irregular;
\
flat or low convex in elevation becoming convex; with an entire margin becoming undulate.
In alkaline-broth (Medium A), growth (37 °C) is even, sediment forming with no surface pellicle.
Grows well at 15°C to 45°C, no growth at 10°C or 50°C.
KOH test: positive
Aminopeptidase: negative
Oxidase: pos itive
Catalase: positive
NaCl tolerance: 0% to < 12%, no growth at 15%
Hydrolysis of Gelatin: positive
Hydrolysis of Starch: weakly positive
Major polar lipid components: phosphatidylglycerol diphosphatidylglycerol
.. phosphatidylglycerol phosphate phosphatidylethanolamine glycolipid- (a-naphthol positive) Major ubiquinone: Q9
Major fatty acids: C15:0 iso, C15:0 anteiso, C16.:0
G+C: 41.0 ± 0.9 mol% (HPLC)
Chemoorganotroph. Grows well on complex substances such as yeast extract. Grows on some simple sugars (e.g., glucose, ribose, maltose and fructose), organic acids (e.g., acetate, 25 lactate, citrate and fumarate), some fatty acids (e.g., propionate and caprate) and amino acids (e.g., proline, histidine and alanine).
Non-Clusterina Strains 30 The strains which do not fall into the clusters defined here are also novel bacteria not previously known or described. These strains, coded wN2, 4E.1, 5E.1, 92124.4 and wBn5, may represent rarer varieties of alkaliphilic bacteria and are probably members of clusters of bacteria representing new genera and species at 35 present not described. A description of these "non-clustering" strains has been made so as to be able to distinguish these organisms from all other bacteria previously known and described.
Strain wN2
40 An aerobic, Gram-negative, motile, rod-shaped bacterium,
frequently in pairs.
Obligate alkaliphile, grows best between pH 9 and pH 10.
40
On alkaline-agar, (Medium A) forms smooth, translucent, beige colored colonies, 1-2 mm in diameter. The colonies are circular, convex with an entire margin.
In alkaline-broth (Medium A), growth (37°C) is flocculent with a ring or surface pellicle and formation of a sediment.
Grows well at 20"C to 30°c. No growth at 15°C or 40°C. KOH test: positive
Aminopeptidase: Oxidase: Catalase:
NaCl tolerance:
Hydrolysis of Gelatin: Hydrolysis of Starch: Major ubiquinone:
G+C:
weak positive weak positive positive obligate halophile, growth at 4% NaCl no growth at 0% or 8% NaCl slow positive positive Q9
64.1 (Th)
Chemoorganotroph. Metabolically unreactive. No growth on simple sugars or organic acids. Grows on complex substrates such as yeast extract and peptones, and on some amino acids.
Strain 4E.1
An aerobic, Gram-negative, motile, rod-shaped bacterium, 1.7 - 5.2 /xm x 0.75 fim.
Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.
On alkaline-agar, (Medium A) forms smooth, opaque, beige or brown colored colonies, 2-4 mm in diameter. The colonies are circular in form, convex in elevation, with an entire margin.
In alkaline-broth (Medium A), growth (37°C) is heavy and flocculent with a sediment and surface pellicle.
Grows well between 20°C and 37°C. Grows very slowly at 10°C and not at all at 8°C. No growth at 40°C or above.
KOH test: positive
Aminopeptidase: Oxidase: Catalase:
NaCl tolerance:
positive very weakly positive, can appear negative positive
0% to 12%, may grow weakly at 15%
no growth at 20%
Hydrolysis of Gelatin: negative
Hydrolysis of Starch: negative
Chemoorganotroph. Does not grow on simple sugars, except for ribose. Grows well on complex substrates such as yeast extract,
d on organic acids (e.g., succinate, pyruvate, citrate,
lonate, acetate and lactate), fatty acids (e.g., propionate,
valerate and suberate), and amino acids (e.g., proline, serine, histidine and lysine).
Strain 5E.1
An aerobic, Gram-negative, rod-shaped bacterium, 3.0 - 5.3
fm x 1.3 /zm.
Obligate alkaliphile, grows best between pH 9 and pH 10.5.
On alkaline-agar, (Medium A) forms smooth, opaque, brown colored colonies, 3-4 mm in diameter. The colonies are fairly
irregular in form, generally flat to slightly umbonate in elevation with a lobate margin.
In alkaline-broth (Medium A), growth (37°C) is moderate to heavy, becoming flocculent"with a sediment and surface pellicle.
Grows well between 20°C and 40°C. Grows slowly at 10°C. No
growth at 45°C.
KOH test: positive
Aminopeptidase: positive
Oxidase: negative
Catalase: positive
NaCl tolerance: 0% to 12%, may grow weakly at 15%
no growth at 20%
Hydrolysis of Gelatin: positive
Hydrolysis of Starch: weakly positive
Major polar lipid components: phosphatidylglycerol
diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylglycerol sulphate phosphatidylethanolamine glycolipid (a-naphthol positive) 30 Major ubiquinone: Q8
Major fatty acids: C16:0, C18:l
Chemoorganotroph. Does not grow on simple sugars. Grows well on complex substrates such as yeast extract, organic acids (e.g., 35 pyruvate, citrate, acetate and lactate), fatty acids (e.g., propionate, caprate and valerate) and amino acids (e.g., proline, alanine and lysine).
Strain 92IM.4
An aerobic, Gram-negative, rod-shaped bacterium, 2.0 - 3.5 x 0.5 - 1.0 im.
V Obligate alkaliphile, no growth below pH 7.5.
|f 27 AUG 1991'
23
On alkaline-agar, (Medium A) forms smooth, cream colored colonies, initially translucent but becoming opaque. The colonies develop from circular, entire to irregular, lobate in form, with a convex elevation.
In alkaline-broth (Medium A), growth (37°C) is slow, slight, flocculent with a sediment but no surface pellicle.
Grows between 10°C and 40°C, no growth at 8°C or 45"C.
positive negative
Oxidase: positive positive
0% to 15%, growth at 15% is slow no growth at 20% positive
Hydrolysis of Starch: weakly positive
Q9
C15:0 iso, C15:0 anteiso, C16:0, C17:0 iso
Chemoorganotroph. Grows on complex substrates such as yeast
extract and peptones, and a variety of sugars, organic acids and amino acids.
KOH test: Aminopeptidase: Oxidase:
Catalase:
NaCl tolerance:
Hydrolysis of Gelatin: Hydrolysis of Starch: Major ubiquinone: Major fatty acids:
40
Strain wBn5
An aerobic, Gram-negative, small, rod-shaped bacterium, frequently forming short chains of cells.
Obligate alkaliphile, no growth below pH 8.
On alkaline-agar, (Medium A) forms smooth, circular, convex colonies with an entire margin, about 1 mm in diameter. The colonies are initially cream/beige, transparent becoming opaque, brown.
In alkaline-broth (Medium A), growth (37°C) is initially evenly turbid with a sediment but no surface pellicle becoming after 4 days flocculent with formation of a pellicle.
Grows at 30°C and 37°C. No growth at 40#C.
KOH test:
Aminopeptidase:
Oxidase:
Catalase:
NaCl tolerance:
Hydrolysis of Gelatin: Hydrolysis of Starch: Major ubiquinone:
G+C:
positive positive positive positive obligate halophile. Growth at 4% NaCl no growth at 0% or 8% slow positive negative Q8, Q9 54.6 mol% (T„)
i
o T 0 9 0 7
Chemoorganotroph. Grows on a range of complex substrates such as yeast extract and peptones, as well as sugars, organic acids, fatty acids and amino acids.
Cluster Definition bv the Calculation of the Minimum
Number of Discriminatory Tests, and the Construction of a Probability Matrix for the Identification of
Gram-Negative Alkaliphiles
One of the purposes of a numerical classification study is
to use the phenetic data, which defines the clusters at a
• selected similarity level, for the assignment or identification of unknown strains. The classification 'test data can be used to determine the minimum set of tests which are required to define the clusters at the 73% (SG) similarity level, and to identify
those characters which are most diagnostic (predictive) for the individual clusters. In other words, the minimum number of tests required to assign an unknown organism to a pre-determined cluster with a high degree of predictability.
From the minimum discriminatory tests, a probability matrix
can be constructed for the identification of unknown strains. The analysis is achieved by using a combination of the CHARSEP and
DIACHAR (TAXPAK) and MCHOICE (not on TAXPAK but available by
Data-Mail from the University of Leicester, U.K.) programs. An evaluation of the identification matrix is provided by using the
MOSTTYP and OVERMAT programs. Practical examples of the use of these programs for the probabilistic identification of bacteria have been published by Williams, S.T., et al., (1983), J. Gen.
Microbiol., 129. pp. 1815-1830; and Priest, F.G. and Alexander,
B., (1988), J. Gen. Microbiol., 134/ PP« 3011-3018; ibid, (1990),
136/ PP. 367-376.
A "n x t" table was constructed from the test data using characters 6 to 10 and 13 to 104 (Appendix C) scored in binary notation (positive = 1, negative = 0). This data matrix was supplemented with the following four extra character states:
[105] Bright yellow colonies (character number 1, Appendix C)
iN"F%J106] Translucent colonies (grown on Medium A, Appendix A)
0^107] Lipase (lipolytic activity on olive oil (Medium M))
^ \L08] Oxidase positive within 10 sees, (test 9, Appendix B)
oV
2 7 AUG 199\ :)
2392
The data matrix is first examined using the CHARSEP program which calculates separation indices and thus the diagnostic value of the individual characters for discriminating between the clusters. Tests with a VSP index > 25% (Sneath, P.H.A., (1979), 5 Computers and Geosciences, 5, 349-357) are accepted, characters with a low diagnostic value (VSP < 25%) were rejected. A preference is made for characters with the highest VSP indices, provided that the criteria in the DIACHAR and MCHOICE programs are also met. In this example, 38 tests have a VSP index >25%, 10 and 9 of the 24 characters finally chosen have a VSP index >50% (Table 11).
The data matrix is next re-examined by means of the DIACHAR program, which determines the most diagnostic character states of each of the clusters. The number of character states was set at 15 10. This result allows the choice of mutually exclusive character states between the clusters. As many of these tests as possible are retained in the final identification matrix of minimum discriminatory tests; in this example between 6 and 9 diagnostic characters per cluster. The remaining, unused tests are also 20 noted and may be applied as additional tests for the confirmation of identification (Table 12).
The MCHOICE program ranks the tests in groups which can be displayed in the form of a dendrogram using the MDEND subroutine. The groups identify tests with similar discriminatory 25 value, thus allowing the rejection of tests which fail to make a significant discrimination as well as allowing choices to be made between tests of equal or very similar diagnostic value.
Table 13 shows the set of 24 tests which is the minimum number required to define the clusters and which can be used for 30 the assignment of unknown strains. In addition, Table 13 shows the identification matrix which consists of the percentage of positive characters which define the clusters on the basis of the 24 minimum discriminatory tests. This is computed by the IDMAT program.
Table 14
Separation Values of Characters used for the Minimum Discriminatory Tests
CHARACTER
VSP Index
[23]
N-acetylglucosamine
.4
[26]
Saccharose
44.8
[27]
Maltose
41.4
[32]
Lactate
51.6
[41]
Propionate
60.9
[43]
Valerate
63.4
[44]
Citrate
45.1
[45]
Histidine
38.0
[47]
Glycogen
31.7
[51]
3 -hydroxybutyrate
66.1
[52]
4 -hydr oxybenz oat e
38.0
[58]
Leucine arylamidase
36.6
[59]
Valine arylamidase
50.5
[64]
Phosphohydrolase
52.8
[65]
a-galactosidase
33.9
[85]
Ampicillin
36.8
[92]
Fusidic Acid
68.7
[93]
Methicillin
58.3
[99]
Polymixin
62.8
[102]
Vancomycin
48.3
239
Table 15
Discriminatory Tests for Each of the Six Clusters fSc) Cluster 1: cream, circular, opaque, mucoid colonies.
Positive Negative
[58] Leucine arylamidase (91%)
[59] Valine arylamidase (91%) [64] Phosphohydrolase (91%) [99] Polymixin (89%)
[23 [27 [41 [42 [43 [44 [45 [47 [52 [65
N-acetylglucosamine (9%)
Maltose (9%)
Propionate (9%)
Caprate
Valerate (9%)
Citrate (9%)
Histidine
Glycogen (9%)
4-hydroxybenzoate a-galactosidase
Cluster 2: small, cream, translucent colonies.
Positive Negative
[21]
Starch
[23]
N-acetylglucosamine
[31]
Acetate
[26]
Saccharose
[41]
Propionate
[45]
Histidine
[43]
Valerate
[50]
2-ketogluconate
[53]
Proline
[52]
4-hydroxybenzoate
[107]
Lipase
[68]
a-glucosidase
[108]
Oxidase (within 10 sees.)
[69]
B-glucos idase
[92]
Fusidic Acid
Cluster 3: cream, opaque colonies. Positive
[31 [32 [41 [43 [44 [51 [53 [58
Acetate Lactate
Propionate (94%)
Valerate (97%)
Citrate (94%) 3-hydroxybutyrate (94%) Proline
Leucine arylamidase (94%)
Negative
[64] Phosphohydrolase (3%)
[65] a-galactosidase (3%) [92] Fusidic Acid (3%) [96] Tetracycline (3%) [102] Vancomycin
[104] Bacitracin
Cluster 4: beige to brown, opaque colonies.
Positive Negative
[32] Lactate
[33] Alanine
[48] 3-hydroxybutyrate
[59] Valine arylamidase
[99] Polymixin
[45] Histidine
[85] Ampicillin
[86] Naladixic acid
[88] Trimethoprim
[89] Penicillin G [93] Methicillin
Table 15 (continued) Discriminatory Tests for Each of the Six Clusters fSJ Cluster 5: bright yellow [105], opaque colonies.
Positive
[64] Phosphohydrolase [65 ] a-galactosidase [66] B-galactosidase [85] Ampicillin
[92] Fusidic Acid
[93] Methicillin [96] Tetracyclinee [102] Vancomycin [104] Bacitracin
Negative
[23] N-acetylglucosamine
[32] Lactate
[33] L-alanine
[34] Mannitol
[41] Propionate
[42] Caprate [43] Valerate
[45] Histidine
[48] 3-hydroxybenzoate
[51] 3-hydroxybutyrate
[52] 4-hydroxybenzoate [99] Polymixin
Cluster 6: cream, irregular, flat colonies.
Positive Negative
[21]
Starch
[17]
Pyruvate
[23]
N-acetylglucosamine
[52]
4-hydroxybenzoate
[26]
Saccharose
[58]
Leucine arylamidase
[27]
Maltose
[59]
Valine arylamidase
[31]
Acetate
[65]
a-galactos idase
[33]
Alanine
[99]
Polymixin
[44]
Citrate
[47]
Glycogen
[51]
3-hydroxybutyrate
[89]
Penicillin G
[92]
Fusidic Acid
[93]
Methicillin
[96]
Tetracyclinee
[104]
Bacitracin
Note: The numbers in square brackets proceeding the character ^ state refers to the character states and unit tests in Appendices B and C. The percentage in parenthesis refers to positive character states.
3 9297
Table 16
A Probability Matrix for the Identification of Alkaliphiles; Percentage Distribution of Positive Discriminatory Characters Which Define the Clusters of Gram-Negative Alkaliphilic Bacteria at the 73% Level (Sc)
TEST
CLUSTER
1
2
3
4
6
[23]
N-acetylglucosamine
13
0
26
0
100
[26]
Saccharose
0
74
100
[27]
Maltose
0
68
60
50
100
[32]
Lactate
38
50
100
100
0
40
[41]
Propionate
0
100
91
60
0
80
[43]
Valerate
13
100
97
80
0
40
[44]
Citrate
13
50
94
50
100
[45]
Histidine
0
0
71
0
0
80
[47]
Glycogen
0
13
26
100
[51]
3 -hydroxybutyrate
13
94
100
0
100
[52]
4 -hydroxybenzoate
0
0
71
80
0
0
[58]
Leucine arylamidase
88
63
94
60
50
0
[59]
Valine arylamidase
88
65
100
0
[64]
Phosphohydrolase
88
13
3
75
40
[65]
a-galactosidase
0
0
3
75
0
[85]
Ampicillin
50
63
56
0
100
80
[92]
Fusidic Acid
0
3
100
100
[93]
Methicillin
50
13
50
0
100
100
[99]
Polymixin
88
50
81
100
0
0
[102] Vancomycin
13
13
3
100
75
[105]
Yellow colony
0
0
0
0
100
0
[106]
Translucent colony
0
100
3
0
0
0
[107]
Lipase
0
100
21
0
0
0
[108]
Oxidase (10 sees)
88
6
0
0
0
Evaluation of the Discriminatory Tests and Assessment of the Reliability of Identification The evaluation of the discriminatory tests has two aspects. Firstly, the validity of the tests can be analyzed using 5 practical examples, which can be further evaluated using statistical theory, or the tests can be directly subjected to theoretical assessment using statistical methods.
Illustration 1
A Practical Evaluation of the Discriminatory Tests
Many workers assess the accuracy of the discriminatory tests only by redetermining the character states of selected cluster representatives. This approach has been used here for the centrotype strains (see below). A far more stringent approach 15 which is seldom applied, is to examine all the strains which were used in the original numerical taxonomic analysis. When subjected to cluster analysis using only the data acquired from the derived set of minimum discriminatory tests, the reconstructed dendrogram can be compared with the original. Using only the 24 20 discriminatory tests previously described (Table 16), the data (two-state, binary form) for all 70 of the novel Gram-negative alkaliphilic bacteria were subjected to cluster analysis by the Sq/UPGMA method. The reconstructed dendrogram is reproduced in Figure 3. This reconstructed dendrogram compares very favorably 25 with the original dendrogram (Figure 1).
Although there has been some rearrangement of position of the clusters, their composition is largely unchanged and they are defined at approximately the same similarity level as the original. Cluster 4 however, has combined with Cluster 3, with a 30 single strain moving to Cluster 1. This further serves to emphasize the difficulty of defining Cluster 4 on phenetic data alone. It has been stressed several times that supplementary chemotaxonomic data are required to make the proper distinction between Cluster 3 and Cluster 4.
In both the original dendrogram and the reconstruction
N r (Figure 3), Cluster 3 appears to comprise several sub-clusters
AUG 19911
•V'
2Z*cv"
A
/
above the 73% similarity level. The fine structure of cluster 3 is also supported by the chemotaxonomic data (see above) .
Illustration 2
A Theoretical Evaluation of the Discriminatory Tests
An assessment of cluster overlap is achieved using the OVERMAT program. This program examines the matrix constructed from the percentage positive values for the selected character states against a critical overlap value by considering the 10 clusters defined by the coordinates of the centroid and the cluster radius (twice root mean square of the distances of the strains from the centroid). If there is significant overlap between the clusters, unknown strains" may not identify with sufficient confidence to any one of them (Sneath, P.H.A. and 15 Sokal, R.R., supra, p. 394-400). At a chosen critical overlap value of 2.5% (which is a more stringent condition than is used by most workers: see Priest, F.G. and Alexander, B., (1988), supra; and Williams, S.T. et al., (1983), supra) there was no significant overlap between the clusters (95% confidence level) 20 except between Cluster 3 and Cluster 4 where the actual overlap was calculated to be 4%. However, chemotaxonomic data (see above) was not taken into account when constructing the identification matrix. On the basis of quinone analyses, strains from Cluster 3 can be distinguished from the strains of Cluster 4.
Illustration 3
A Theoretical Assessment of the Reliability of Identification The hypothetical median organism (HMO) is another estimate of the "average" organism in a cluster (Sneath, P.H.A. and Sokal, 30 R.R., supra, pp. 194 et sea.). A HMO is not a real strain but a hypothetical organism possessing the most common state for each character. The MOSTTYP program calculates HMO's for each cluster in the identification matrix and then attempts to identify them. In other words, MOSTTYP is a program to evaluate an identification matrix by calculating identification scores of the ost typical strains against the clusters. A good identification trix should give a high probability of a HMO being reassigned
23929
to its own cluster. The results of this analysis were very satisfactory (Table 17), especially since MOSTTYP was programed to consider only the first 20 diagnostic tests of the identification matrix (Table 16), i.e. excluding tests 105-108. Each HMO was reassigned to its original cluster with Willcox probabilities of 0.998-1.000 (Willcox, W.R. et al., (1973) J. Gen. Microbiol., 77, 317-330). The Taxonomic Distances were all low and the standard errors of the Taxonomic Distance were all negative, indicating that the HMO's were all closer to the centroid of the cluster than the average for the cluster (Table 17).
Table 17
Identification Scores for the Hypothetical Median Organism of each cluster provided bv the MOSTTYP Program
Identification Score
CLUSTER
Willcox Probability
Taxonomic Distance
Standard Error of Taxonomic Distance
1
2
3
4
6
0.999 0.999 1.000 0.998 1.000 1.000
0.194 0.236 0.231 0.195 0.217 0.182
2.742 2.214 2.115 2.998 1.839 2.502
Illustration 4 A Practical Evaluation of Identification Score Identification of strains using the minimum set of discriminatory tests is achieved using the MATIDEN program in TAXPAK. The program compares presence-absence data for an unknown strain against each cluster in turn in an identification matrix of percentage positive characters. Identification coefficients are computed, namely Willcox probability, Taxonomic Distance and the Standard Error of the Taxonomic Distance. The results are displayed, showing the identification scores to the best cluster and to the two next best alternative clusters. Additionally, the atypical results ("characters against") are recorded. In an analysis using data from real strains, the centrotypes were eassigned to their original clusters with Willcox probabilities
A. £ r p * V
239297
of 0.9996-1.000 (Table 18). The Taxonomic Distances were low. The Standard Errors of the Taxonomic Distance were all negative indicating that the centrotypes were closer to the centroid of the cluster than the average for the cluster.
Table 18
Identification Scores for the Centrotype Organisms of Each Cluster Provided bv the MATIDEN Program
Identification Score
Assigned Willcox Taxonomic Standard
Cluster Strain to Cluster Probability Distance (D) Error of D
1
2
3
4
6
2E.1
45E.3
28N.1'
wB4CT
17N.1'
64N.4
ct ct ct ct
1
2
3
4
6
1.000
1.000
1.000
0.9996
0.9999
1.000
0.309 0.226 0.305 0.265 0.255 0.211
0.283 1.749 0.622 1.092 0.478 1.126
Illustration 5 Identification of Unknown Isolates The identification matrix was assessed for the ability to 25 assign unknown Gram-negative alkaliphiles to the clusters defined herein. The criteria for a successful identification were:
(a) bacteria isolated from a habitat similar to, but geographically separate from, the East African soda lakes;
(b) a Willcox probability greater than 0.95 and low values for 30 Taxonomic Distance and its standard error (< 3) ;
(c) an identification score to the best cluster significantly better than those against the two next best alternatives;
(d) "characters against" the best cluster should be zero or few in number.
Unknown microorganisms may be examined using the minimum tests listed in Table 16. The character states are determined and identification scores obtained using the MATIDEN program. This program compares the character states of the unknown with the identification matrix determined for all of the predetermined 40 clusters, computes the best match and assigns t&Sf*unknown to the most appropriate cluster.
-\<27 AUG 1991
rr ( V
239297
A Willcox probability is calculated to determine the acceptability of identification. Willcox probabilities of 0.85 and 0.95 have been accepted as criteria for a successful identification (Williams, S.T., et al. (1983), supra; Priest, 5 F.G. and Alexander, B., (1988), supra). The Taxonomic Distance of the unknown from the cluster centroid is calculated and may be compared to the radius of the cluster. The Standard Error of the Taxonomic Distance should be less than the upper value of +3.0 suggested by Sneath, P.H.A. ((1979), pp. 195-213). Moreover, 10 physical characteristics, additional biochemical data and chemotaxomomic markers may be used to further confirm the identity of the unknown in a particular cluster.
The results provided by these five illustrations, together 15 with the statistical data provided by the numerical taxonomic analysis and the chemotaxonomic data, indicate a robust classification which identifies 6 major groups of new, Gram-negative, alkaliphilic bacteria.
Production and Application of Alkali-Tolerant Enzvmes
The alkaliphilic microorganisms of the present invention produce a variety of alkali-tolerant enzymes. Examples of enzyme activities present in representative strains of the Gram-negative bacteria of the present invention may be found in Appendices D 25 and E. These enzymes are capable of performing their functions at an extremely high pH, making them uniquely suited for their application in a variety of processes requiring such enzymatic activity in high pH environments or reaction conditions.
Examples of the various applications for alkali-tolerant 30 enzymes are in detergent compositions, leather tanning, food treatment, waste treatment and in the textile industry. These enzymes may also be used for biotransformations, especially in the preparation of pure enantiomers.
The alkaliphilic bacteria of the present invention may 35 easily be screened for the production of alkali-tolerant lipases, proteases and starch-degrading enzymes, inter alia, using the methods described herein.
iL 0 v
CI
The broth in which alkaliphilic bacteria are cultured typically contains one or more types of enzymatic activity. The broth containing the enzyme or enzymes may be used directly in the desired process after the removal of the bacteria 5 therefrom by means of centrifugation or filtration, for example.
If desired, the culture filtrate may be concentrated by freeze drying, before or after dialysis, or by ultrafiltration. The enzymes may also be recovered by precipitation and filtration. Alternatively, the enzyme or enzymes contained in the 10 broth may be isolated and purified by chromatographic means or by gel electrophoresis, for example, before being applied to the desired process. The. exact methods used to-, treat the culture filtrate and/or to extract and/or purify the alkali-tolerant enzymes is not critical to the present invention, and may be 15 determined by one skilled in the art.
The genes encoding alkali-tolerant enzymes of interest may be cloned and expressed in organisms capable of expressing the desired enzyme in a pure or easily recoverable form.
The following examples are provided to illustrate methods for the identification of Gram-negative alkaliphilic bacteria of the present invention, as well as methods of screening these alkaliphilic bacteria for the presence of various alkali-tolerant enzymes and methods for the subsequent production and application 25 of these enzymes in industrial processes. These examples are not to be construed so as to limit the scope of the present invention.
Example 1
Identification of Unknown Isolates
Six strains of Gram-negative, alkaliphilic bacteria were isolated from Mono Lake, a hypersaline, alkaline lake situated in California, U.S.A. (Javor, B., in Hypersaline Environments. Springer Verlag, Berlin and Heidelberg (1988), pp. 303-305). The 35 strains were isolated from samples of partially submerged soda-%ncrusted wood, tufa and soda-soil collected from the environs of Mono Lake (California, U.S.A.) in May, 1990 by enrichment culture
A
f /
at 37 "C in Medium A (Appendix A). The six strains are described in Table 19. The strains were examined using 21 of the 24 minimum tests listed in Table 16. The character states were determined and identification scores obtained using the MATIDEN program. The 5 results are outlined in Table 20.
Table 19
Alkaliphilic Strains from Mono Lake
Colony
Cell Shape
Strain
Sample
Color
Form
Elevation
Margin
ML005
tufa beige circular convex entire rod
ML104
wood pink/beige circular convex entire rod
ML201
wood yellow circular convex entire rod
ML203
wood pink/beige circular convex entire rod
ML206
wood yellow circular convex entire short
rod
ML301
soil beige circular convex entire rod
-
W £L
Table 20
Six Unknown Strains against the
Identification
Matrix
A.
Reference Number of unknown is
ML005
Value in
Percent in
•
Character unknown Best Taxon Next
Best Taxon
[23
N-acetylglucosamine
+
26
[26
Saccharose
+
74
[27
Maltose
+
68
60
[32
Lactate
+
99
99
[41
Propionate
+
91
60
[43
Valerate
+
97
80
[44
Citrate
+
94
[45
Histidine
+
71
1
[47
Glycogen
+
26
[51
3-Hydroxybutyrate
+
94
99
[52
4-Hydroxybenzoate
—
71
80
[58
Leucine arylamidase
+
94
60
[59
Valine arylamidase
+
65
99
[64
Phosphohydrolase
—
3
[65
a-Galactosidase n.t.
3
[85
Ampicillin
-
56
1
[92
Fusidic Acid
-
3
[93
Methicillin
-
50
1
[99
Polymix
+
81
99
[102
Vancomycin
—
3
[105
Yellow colony
—
1
1
[106
Translucent colony n.t.
3
1
[107
Lipase n.t.
21
1
[108
Oxidase (10 sec.)
+
6
1
n.t. = not tested
Isolate ML005 best identification is Cluster 3. Scores 40 for coefficients: 1 (Willcox probability), 2 (Taxonomic Distance) , 3 (Standard Error of Taxonomic Distance) .
45
Cluster 3 Cluster 4 Cluster 2
1
0.9999 0.8266 X 10 0.4086 X 10
-4 -7
2
0.407 0.526 0.584
3
1.475 4.590 6.296
50
Characters against [108] Oxidase (10 sec.)
Cluster 3 % in Taxon Value in unknown
Additional characters that assist in separating
Cluster 3 from
AUG 1991
*
06] Translucent colony
07] Lipase
%
3 21
Cluster 4 %
99 99
40
45
50
55
y"\ •• -J
' % i V U /
B. Reference Number of unknown is ML104
Character
Value in unknown
Percent in: Best Taxon Next
Best Taxon
[23]
N-acetylglucosamine
13
1
[26]
Saccharose
—
1
[27]
Maltose
-
1 '
[32]
Lactate
-
38
50
[41]
Propionate
-
1
99
[43]
Valerate
—
13
99
[44]
Citrate
-
13
50
[45]
Histidine
-
1
1
[47]
Glycogen
—
1
13
[51]
3 -Hydroxybutyrate
—
13
[52]
4-Hydroxybenz oate
—
1
1
[58]
Leucine arylamidase
+
88
63
[59]
Valine arylamidase
+
88
[64]
Phosphohydrolase
+
88
13
[65]
a-Galactosidase n.t.
1
1
[85]
Ampicillin
-
50
63
[92]
Fusidic Acid
—
1
[93]
Methicillin
-
50
13
[99]
Polymix
+
88
50
[102]
Vancomycin
—
13
13
[105]
Yellow colony
—
1
1
[106]
Translucent colony n.t.
1
99
[107]
Lipase n.t.
1
99
[108]
Oxidase (10 sec.)
+
88
n.t. = not tested
Isolate ML104 best identification is Cluster 1. Scores for coefficients: 1 (Willcox probability), 2 (Taxonomic Distance) , 3 (Standard Error of Taxonomic Distance).
Cluster 1 Cluster 2 Cluster 4
Characters against (none)
1
0.9999 0.825 x 10' 0.407 x 10"
2
0.271 0.473 0.534
3
-1.108 3.797 4.767
Cluster 1 % in Taxon Value in unknown
Additional characters that assist in separating
Cluster 1 from
%
[106] Translucent colony 1
[107] Lipase 1
(none)
cluster 1
from
%
Cluster 2 %
99 99
Cluster 4
%
<•* //
40
45
50
55
C. Reference Number of unknown is ML201
Value in
Percent in:
Character unknown
Best Taxon Next
Best Taxon
[23]
N-acetylglucosamine
_
1
13
[26]
Saccharose
—
[27]
Maltose
-
50
[32]
Lactate
-
1
38
[41]
Propionate
—
1
1
[43]
Valerate
—
1
13
[44]
Citrate
-
50
13
[45]
Histidine
-
1
1
[47]
Glycogen
—
1
[51]
3 -Hydroxybutyrate
—
1
13
[52]
4 -Hydroxybenzoate
—
1
1
[58]
Leucine arylamidase
+
50
88
[59]
Valine arylamidase
+
88
[64]
Phosphohydrolase
+
75
88
[65]
a-Galactosidase n.t.
75
1
[85]
Ampicillin
+
99
50
[92]
Fusidic Acid
+
99
[93]
Methicillin
+
99
50
[99]
Polymix
-
1
88
[102]
Vancomycin
+
99
13
[105]
Yellow colony
+
99
1
[106]
Translucent colony n.t.
1
1
[107]
Lipase n.t.
1
1
[108]
Oxidase (10 sec.)
—
1
n.t. = not tested
Isolate ML201 best identification is Cluster 5. Scores for coefficients: 1 (Willcox probability) , 2 (Taxonomic Distance) , 3 (Standard Error of Taxonomic Distance).
1 2 3
Cluster 5 0.9999 0.267 -0.176
Cluster 1 0.835 x 10"4 0.437 2.435
Cluster 2 0.177 x 10'11 0.641 7.593
Cluster 5 in Taxon Value in unknown
Characters against (none)
Additional characters that assist in separating
[65] a-Galactosidase
Cluster 5 %
75
Cluster 5 %
[65] a-Galactosidase 75
[1061 Translucent colony [107] Lipase
•v
27 AUG 1991*:
from from
Cluster 1 %
1
Cluster 2 %
1 99 99
40
45
50
*39297
D. Reference Number of unknown is ML203
Value in
Percent in:
Character unknown
Best Taxon Next
Best Taxon
[23]
N-acetylglucosamine
+
26
[26]
Saccharose
+
74
[27]
Maltose
+
68
60
[32]
Lactate
+
99
99
[41]
Propionate
+
91
60
[43]
Valerate
+
97
80
[44]
Citrate
+
94
[45]
Histidine
+
71
1
[47]
Glycogen
+
26
[51]
3 -Hydroxybutyrate
+
94
99
[52]
4-Hydroxybenzoate
+
71
80
[58]
Leucine arylamidase
+
94
60
[59]
Valine arylamidase
+
65
99
[64]
Phosphohydrolase
-
3
[65]
a-Galactosidase n.t.
3
[85]
Ampicillin
-
56
1
[92]
Fusidic Acid
-
3
[93]
Methicillin
-
50
1
[99]
Polymix
+
81
99
[102]
Vancomycin
+
3
[105]
Yellow colony
-
1
1
[106]
Translucent colony n.t.
3
1
[107]
Lipase n.t.
21
1
[108]
Oxidase (10 sec.)
+
6
1
n.t. = not tested
Isolate ML203 best identification is Cluster 3. Scores for coefficients: l (Willcox probability), 2 (Taxonomic Distance) , 3 (Standard Error of Taxonomic Distance).
Cluster 3 Cluster 4 Cluster 2
1
0.9989 0.1090 x 10" 0.8137 x 10'
2
0.437 0.526 0.650
3
2.076 4.590 7.793
Characters against
[102] Vancomycin [108] Oxidase (10 sec.)
Cluster 3 % in Taxon Value in unknown
3 6
Additional characters that assist in separating
Cluster 3 from %
(none)
+ +
Cluster 4
40
45
50
- 66 •
23
929
E.
Reference Number of unknown is ML206
Value in
Percent in:
Character unknown
Best Taxon
Next
Best Taxon
[23
N-acetylglucosamine
—
1
13
[26
Saccharose
+
[27"
Maltose
-
50
[32"
Lactate
-
1
38
[41
Propionate
-
1
1
[43
Valerate
—
1
13
[44
Citrate
-
50
13
[45
Histidine
-
1
1
[47
Glycogen
+
1
[51
3-Hydroxybutyrate
—
1
13
[52
4-Hydroxybenzoate
—
1
1
[58
Leucine arylamidase
+
50
88
[59
Valine arylamidase
+
88
[64"
Phosphohydrolase
+
75 '
88
[65"
a-Galactosidase n.t.
75
1
[85;
Ampicillin
+
99
50
[92"
Fusidic Acid
+
99
[93"
Methicillin
+
99
50
[99"
Polymix
-
1
88
[102'
Vancomycin
+
99
13
[105"
Yellow colony
+
99
1
[106"
Translucent colony n.t.
1
1
[107'
Lipase n.t.
1
1
[108'
Oxidase (10 sec.)
••
1
n.t. = not tested
Isolate ML206 best identification is Cluster 5. Scores for coefficients: l (Willcox probability), 2 (Taxonomic Distance) , 3 (Standard Error of Taxonomic Distance).
Cluster 5 Cluster 1 Cluster 6
1
0.9999 0.2530
x 10
0.2531 x 10
-5
•13
2
0.345 0.512 0.652
3
1.766 4.013 10.883
Characters against (none)
Cluster 5 % in Taxon Value in unknown
Additional characters that assist in separating
Cluster 5 from %
[65] a-Galactosidase 75
Cluster 1
9.
40
45
50
239297
F. Reference Number of unknown is ML3 01
Character
Value in unknown
Percent in: Best Taxon Next
Best Taxon
[23]
N-acetylglucosamine
_
26
1
[26]
Saccharose
+
74
1
[27]
Maltose
+
68
1
[32]
Lactate
+
99
50
[41]
Propionate
+
91
99
[43]
Valerate
+
97
99
[44]
Citrate
+
94
50
[45]
Histidine
+
71
1
[47]
Glycogen
+
26
13
[51]
3-Hydroxybutyrate
+
94
[52]
4-Hydroxybenzoate
—
71
1
[58]
Leucine arylamidase
+
94
63
[59]
Valine arylamidase
+
65
[64]
Phosphohydrolase
-
3
13
[65]
a-Galactosidase n.t.
3
1
[85]
Ampicillin
+
56
63
[92]
Fusidic Acid
—
3
1
[93]
Methicillin
+
50
13
[99]
Polymix
+
81
50
[102]
Vancomycin
—
3
13
[105]
Yellow colony
—
1
1
[106]
Translucent colony n.t.
3
99
[107]
Lipase n.t.
21
99
[108]
Oxidase (10 sec.)
+
6
88
n.t. = not tested
Isolate ML301 best identification is Cluster 3. Scores for coefficients: 1 (Willcox probability), 2 (Taxonomic Distance) , 3 (Standard Error of Taxonomic Distance) .
Cluster 3 Cluster 2 Cluster 4
1.000
0.2841 x 10" 0.9313 x 10"
2
0.429 0.603 0.623
3
1.918 6.731 6.704
Characters against [108] Oxidase (10 sec.)
Cluster 3 % in Taxon Value in unknown
Additional characters that assist in separating
Cluster 3 from Cluster 2
% %
[106] Translucent colony 3 99
[107] Lipase 21 99
.TEA/
•'V A
91
mlt
239297
Example 2 Production of Proteolytic Enzymes Two alkaliphilic strains (1E.1 and 9B.1) were tested for the production of proteolytic enzyme(s) in 7 different media 5 poised at an alkaline pH. The experiments were carried out in 2 liter shake flasks with a baffle, each of the flasks contained 400 ml of the nutrient media R to X (Appendix A) . The flasks were placed in an orbital incubator rotating at 280 revolutions per minute at a constant temperature of 37°C.
Samples of culture media were removed from the flasks at intervals of 1, 2, 3, 4, 5, 6 and 8 days for the determination of enzyme content which is expressed in Alkaline Delft Units (ADU - as described in British Patent Specification 1,353,317) .
Table 21 presents the maximum enzyme yields and the pH
of the cultivation medium at the moment at which the measurement of enzyme levels were made.
Table 21
Production of Proteolytic Enzymes
STRAIN
1E.1CT
STRAIN
9B.1
MEDIUM
ADU/ml pH Of
ADU/ml pH of
MEDIUM
MEDIUM
R
100
8.2
14
9.7
S
140
8.5
49
9.1
T
111
8.7
6
9.1
U
6
9.7
4
9.7
V
51
9.5
7
9.6
w
94
9.2
7
9.3
X
100
9.6
28
9.6
The results of the test clearly indicate the presence of proteolytic enzymes, produced by the alkaliphilic bacteria of the present invention, in the culture broth.
259297
Example 3
Wash. Performance Test Using Proteolytic Enzymes
Enzyme preparations from the alkaliphilic bacteria were tested in a specially developed mini-wash test using cotton 5 swatches (2.5 x 2.5 cm) soiled with milk, blood and ink (obtained from EMPA, St.Gallen, Switzerland, and designated EMPA 116). Prior to the wash test the swatches were pre-treated with a solution containing an anionic surfactant, sodium perborate and a bleach activator (TAED) at ambient 10 temperature for 15 minutes. After this treatment the test swatches were rinsed in running demineralized water for 10 minutes and air-dried. This treatment results in the fixation of the soil, making its removal more difficult.
The washing tests were performed in 100 ml Erlenmeyer 15 flasks provided with a baffle and containing 30 ml of a defined detergent composition plus 300 ADU protease to be tested. In each flask were placed two pre-treated EMPA 116 test swatches. The flasks were placed in a reciprocal shaking water bath (2 cm stroke) and agitated at 320 revolutions per 2 0 minute. The tests were carried out at 40°C for 3 0 minutes. After washing, the swatches were rinsed in running demineralized water for 10 minutes and air-dried. The reflectance of the test swatches was measured at 680 nm with a Photovolt photometer (Model 577) equipped with a green 25 filter.
The wash performance of the supernatant fraction of cultures of various alkaliphilic bacteria in European powder detergents was determined according to the method specified above. The supernatant fractions were subjected to various 30 treatments so as to produce enzyme-containing preparations.
100 ml Erlenmeyer flasks were charged with powder detergent IEC dissolved in standard tap water of 15° German Hardness so as to give a final concentration of 4 g per liter.
The composition of the powder detergent IEC was as
Component wt %
Linear sodium alkyl benzene sulphonate 6.4 (mean chain length of alkane chain (C11.5))
Ethoxylated tallow alcohol (14E0) 2.3
Sodium soap 2.8
Sodium tripolyphosphate (STPP) 35.0
Sodium silicate 6.0
Magnesium silicate 1.5
Carboxy methyl cellulose 1.0
Sodium sulphate 16.8
Sodium perborate tetrahydrate 18.5
TAED 1.5
Miscellaneous + water up to 100
Standard tap water is composed of CaCl2-2H20, 0.291 g/1; MgCl-6H20, 0.140 g/1 and NaHC03, 0.210 g/1 dissolved in demineralized water.
To each flask, two EMPA 116 swatches were added and
sufficient enzyme-containing preparations to give a final activity of 300 ADU. The final volume of the sud was 30 ml.
By way of comparison, one flask contained no enzyme preparation, which was replaced with water. The trial was repeated either two or three times. The results are shown in
Table 22.
Table 22 Application Washing Trials Performance of Proteolytic Enzvme-Containing Preparations in a Washing Formulation.
Preparation from Strain None
(control) 1E.1ct 9B.1 17N. 1°
24B.1
. CT
Untreated Culture Supernatant
Average Remission of EMPA 116 Test Swatches
Trial 1
11.4 29.2 23.7
Trial 2
11.8
22.2
23.9 11.8
17.3
Trial 3
13 .0 24.7
24.2 18.4
16.3
Preparation from Strain None
(control) IE. 1CT 9B.1 17N • 1CT 24B.1
9 ? 0 *-> n
■C. %J kJ .. / ■:
Freeze Dried Supernatant Fraction
Average Remission of EMPA 116 Test Swatches
Trial 1
.4 15.1 21.7
Trial 2
11.8
28.9 14.9
13.7
17.8
Trial 3
13.0 30.0 17.4 17.9 17.3
Preparation from Strain None
(control) 1E.1ct 9B.1 17N.1" 24B.1
, CT
Dialvzed Supernatant Fractions Average Remission of EMPA 116 Test Swatches
Trial 1
11.4 26.4 18.7
Trial 2
11.8 22.7 16.7 12.0 12.6
Trial 3
13.0
26.3 17.0 12.6
12.4
Ultrafiltration Concentrate of Supernatant Fractions
Preparation Average Remission of EMPA 116 Test Swatches from
Strain Trial 1 Trial 2 None
(control) 10.4 11.4
1E.1CT 14.6 26.0
9B.1 15.5 16.1
Acetone Precipitates of Supernatant Fractions
Preparation Average Remission of EMPA 116 Test Swatches from
Strain Trial 1 Trial 2 None
(control) 10.4 11.4
1E.1CT 13.4 23.4
9B.1 12.6 14.7
The results of the trials demonstrate the efficacy of the proteolytic enzymes produced by the strains of the present invention, provided in various forms, in detergent formulations and the improved washing performance obtained.
\27AUGi99rlj
72
2
Example 4
Production of Starch Degrading Enzvmes Strain 1E.1CT was tested for the production of starch degrading enzymes on a starch containing medium poised at an 5 alkaline pH.
500 ml Erlenmeyer flasks were charged with 100 ml of alkaline medium (Medium Y, Appendix A) containing 2% soluble starch. The flasks were inoculated (5%) with cells of strain 1E.1ct grown for 24 hours on Medium A (37"C). As controls, 10 similar flasks of alkaline medium not containing starch were also inoculated.
The flasks were placed in an orbital shaking incubator rotating at 280 revolutions per minute, at a constant temperature of 37°C for 24 hours. The fluid containing the 15 enzyme activity was separated from the cells by centrifugation for 10 minutes at 4000 r.p.m.
The enzyme activity of the supernatant was determined using the reducing sugar assay of Nelson and Somogyi (Methods in Microbiology, volume 5B, pp. 3 00-3 01; (eds. J.R. Norris 20 and D.W. Ribbons), Academic Press, London, 1971).
Determination of Starch Degrading Enzyme Activity bv the
Reducing Sugar Assay Solutions
Reagent 1
144 g Na2S04 is dissolved by gentle warming in 500 ml demineralized water. 12 g potassium sodium tartrate tetrahydrate, 24 g Na2co3 and 16 g NaHC03 are added to the solutions. The total volume of the solution is brought to 800 30 ml by the addition of demineralized water.
Reagent 2
36 g Na2S04 is dissolved by gentle warming in 100 ml demineralized water and 4 g CuS04-5H20 is added to the warmed solution. The total volume of the solution is brought to 200 35 ml by the addition of demineralized water.
Directly before use, Reagents 1 and 2 ratio of 4:1 (Reagent l : Reagent 2).
are m±j
1 Efil
2
73
Reagent 3
g ammonium molybdate tetrahydrate is dissolved in 450 ml demineralized water and 21 ml concentrated sulphuric acid is added with thorough mixing. 3 g Na2HAs04 • 7H20 are dissolved 5 in 25 ml demineralized water and this solution is added to the molybdate solution. The total solution is warmed for 48 hours at 37"C and any precipitate is filtered off.
the total volume is brought to 100 ml. Before use, the solution is diluted 10 fold with demineralized water. Substrate
0.25% soluble starch (Merck, product number 1257) dissolved in 0.1 M Na2C03-NaHC03 buffer, pH 10.1.
0.9 ml starch substrate solution, pH 10.1 is placed in a test-tube. The test-tube is placed in a water bath at 25°C and allowed to equilibrate. The enzyme reaction is started by 20 adding 0.1 ml of the enzyme-containing culture supernatant. The reaction is allowed to proceed for 30 minutes. The reaction is stopped by adding 1 ml of Reagent 1/2 and heating for 10 minutes at 100°C. The mixture is cooled on ice for 5 minutes and then 0.5 ml of Reagent 3 is added and the blue 25 color is allowed to develop during 30 minutes at room temperature. The mixture is diluted by adding 1.0 ml demineralized water and the extinction is measured at 500 nm in a spectrophotometer. The reducing sugars are measured as glucose equivalents from a standard curve.
One unit of starch degrading enzyme activity is defined as 1 fig of reducing sugars measured as glucose released per milliliter per minute at pH 10.1 and 25°C.
The number of starch degrading enzyme units formed is shown in Table 23.
Standard
100 mg glucose is dissolved in demineralized water and
Assay
Table 23
Production of Starch Degrading Enzymes bv Strain 1E.1
OPTICAL
MEDIUM
DENSITY
FINAL
ENZYME
at 550 nm pH
units per liter plus starch
2.25
9.4
1150
no starch
0.75
.3
660
The results of the test clearly indicate the presence of starch degrading enzymes, produced by the alkaliphilic bacterial strain of the present invention, in the culture broth.
Example 5
Stability of Starch Degrading Enzvmes in Detergent
The ability of the starch degrading enzymes from strain 1E.1ct to withstand detergents, which is essential for their 5 application in laundry detergents or textile desizing, is demonstrated.
100 ml Erlenmeyer flasks provided with a baffle were each charged with 30 ml of 0.1 M Na2C03/NaHC03 buffer, pH 10.1 containing 0.12 g of sodium dodecyl sulphate (equivalent to 4 10 g per liter) . To one half of the flasks 0.3 g potato starch (equivalent to 1%) was added.
Each flask was dosed with enzyme-containing supernatant from strain 1E.1CT by adding 0.5, 1.0 or 2.0 ml (see Table 23) . As a control, the supernatant fluid was replaced with 15 1.0 ml water. Immediately after adding the enzyme, a 0.1 ml sample was removed (time = zero hours) for the measurement of enzyme activity.
The flasks were incubated with shaking at 25'C for 2.5 hours at which time a second 0.1 ml sample was removed for 20 the measurement of enzyme activity.
As a comparison the experiment was repeated using a conventional a-amylase derived from Bacillus subtilis.
Enzyme activity was determined using the reducin^^^^^.
method previously described. ff
*'V <\\
ij • \
\ AUG 19$] vj
The results are recorded in Table 24.
Table 24
o
;• ; . > y' " ' ^ \ ^
? ^ u
Stability of Starch Degrading Enzymes from Strain 1E.1CT in
Detergents
ENZYME-CONTAINING
SUPERNATENT
ADDED
ENZYME UNITS RECOVERED
(ml)
CONDITIONS
PH
0 h.
2.5 h.
o *
.4
0
0
0.5
SDS
.3
26
1.0
.3
44
48
2.0
.3
109
113
0 *
.3
0
0
0.5
SDS +
.2
12
17
1.0
STARCH
.1 '
36
48
2.0
.2
79
120
Standard
§ SDS
.4
0
0
Standard
§ SDS + STARCH
.2
0
0
* replaced with 1 ml water
§ 2.8 RAU Bacillus subtilis a-amylase - One RAU (Reference 25 Amylase Unit) is defined as the quantity of enzyme that will convert 1 mg of starch per minute at pH 6.6 and 30°C into a product which upon reaction with iodine has an equal absorbance at 620 nm. as a solution containing 25 g CoC12-6H20, 3.84 g K2Cr207 and 1 ml 1 M HC1 in 100 ml distilled 30 water.
The results of this test clearly demonstrate the stability of the starch degrading enzymes, produced by the alkaliphilic bacterial strain of the present invention, in the presence of detergent.
40
Example 6 Production of Lipolytic Enzvmes Eleven of the new strains which clearly exhibited lipase activity (Appendix D) were tested further for the production of lipolytic enzymes. The eleven strains are examples from Cluster 2 and Cluster 3 (Figure l).
to The experiments were carried out in 100 ml conical
'*7*
flasks containing 30 ml sterile alkaline nutrient medium, pH 9.6, inoculated with the appropriate bacterial strain. Three different media were used, designated medium Z to BB
(Appendix A) . The flasks were placed in an orbital shaking incubator (300 rpm) at 30°C for 48 hours.
The cells were separated from the culture broth by centrifugation and the supernatant dialyzed against 50 5 volumes 0.1 mM Tris-HCl buffer pH 9, with 3 changes of buffer over 24 hours. The dialysate was freeze dried to give a lipase preparation (Table 25).
The lipase preparations obtained according to this example were used for the washing test described in Example 10 7, below.
Table 25 Production of Lipase
PRODUCTION LIPASE LIPASE
STRAIN MEDIUM TLU/ml* TLU/g
39E.3 BB 1.3 134
40E.3 Z 1.2 118
4IE. 3 Z 1.1 82
42E.3 Z 1.2 76
44E.3 Z 1.2 99
45E. 3CT AA 1.4 98
48E.3 BB 2.0 152
49N.3 BB 1.5 123
ON. 3 BB 2.0 100
IN. 3 BB 1.0 98
52N.3 BB 1.2 128
* TLU = True Lipase Unit as defined in U.S. Patent 4,933,287.
The results of this test clearly demonstrate the presence of lipolytic enzymes, produced by alkaliphilic bacteria of the present invention, in the culture broth and in a freeze-dried preparation of the dialyzed culture broth.
t %
\27AU6i99i"i
r
Example 7 Lipase Washing Test The lipase preparations from Example 6 were tested for performance under washing conditions in TIDER powder (1.5 5 g/1), a detergent product from Procter & Gamble.
The washing test (SLM-test) was carried out as described in U.S. Patent 4,933,287, which is hereby incorporated by reference. As control, a lipase derived from Pseudomonas alcaligenes strain Ml (CB3 473*85) as described in U.S. 10 Patent 4,933,287 was used. The results are shown in Table 26.
Table 26
Lipase Washing Test 15 Detergent: TIDE8 (powder), 1.5 g/1
Ligase Cci
PH
2 TLU/ml
'5 M (sodium tripolyphosphate added) 9.5
RECOVERY (%)
STRAIN
TRIGLYCERIDES
TOTAL
39E.3
55.3
73.4
40E.3
82.0
89.9
4IE. 3
44.7
78.7
42E.3
55.5
74.8
44E.3
6.6
76.9
45E. 3CT
81.6
92.9
48E.3
76.5
82.9
49N.3.
72.4
82.0
50N.3
50.5
76.6
51N.3
80.4
87.6
52N.2
77.0
84.7
Ml
88.5
91.5
control*
98.7
98.7
40
* Standard tap water as defined in U.S. Patent 4,933,287
The decrease in the percent recovery of triglycerides and total lipids, as compared to the control, clearly indicate the ability of the lipolytic enzymes, produced by the alkaliphilic bacteria of the present invention, to break 45 down and remove triglycerides and their degradation products embedded on a fabric sample, as well as thei^r improved performance as compared to a known lipase.
CT 7 AUG 1991
78
Appendix A Media Used in the Present Invention
23929 7
MEDIUM A
Glucose 10.0 gl"1
Peptone (Difco: Detroit, MI, USA) 5.0 gl*1
Yeast Extract (Difco) 5.0 gl*1
K2HP04 1.0 gl*1
MgS04-7H20 0.2 gl*1
NaCl 40.0 gl*1
Na2C03 10.0 gl*1
* Agar 20.0 gl"1
* (when required for a solid medium)
medium b
Glucose
.0
gl"
Peptone (Difco)
.0
gi"
Yeast Extract (Difco)
.0
gi"
k2hpo4
1.0
gi
MgS04 • 7H20
0.2
gi*
NaCl
40.0
gi
Na2C03 Novobiocin
.0
gi'
50.0
mgl
Agar
.0
gl"
medium c
Glucose 10.0 gl"1
Peptone (Difco) 5.0 gl"1
Yeast Extract (Difco) 5.0 gl"1
K2HP04 1.0 gl*1
MgS04-7H20 0.2 gl*1
NaCl 40.0 gl*1
Na2C03 10.0 gl*1
Lactalbumin 10.0 gl*1
Agar 20.0 gl*1
medium d
Glucose 10.0 gl"1
Peptone (Difco) 5.0 gl"1
Yeast Extract (Difco) 5.0 gl"1
K2HP04 1.0 gl"1
MgS04-7H20 0.2 gl"1
NaCl 40.0 gl*1
Na2C03 10.0 gl*|
Casein 20.0 gl
Agar 20.0 gl*1
MEDIUM E
79
239297
Soluble Starch
.0
91"
Peptone (Difco)
.0
gi*
Yeast Extract (Difco)
.0
gi*
KjHPO^
1.0
gi*
MgS04 • 7H20
0.2
gi*
NaCl
40.0
gi"
Na2C03
.0
gi*
Lactalbumin
.0
gi"
Agar
.0
gi*1
MEDIUM F
Soluble Starch
.0
g!"
peptone (Difco)
.0
gi,
Yeast Extract (Difco)
.0
gi'
k2hpo4
1.0
gi'
MgS04 • 7H20
0.2
gi'
NaCl
40.0
gi"
Na2C03 Casein
.0
gi*
.0
gi"
Agar
.0
gi"1
MEDIUM G
Oxbile (Oxoid: Basingstoke, U.K.)
.0
gi'
(NH4)2S04
.0
gl"
MgS04 • 7H20
0.2
gi'
Yeast Extract (Difco)
0.5
gi'
Lactalbumin
.0
gi"
Agar
.0
gi"
Adjusted to pH 8.5 with 50% Na2C03
solution
MEDIUM H
Oxbile (Oxoid)
.0
gi"
(NH4)2S04
.0
MgS04-7H20
0.2
g!"
Yeast Extract (Difco)
0.5
gl"
Casein
.0
gi*
Agar
.0
gi*1
Adjusted to pH 8.5 with 50% Na2C03
solution
MEDIUM I
Sabouroud Dextrose Agar (Oxoid) 65.0 gl"
NaCl 40.0 gl'
Na2C03 2 0 . 0 gl"
Cycloheximide 50.0 mgl"
Penicillin G 25000 IU1"
Streptomycin 25 mgl"
(,*-7 SEP (383
80
MEDIUM J
239297
Bacto Potato Dextrose Agar (Difco) 39.0 gl"1
NaCl 40.0 gl"1
Na2C03 20.0 gl'1
Novobiocin 50.0 mgl*1
MEDIUM K
Bacto Potato Dextrose Agar (Difco)
39.0
gi"
NaCl
40.0
gl'
Na2C03
.0
gi'
Cycloheximide
50.0
mgl
Penicillin G
25000
IU1"
Streptomycin
.0
mgl*
MEDIUM L
Glucose 0.2 gl*1
Peptone (Difco) 0.1 gl"1
Yeast Extract (Difco) 0.1 gl"1
K^HP04 1.0 gl*1
MgS04 • 7H20 0.2 gl"1
NaCl 40.0 gl'1
Na2C03 10.0 gl*1
Casein 20.0 gl*1
Agar 20.0 gl"1
MEDIUM M (pH 9.6)
Oxbile (Oxoid) 2.0 gl'1
(NH4)2S04 1.0 gl*1
MgS04-7l^0 0.04 gl"
Yeast Extract (Difco) 0.1 gl"
Olive Oil 10.0 ml l*1
Na2C03 6.1 gl"1
Agar 20.0 gl'1
MEDIUM N (pH 9.6)
Oxbile (Oxoid) 2.0 gl"1
(NH4)2S04 1.0 gl"1
MgSOA-7HzO 0.04 gl*1
Yeast Extract (Difco) 0.1 gl'1
Olive Oil 10.0 ml l'1
Na2C03 6.1 gl"1
Tergitol 7 (Fluka: Buchs. CH) 500 ppm
Agar 20.0 gl*1
MEDIUM O (pH 9.6)
Oxbile (Oxoid)
(NH4)2S04 MgS04 • 7H20
Yeast Extract (Difco)
Olive Oil Na2C03
NaCl & <• r-,
Agar / « *
X
v *\\
OiJ
-7 SEP 1993 j
2.0
gi"
1.0
gi*
0.04
gi*
0.1
gi
.0
ml 1
6.1
gl'
40.0
gi"
.0
gi
81
239297
MEDIUM P (pH 9.6)
Oxbile (Oxoid) 10.0 gl"1
(NH4)2S04 5.0 gl"1
MgS04-7H20 0.2 gl*1
Yeast Extract (Difco) 0.5 gl"1
Olive Oil 10.0 ml l"1
NaCl 40.0 gl'1
Agar 20.0 gl"1
Adjusted to pH 9.6 with 50% Na2C03 solution
MEDIUM O (pH 9.6)
Oxbile (Oxoid) 10.0 gl*1
(NH4)2S04 5.0 gl*1
MgS04-7H20 0.2 gl'1
Yeast Extract (Difco) 0.5 gl*1
Olive oil 10.0 ml l*1
Agar 20.0 gl*1
Adjusted to pH 9.6 with 50% Na2C03 solution
MEDIUM R (pH 9.5)
Fresh Yeast 82.5 gl*1
Glucose 3.3 gl"1
K2HP04 1.6 gl"1
I^COj 0.6 gl"1
KHC03 1.76 gl"1
CaCl2 0.05 gl"1
MgS04-7H20 0.05 gl'1
FeS04 0 . 0 05 gl*1
MnS04 0.0066gl*1
MEDIUM S
Fresh Yeast
8.25 gl"1 1.32 gl"1 1.6 gl"1 0.05 gl"1 0.05 gl'1 0.005 gl"1
Glucose
K2HP04
caci2
MgS04 • 7HpO
FeS04
MnS04
o.ooeegi*1 40.0 gl*1
NaCl
Adjusted to pH 10.5 with 40% Na2C03
solution
MEDIUM T (pH 10.1)
Glucose 10.0 gl"1
Peptone (Difco) 5.0 gl"1
Yeast Extract (Difco) 5.0 gl"1
K2HP04 1.0 gl"1
MgS04-7H20 0.2 gl"1
NaCl 40.0 gl*1
Na2C03 10.0 gl"1
/< °-//V
!
~7sEF!??3
82
MEDIUM U
Oxbile 10.0 gl*1
(NH4)2S04 5.0 gl*1
MgS04 • 7H20 0.2 gl*1
Yeast extract (Difco) 0.5 gl*1
Casein 10.0 gl*1
Adjusted to pH 9.8 with 40% Na2C03 solution
MEDIUM V
Tryptone Soya Broth (Oxoid) 30.0 gl*1
Adjusted to pH 9.9 with 40% Na?CO, solution
MEDIUM W (pH 10.1)
Soluble starch
.0
gl'
Peptone (Difco)
.0
gi"
Yeast extract (Difco)
.0
gi*
KH2P04 • ■
1.0
'gi*.
MgS04 • 7H20
0.2
gi*.
NaCl
40.0
gi*.
Na2C03
.0
gi*
MEDIUM X
MEDIUM Y
Skim milk (Difco) 100.0 gl*1
Adjusted to pH 10.8 with 25% Na2C03 solution
Yeast Extract (Difco) 1.0 g
KN03 10.0 g
KH2P04 1.0 g
MgS04 • 7H20 0.2 g
Na2C03 10.0 g
NaCl 40.0 g
Soluble starch (Merck) 20.0 g
Demineralised water 1 liter
MEDIUM Z
Brain Heart Infusion (Difco) 20.0 g Na2EDTA (Komplexion III, Siegfried AG,
Switzerland) 1.0 g
FeS04-7H20 0.006 g
MnS04-H20 0.003 g
CaCl2 • 2H20 1.0 g
MgS04-7H20 0.25 g
Tween 80 5.0 g
Soya oil 5.0 g
Distilled water to 1 liter, pH of medium adjusted to pH 9.6 with 25% Na2C03 solution.
,-v'v th O
, / % c tf \
'•v
\\ -7 SEP 1993 "/
P | Mf
I
83
239297
MEDIUM AA
Yeast Extract (Difco) 20.0 g
KH2P04 5.0 g
FeS04-7H20 0.006 g
MnS04-H20 0.003 g
CaCl2 • 2H20 i.o g
MgS04-7H20 0.25 g
Tween 80 5.0 g
Soya oil 5.0 g Distilled water to 1 liter, pH of medium adjusted to pH 9.6 with 25% Na2C03 solution.
medium bb
Brain Heart FeSO,
Infusion (Difco)
■7h20
•h2o
■2h20
MnS04 CaCl2
MgS04-7H20 Tween 80 Soya oil
Distilled water to 9.6 with 25% Na2C03
.0 0.006 0.003 1.0 0.25 5.0 5.0
g g g g g g g
1 liter, pH of medium adjusted to pH solution.
k
\ " 7SEPI993
I
•I
I
23929
84
Appendix B
Methods for Unit Tests
1. Character numbers 1 to 5
Colony color, form, elevation, margin, size
A suspension of bacteria was spread over an alkaline nutrient agar (Medium A) and cultivated at 37"C.
Colonies were examined after 48 hours.
2. Character number 6 and 7
Cell morphology. Gram's strain reaction
Bacteria cells grown in alkaline nutrient broth (Medium A, without agar) for 24 hours were spun down in a centrifuge and resuspended in a small amount of alkaline nutrient broth and allowed to air-dry on a microscope slide. Or, bacteria were cultivated for 24 - 48 hours on an alkaline nutrient agar (Medium A) so as to form colonies. Colonies of bacteria were suspended in physiological saline and a few drops allowed to air-dry on a microscope slide. The Gram's staining test was performed using the Dussault modification (Journal of Bacteriology, 70, 484-485, 1955) with safranin as counterstain.
3. Character number 8 Oxidase reaction
Filter paper moistened with a 1% aqueous solution of N,N,N1,N1-tetramethyl-p-phenylenediamine or oxidase identification discs (bioMerieux: Charbonieres-les-Bains, France) were smeared with a young bacterial culture from alkaline nutrient agar. A purple color within 1 minute was recorded as a positive reaction. E. coli. used as a control, did not give a positive reaction within one minute.
4. Character number 9 Skim milk test
A minimal medium composed (g/1 distilled water) of yeast extract, 1.0; KN03, 10.0; K2HP04, 1.0;MgS04-7H20, 0.2; NaCl, 40.0; Na2C03, 10.0; agar, 20.0 was supplemented with 5.0 g/1 skim milk powder, sterilised by autoclaving and poured into Petri dishes. Bacteria were inoculated and incubated at 37'C. Areas of clearing around bacterial colonies in an otherwise opaque agar were recorded as a positive reaction. Non-alkaliphilic reference strains were tested in an identical fashion using media of the same composition but without Na2C03 so as to give a pH of 6.8-7.0.
. Character number 10 Gelatin hvdrolvsis
Charcoal-gelatin discs (bioMerieux) or "chargels" (Oxoid) were incubated at 37 "C in an alkaline nutrient broth (Medium A) together with bacteria. A black sediment indicated a positive reaction.
? p • ••
85
239
Appendix B (continued)
Character number 11
NaCl tolerance
Two methods were applied.
(a) Bacterial strains were cultivated at 37°C on an alkaline nutrient agar (Medium A) containing 0%, 4%, 8%, 12% or 15% (w/v) NaCl. The agar plates were examined for bacterial growth after 48 hours.
(b) Bacterial strains were cultivated at 37'C in an alkaline nutrient broth (Medium A) containing 0%, 4%, 8%, 12%, 15% or 25% NaCl. Bacterial growth was monitored by optical density measurements using a Klett meter (green filter) at 0, 12, 24, 48, 72 and 144 hours.
Character number 12 Minimum pH for growth
Nutrient agar, pH 6.8-7.0 (Medium A without sodium carbonate) was poured into square Petri dishes. A strip of solidified agar was removed from one end and replaced with molten 4% (w/v) agar containing 3.6 % (w/v) Na2C03 and 0.8 % (w/v) NaOH. A pH gradient from pH 10.5 to pH 7 across the plate was allowed to develop overnight. Bacteria were inoculated by streaking along the pH gradient and cultivated at 37°C for 48 hours. The pH at the point where bacterial growth ceased was measured with a flat head electrode and with "Alkalite" pH strips (Merck: Darmstadt, W. Germany).
Character numbers 13 - 21 Carbohydrate utilisation
A minimal medium composed (g/1 distilled water) of yeast extract, 1.0; KN03, 10.0; KgHPC^, 1.0; MgS04-7H20, 0.2; NaCl, 40.0; Na2C03, 10.0; agar, 20.0 was supplemented with 2.0 g/1 of the carbohydrate under test and poured into square Petri dishes. Bacteria were inoculated, using a 25 point multipoint inoculator, from 1.0 ml of a bacterial suspension cultivated for 48 hours in an alkaline nutrient broth (Medium A) . The agar-plates were incubated at 37'C for 48 hours. The results were recording by comparing bacterial growth on minimal nutrient medium containing a carbohydrate supplement with growth on a minimal medium without the carbohydrate under test.
Non-alkaliphilic reference strains were tested in an identical fashion using media of the same composition but without Na2C03 so as to give a pH of 6.8 - 7.0.
Character numbers 22 -53 Growth on carbon substrates
Use was made of the commercially available test strip ATB 32 GN (API-bioMerieux: La Balme les Grottes, France) . The strips were used according to the manufacturer's instructions but with an addition of 1.0 ml of a solution containing 4% NaCl and 1% Na2C03 to the vials of basal medium provided. The strips were incubated at 37°C for 48 hours. Non-alkaliphilic reference strains were incubated in. the standard basal medium.
I, -7SEhf3:
86
239297
W9B
Appendix B (continued)
. Character numbers 54 -72 Enzvmatic activities
Use was made of the commercially available test strip APIZYM (API-bioMerieux) which was used according to the manufacturer's instructions, except that the alkaliphilic bacterial cells were suspended in alkaline nutrient broth (Medium A). The strips were incubated at 37"C for 4 hours.
11. Character numbers 73 - 82
Amino acids as carbon and nitrogen source
The same technique was employed as for tests 14-21 except that KNOj was omitted from the minimal nutrient medium.
12. Character numbers 83 - 104 Antibiotic sensitivity
A light suspension of bacteria in alkaline nutrient broth was spread. . on the surface of alkaline nutrient agar (Medium A) and allowed to dry. Commercially available antibiotic susceptibility test discs (Oxoid or Mast Laboratories: Merseyside, U.K.) were applied to the agar surface. The bacteria were cultivated at 37*c for 48 hours. Clear zones around the antibiotic disks indicated sensitivity.
87
Appendix C
Unit Tests for Analysis bv Numerical Taxonomy
CHARACTER NUMBER
TEST
DESCRIPTION
COMPUTER CODE
2392'
6
8
11
Colony color
Colony form
Colony elevation
Colony margin
Colony size Cell morphology
Gram's stain
Oxidase test
Skim milk test
Gelatin hydrolysis
NaCl tolerance white = l cream = 2
beige = 3
yellow = 4
orange = 5
pink = 6
brown = 7
red - 8
circular = 1
irregular = 2
punctiform = 3
filamentous = 4
convex raised umbonate flat entire undulate lobate fimbriate
= l
= 2
= 3
= 4
= 1
= 2
= 3
= 4
diameter in millimeters rod coccus negative positive negative positive negative positive negative positive
= 1
= 2
= 1
= 2
= 1
= 2
= 1
= 2
= 1
= 2
growth at 0% - 4% = growth at 0% - 8% = growth at 0% - 12% = growth at 0% - 15% = growth only at 0% = growth only at 4%-15%
1
2
3
4
= 6
/A
•'v
\ ~ 7 SEP 1993
13
14
16
17
18
19
21
I _
22
23
24
26
27
28
29
31
32
33
34
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
Appendix C (continued)
TEST
DESCRIPTION
COMPUTER CODE
Minimum pH for growth on nutrient agar pH 7.5 = 7.5
pH 8.0 = 8.0
pH 8.5 = 8.5
pH 9.0 = 9.0
pH 9.5 = 9.5
pH 10.0 = 10.0
pH 10.5 = 10.5
Carbohydrate utilisation
Formate
Fumarate
Succinate
Galactose
Pyruvate
Fructose enhanced growth = 2 equal growth = l gr.owth inhibited = 0
Lactose
Xylose
Starch
Growth on carbon substrates Rhamnose
N-acetylglucosamine
D-ribose
Inositol
D-saccharose
Maltose
Itaconate
Suberate
Malonate
Acetate
DL-lactate positive = 2
L-alanine negative = 1
Mannitol
D-glucose
Salicin
D-melibiose
L-fucose
D-sorbitol
L-arabinose
Propionate
Caprate
Valerate
Citrate
Histidine
-ketogluconate
Glycogen
3-hydroxybenz oate L-serine
2-ketogluconate
3-hydroxybutyrate
4-hydroxybenz oate L-proline r-7 SEP 1993 i
V
Appendix C (continued)
CHARACTER NUMBER
TEST
DESCRIPTION
COMPUTER CODE
54 - 72
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73 - 82
73
74
75
76
77
78
79
80
81
82
83 - 104
Enzymatic activities Alkaline phosphatase Esterase (C4)
Esterase lipase (C8)
Lipase (C14)
Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin Chymotrypsin Acid phosphatase Naphthol-AS-BI-phosphohydrolase a-galactosidase fi-galactosidase fl-glucuronidase a-glucosidase B-glucosidase N-acetyl-B-glucosaminidase a-mannos idase a-fucosidase
Amino acids as carbon and nitrogen source Serine Proline Asparagine Arginine Alanine Lysine Methionine Phenylalanine Glycine Valine
Antibiotic sensitivity positive = 2 negative = 1
enhanced growth= 2 equal growth = 1 no growth = 0
83
Gentamycin
m g
84
N itrofuranto in
50
Hg
85
Ampicillin
Mg
86
Nalidixic Acid
Mg
87
Sulphamethoxazole 50
Mg
88
Trimethoprim
2-5
fig
89
Penicillin G
1
IU
90
Chloramphenicol
Mg
91
Erythromycin
m g
92
Fusidic Acid
Mg
93
Methicillin
Mg
94
Novobiocin
Mg
95
Streptomycin
m g
96
Tetracycline
Mg
97
Sulphafurazole
100
m g
98
Oleandomycin
Mg
99
Polymixin
300
IU
100
Rifampicin
2
Mg
101
Neomycin
m g antibiotic sensitive inhibition of growth= 2
antibiotic insensitive, no growth inhibitions 1
" 7 SEP J993
90
Appendix C (continued)
23929
CHARACTER TEST COMPUTER
NUMBER DESCRIPTION CODE
102 Vancomycin 3 0 /xg
103 Kanamycin 3 0 /zg
104 Bacitracin 10 IU
/* CA
IN -*\
'z o]
"7 SEP J993
91
23929
Appendix D
Screening for Proteolytic. Amvlolvtic and Lipolytic Activity
Proteolytic Activity Cluster 1
STRAIN LACTALBUMIN CASEIN BLOOD GELATIN
1E.1ct
+
+
+
+
2E.1
-
-
-
-
wB2
-
-
-
+
wB5
-
-
-
-
wBs4
-
+
+
+
10B.1
+
+
+
+
20N.1
+
+
-
+
27M.1
-
+
-
—
wNk2
—
+
-
+
Cluster 2
STRAIN
LACTALBUMIN
CASEIN
BLOOD
GELATIN
39E.3
n.t.
n.t.
n.t.
+
4IE. 3
n.t.
n.t.
n.t.
+
45E. 3CT
n.t.
n.t.
n.t.
+
47E.3
n.t.
n.t.
n.t.
+
5IN. 3
n.t.
n.t.
n.t.
+
52N.3
n.t.
n.t.
n.t.
+
42E.3
n.t.
n.t.
n.t.
+
50N.3
n.t.
n.t.
n.t.
+
Cluster 3
STRAIN
LACTALBUMIN
CASEIN
BLOOD
GELATIN
6B.1
+
+
-
+
7B.1
+
+
-
-
8B.1
-
+
-
+
38E.2
n.t.
n.t.
n.t.
-
56E.4
+
n.t.
n.t.
-
25B.1
n.t."
+
n.t.
+
26N.1
-
+
-
+
11C.1
+
+
-
+
WB.I
-
-
-
+
12C.1
+
—
—
2 8N. 1CT
-
—
—
-
6IN. 4
+
n.t.
n.t.
-
36E.2
n.t.
n.t.
n.t.
-
40E.3
n.t.
n.t.
n.t.
+
65B.4
+
n.t.
n.t.
-
94LM.4
n.t.
n.t.
n.t.
+
19N.1
-
+
-
+
24B.1
+
+
-
+
21M.1
+
+
+
+
/n
I'N
" 7 SEf f
239297
Proteolytic Activity (continued)
Cluster 3
STRAIN LACTALBUMIN CASEIN BLOOD GELATIN
29C.1
_
35E.2
n.t.
n.t. •
n.t.
—
37E.2
n.t.
n.t.
n.t.
-
48E.3
n.t.
n.t.
n.t.
+
78LN.4
n.t.
+
n.t.
+
73aC.4
n.t.
+
n.t.
+
75C.4
n.t.
+
n.t.
+
73bC.4
n.t.
+
n.t.
+
74C.4
n.t.
+
n.t.
— ■
77LN.4
n.t.
+
n.t.
-
WNl
-
-
—
-
49N.3
n.t.
n.t.
n.t.
+
44E.3
n.t.
n.t.
n.t.
+
58E.4
n.t.
n.t.
n.t.
57E.4
+
n.t.
Cluster 4
n.t.
+
STRAIN
LACTALBUMIN
CASEIN
BLOOD
GELATIN
WE5 WB4CT
-
-
-
+
WNkl
—
-
—
+
wEll
—
-
—
—
WE12
Cluster 5
+
STRAIN
LACTALBUMIN
CASEIN
BLOOD
GELATIN
9B.1
—
+
+
16N.1 17N.1CT
+
n.t.
n.t.
+
+
+
—
+
22M.1
+
+
Cluster 6
+
STRAIN
LACTALBUMIN
CASEIN
BLOOD
GELATIN
18N.1
+
+
+
59E.4 64B.4CT
+ +
n.t. n.t.
n.t. n.t.
+ +
63N.4
+
n.t.
n.t.
+
53E.4
+
n.t.
n.t.
+
\"7 SEP 1993
STRAIN
93
Proteolytic Activity (continued) Non-Clusterina Strains LACTALBUMIN CASEIN BLOOD
239297
GELATIN
wN.2
4E.1
5E.1
92LM.4
wBn5
n.t. = not tested n.t.
n.t.
n.t.
+ +
Amvlolytic and Lipolytic Activity*
Cluster 1
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
1E.1ct
+
+
+
2E.1
+
-
+
—
WB2
-
-
+
—
WB5
-
-
+
—
wBs4
-
-
+
—
10B.1
+
-
+
—
2 ON. 1
-
-
+
—
27M.1
-
-
+
, —
WN1C2
+
—
+
—
Cluster 2
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
39E.3
+
+
+
41E.3
+
+
+
—
45E.3CT
+
+
+
+
47E.3
+
+
-
—
51E.3
+
+
+
+
52E.3
+
+
+
+
42E.3
+
+
+
+
5ON. 3
+
+
+
+
Cluster 3
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
6B.1 7B.1 8B.1 38E.2
+ +
+ + + +
+ +
/« °
V
7 SEP? 993
239297
Amvlolytic and Lipolytic Activity' (continued)
Cluster 3
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
56E.4
—
+
+
+
25B.1
-
-
+
+
26N.1
-
-
+
-
11C.1
-
-
+
-
wBl
-
-
+
-
12C.1
+
-
-
-
28N. 1CT
+
-
+
-
6 IN. 4
+
-
-
-
36E.2
-
-
-
-
40E.3
+
+
+
+
65B.4
+
-
+
-
94LM.4
+
-
+
-
19N.1
+
-
+
+
24B.1
-
-
+
-
21M.1
+
-
-
-
29C.1
+
-
+
+
35E.2
-
-
+
-
37E.2
-
-
+
1 -
48E.3
-
+
+
' -
78LN.4
-
-
•f
1 —
73aC.4
-
-
+
75C.4
+
-
-
—
73bC.4
+
-
+
-
74C.4
+
-
+
-
77LN.4
-
-
.+
+
WNl
+
-
+
—
49N.3
+
+
+
-
44E.3
+
+
+
+
58E.4
+
+
+
-
57E.4
+
—
+
—
Cluster 4
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
WE5
+
_
—
—
WB4cr
-
-
+
-
WNkl
+
-
+
-
wEll
-
-
+
-
WEI 2
-
-
+
-
tu
!z
K-<
- 7 SEP 1993
95
23929
Amvlolytic and Lipolytic Activity* (continued)
Cluster 5
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
9B.1
+
—
+
_
16N.1
+
-
+
—
17N. 1CT
+
-
+
—
22M.1
+
-
+
—
Cluster 6
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
18N.1
+
—
+
_
59E.4
+
-
+
-
64B.4CT
+
-
+
-
63N.4
+
-
-
-
53E.4
+
—
+
' —
Non-Clusterina Strains
LIPASE
ESTERASE
STARCH
ACTIVITY
LIPASE
LIPASE
STRAIN
HYDROLYSIS
ON OLIVE OIL
ACTIVITY
ACTIVITY
wN2
+
—
+
+
4E.1
-
—
+
-
5E.1
+
—
+
-
92LM.4
+
+
+
-
wBn5
-
—
+
-
* Starch Hydrolysis determined according to Character 21 (Appendix B)
Lipase Activity (Olive Oil) determined on media M - P (Appendix A)
Esterase Lipase Activity determined according to character 56 (Appendix B)
Lipase Activity determined according to Character 57 (Appendix B)
^ E ' 0 .
//'V
V
-7 SEP 1993
96. 239 29 Jr-7SEFt^r>
Appendix E
Percentage Positive States for Characters tin Clusters
CLUSTER
CHARACTER
1
2
3
4
6
[6]
Cell morphology Gram's stain
9
0
12
0
0
0
[7]
0
0
0
0
0
0
[8]
Oxidase test
55
100
80
0
[9]
Skim milk test •
36
11
6
0
[10]
Gelatin
67
100
56
60
100
100
[131
Formate
0
0
40
0
0
[14]
Fumarate
45
78
74
80
75
60
[15]
Succinate
45
89
85
40
67
50
[16]
Galactose
45
11
12
40
0
[17]
Pyruvate
64
89
88
40
75
0
[18]
Fructose
45
11
68
60
50
100
[19]
Lactose
9
0
0
0
0
0
[20]
Xylose
18
11
0
0
[21]
Starch
73
100
91
100
100
[22]
Rhamnose
9
0
0
0
[23]
N-acetylglucosamine
9
0
26
0
100
[24]
D-ribose
27
0
50
0
0
[25]
Inositol
9
0
9
0
50
0
[26]
D-saccharose
27
0
74
100
[27]
Maltose
9
11
74
40
50
100
[28]
Itaconate
0
11
6
0
0
0
[29]
Suberate
9
67
53
40
0
0
[30]
Malonate
0
11
65
0
40
[31]
Acetate
18
100
100
80
100
[32]
DL-lactate
27
56
100
100
0
40
[33]
L-alanine
18
89
82
100
0
100
[34]
Mannitol
0
0
41
0
80
[35]
D-glucose
9
11
71
60
0
60
[36]
Salicin
0
0
3
0
60
[37]
D-melibiose
0
0
3
0
0
0
[38]
L-fucose
0
0
0
0
[39]
D-sorbitol
18
0
41
0
0
40
[40]
L-arabinose
9
0
3
40
0
0
[41]
Propionate
9
100
94
80
0
80
[42]
Caprate
0
78
32
40
0
80
[43]
Valerate
9
100
97
80
0
40
[44]
Citrate
9
56
94
50
100
[45]
Histidine
0
0
71
0
0
80
[46]
-ketogluconate
0
0
21
0
0
0
[47]
Glycogen
9
22
26
100
[48]
3-hydroxybenzoate
9
0
38
0
0
0
[49]
L-serine
0
0
68
60
0
0
[50]
2-ketogluconate
0
0
56
80
[51]
3-hydroxybutyrate
18
33
94
100
0
100
[52]
4-hydroxybenzoate
0
0
71
80
0
0
[53]
L-proline
45
100
100
80
80
[54]
Alkaline phosphatase
100
44
94
40
100
60
[55]
Esterase (C4)
100 ■■
100
100
100
100
100
[56]
Esterase lipase (C8)
100
89
85
100
100
100
97
9Q7
p. pi.
Appendix E (continued^
Percentage Positive States for Characters in Clusters
CHARACTER
CLUSTER
1
2
3
4
6
9
67
26
0
0
0
91
67
94
60
50
0
91
33
65
100
0
73
0
0
0
0
64
11
9
60
0
0
73
0
3
75
0
91
11
94
100
100
60
91
22
3
100
40
0
11
3
100
0
9
0
0
100
0
9
0
3
0
27
0
79
60
100
40
9
0
9
80
75
0
27
0
0
0
0
0
0
0
0
0
0
0
9
0
0
0
0
22
13
29
60
100
50
56
63
65
80
100
100
67
38
61
60
75
100
56
53
80
50
50
67
100
62
40
100
50
44
75
71
80
75
100
60
50
53
nc
50
50
89
100
76
100
100
100
44
13
29
80
50
50
44
50
41
0
50
36
67
3
0
0
18
33
21
0
0
0
45
67
56
0
100
80.
36
78
76
0
0
18
33
38
0
0
0
45
22
0
75
60
27
11
29
0
75
100
100
100
100
100
100
100
91
100
100
100
100
100
18
0
3
100
100
45
11
50
0
100
100
18
0
3
0
0
100
78
97
100
75
100
45
22
3
100
100
40
13
nc
0
0
100
38
94
100
100
100
89
63
78
100
0
0
100
38
78
100
100
100
0
13
0
0
0
0
22
13
0
100
75
11
13
0
0
0
0
33
13
0
100
100
^TE N r o'X
;57] Lipase (C14)
;58] Leucine arylamidase
59] Valine arylamidase
;60] Cystine arylamidase
;61] Trypsin
'62] Chymotrypsin
63] Acid phosphatase
64] Naphthol phosphohydrolase ;65] a-galactosidase
\66] B-galactosidase
;67] B-glucuronidase
;68] a-glucosidase
;69] B-glucosidase
] N-acetyl-B-glucosaminidase
71] a-mannosidase
72] a-fucosidase
73] Serine
74] Proline
75] Asparagine
76] Arginine
77] Alanine
78] Lysine
79] Methionine
8 0] Phenylalanine
81] Glycine
82] Valine
83] Gentamycin
84] Nitrofurantoin
85] Ampicillin
86] Nalidixic Acid
87] Sulphamethoxazole
88] Trimethoprim
89] Penicillin G
90] Chloramphenicol
91] Erythromycin
92] Fusidic Acid
93] Methicillin
94] Novobiocin
95] Streptomycin
96] Tetracycline
97] Sulphafurazole
98] Oleandomycin
99] Polymixin
100] Rifampicin
101] Neomycin
102] Vancomycin
103] Kanamycin
104] Bacitracin v
"4
n'.
- 7 SEPJ993
239297
Claims (33)
1. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of 5 aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: a) forms cream-colored, circular colonies; b) grows optimally between pH 9 and pH 10; c) gives a positive response to the following tests: 10 1) Leucine arylamidase 2) Valine arylamidase 3) Pho sphohydro1ase 4) Polymixin; d) gives a negative response to the following tests: 15 1) N-acetylglucosamine 2) Maltose 3) Propionate 4) Caprate 5) Valerate 20 6) Citrate 7) Histidine 8) Glycogen 9) 4-hydroxybenzoate 10) a-galactosidase. 25
2. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: 30 a) forms small, cream-colored colonies; b) grows optimally between pH 7.8 and pH 11.2; c) gives a positive response to the following tests: 35 1) Starch 2) Acetate 3) Propionate 4) Valerate 5) Proline fa* °A 239297 - 99 - 6) Lipase 7) Oxidase (response within 10 seconds); d) gives a negative response to the following tests: 1) N-acetylglucosamine 5 2) Saccharose 3) Histidine 4) 2-ketogluconate 5) 4-hydroxybenzoate 6) a-glucosidase 10 7) B-glucosidase 8) Fusidic Acid.
3. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of 15 aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: a) forms cream-colored, opaque colonies; b) grows optimally between pH 8.5 and pH 10.7; c) contains ubiquinone 6 as a major respiratory 20 quinone; d) gives a positive response to the following tests: 1) Acetate 2) Lactate 3) Propionate 25 4) Valerate 5) Citrate 6) 3-hydroxybenzoate 7) Proline 8) Leucine arylamidase; 30 e) gives a negative response to the following tests: 1) Phosphohydrolase 2) a-galactosidase 3) Fusidic Acid 4) Tetracyclinee 35 5) Vancomycin ^ ^ 6) Bacitracin. z ' * •\ ~ 7 SEP S993 ^ f r i ■>' 239297 -100-
4. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: 5 a) forms beige to brown-colored, opaque colonies; b) grows optimally between pH 7.5 and pH 10.9; c) contains ubiquinone 9 as a major respiratory quinone; d) gives a positive response to the following tests: 10 1) Lactate 2) Alanine 3) 3 -hydroxybutyrat e 4) Valine arylamidase 5) Polymixin; 15 e) gives a negative response to the following tests: 1) Histidine 2) Ampicillin 3) Naladixic acid 4) Trimethoprim 20 5) Penicillin G 6) Methicillin.
5. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of 25 aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: a) forms bright yellow-colored colonies; b) grows optimally between pH 8 and pH 10.5; c) gives a positive response to the following tests: 30 1) Phosphohydrolase 2) a-galactosidase 3) B-galactosidase 4) Ampicillin 5) Fusidic Acid 35 6) Methicillin / 7) Tetracycl inee 't V 8) Vancomycin jl \ " 7 SEP 1993 239297 - 101 - 9) Bacitracin; d) gives a negative response to the following tests: 10 1) N-acetylglucosamine 2) Lactate 3) L-alanine 4) Mannitol 5) Propionate 6) Caprate 7) Valerate 8) Histidine 9) 3-hydroxybenzoate 10) 3-hydroxybutyrate 11) 4-hydroxybenzoate 12) Polymixin. 15
6. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: 20 a) forms cream to beige-colored, irregular, flat colonies; b) grows optimally between pH 8.2 and pH 10.9; c) gives a positive response to the following tests: I) Starch 25 2) N-acetylglucosamine 3) .Saccharose 4) Maltose 5) Acetate 6) Alanine 30 7) Citrate 8) Glycogen 9) 3-hydroxybutyrate 10) Penicillin G II) Fusidic Acid 35 12) Methicillin 13) Tetracyclinee 14) Bacitracin; ~ 7 SEP1993 35 - 102 - 239297 d) gives a negative response to the following tests: 1) Pyruvate 2) 4-hydroxybenzoate 3) Leucine arylamidase 5 4) Valine arylamidase 5) a-galactosidase 6) Polymixin.
7. A pure bacterial culture useful for production of 10 alkali-tolerant enzymes wherein the bacteria consist of aerobic, Gram-negative, motile, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: a) cells frequently in pairs; b) grows optimally between pH 9 and pH 10; 15 c) on alkaline-agar, forms smooth, translucent, beige colored colonies, 1-2 mm in diameter which are circular, convex with an entire margin; d) in alkaline-broth, growth (37°C) is flocculent with a ring or surface pellicle and formation of a 2 0 sediment; e) grows optimally at 20"C to 30°C; f) no growth at 15°C or 40°C; g) KOH test is positive; h) aminopeptidase test is weak positive; 25 i) oxidase test is weak positive; j) catalase test is positive; k) obligate halophile; 1) grows optimally at 4% NaCl; m) no growth at 0% or 8% NaCl; 30 n) hydrolysis of gelatin test is slow positive; o) hydrolysis of starch is positive; p) does not grow on simple sugars; q) does not grow on organic acids; r) grows on yeast extract and peptones.
8. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist^of...^ ^5 E N ^ A* °\ i'J ~\ r 7 SEP 1973 -• fc'J kfi, ./■ *t ■ • V 23 mz 5, f'A 93 - 103 - aerobic, Gram-negative, motile, rod-shap4d, obligate alkaliphilic bacteria having the following characteristics: a) grows optimally between pH 8.2 and pH 10.9; b) on alkaline-agar, forms smooth, opaque, beige or 5 brown colored colonies, 2-4 mm in diameter which are circular in form, convex in elevation, with an entire margin; c) in alkaline-broth, growth (37°C) is heavy and flocculent with a sediment and surface pellicle; 10 d) grows optimally between 20°C and 37°C; e) no growth at 8°C or at 40"C or above; f) KOH test is positive; g) aminopeptidase test is positive; h) oxidase test is very weakly positive; 15 i) catalase test is positive; j) grows at a NaCl concentration of between 0% and 15%; k) no growth at 20% NaCl; 1) hydrolysis of gelatin test is negative; 20 m) hydrolysis of starch is negative; n) grows on yeast extract; o) grows on organic acids selected from the group consisting of succinate, pyruvate, citrate, malonate, acetate and lactate; 25 p) grows on fatty acids selected from the group consisting of propionate, valerate and suberate; q) grows on amino acids selected from the group consisting of proline, serine, histidine and lysine. 30
9. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: 35 a) grows optimally between pH 9 and pH 10.5; b) on alkaline-agar, forms smooth, opaque, brown-colored colonies, 3-4 mm in diameter which are /■v - * - 104 - 239297 fairly irregular in form, generally flat to slightly umbonate in elevation with a lobate margin; c) in alkaline-broth, growth (37°C) is moderate to 5 heavy, becoming flocculent with a sediment and surface pellicle; d) grows optimally between 20°C and 40°C; e) no growth at 45'C; f) KOH test is positive; 10 g) aminopeptidase test is positive; h) oxidase test is negative; i) catalase test is positive; j) grows at a NaCl concentration 0% to 12%; k) no growth at 20% NaCl; 15 1) hydrolysis of gelatin test is positive; m) hydrolysis of starch is weakly positive; n) does not grow on simple sugars; o) grows on yeast extract; p) grows on organic acids selected from the group 20 consisting of pyruvate, citrate, acetate and lactate; q) grows on fatty acids selected from the group consisting of propionate, caprate and valerate; r) grows on amino acids selected from the group 25 consisting of proline, alanine and lysine.
10. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic 30 bacteria having the following characteristics: a) does not grow below pH 7.5; b) on alkaline-agar, forms smooth, cream colored colonies, initially translucent but becoming opaque; 35 c) on alkaline-agar, the colonies develop from circular, entire and become irregular, lobate in form, with a convex elevation; ;/y \ t iv f L t 'A " 7 SEP 1993 J 239297 - 105 - d) in alkaline-broth, growth (37"C) is slow, slight, flocculent with a sediment but no surface pellicle; e) grows optimally between 10°C and 40"C; f) no growth at 8°C or 45"C; 5 g) KOH test is positive; h) aminopeptidase test is negative; i) oxidase test is positive; j) catalase test is positive; k) grows at an NaCl contration of between 0% to 15%; 10 1) no growth at 20% NaCl; m) hydrolysis of gelatin test is positive; n) hydrolysis of starch is weakly positive; o) grows on yeast extract and peptones; p) grows on sugars; 15 q) grows on organic acids; r) grows on amino acids.
11. A pure bacterial culture useful for production of alkali-tolerant enzymes wherein the bacteria consist of 20 aerobic, Gram-negative, small, rod-shaped, obligate alkaliphilic bacteria having the following characteristics: a) cells frequently form short chains; b) does not grow below pH 8; c) on alkaline-agar, forms smooth, circular, convex 25 colonies with an entire margin, about 1 mm in diameter which are initially transparent, cream/beige in color, the colonies become opaque and brown in color with age; d) in alkaline-broth, growth (37°C) is initially 30 evenly turbid with a sediment but no surface pellicle becoming flocculent with formation of a pellicle; e) grows optimally between 3 0°C and 37°C; f) no growth at 40°C; 35 g) KOH test is positive; / /A °J\ h) aminopeptidase test is positive; /L rf l) oxidase test is positive; |z r-:j 7 SEP 1993 J 239297 - 10 6 - j) catalase test is positive; k) obligate halophile; 1) grows at 4% NaCl; m) no growth at 0% or 8% NaCl; 5 n) hydrolysis of gelatin test is slow positive; o) hydrolysis of starch is negative; p) grows on yeast extract and peptones; q) grows on sugars; r) grows on organic acids; 10 s) grows on fatty acids; t) grows on amino acids.
12. A method for the preparation of alkali-tolerant enzymes comprising: 15 culturing the bacteria of claim 1 in a culture medium; separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium.
13. A method for the preparation of alkali-tolerant enzymes 2 0 comprising: culturing the bacteria of claim 2 in a culture medium; separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium. 25
14. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 3 in a culture medium; separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium. 30
15. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 4 in a culture medium; separating the bacteria from the culture medium; and 35 recovery enzyme activity from the culture medium. / r.\ \\ "7 SEP 1993" I \\ / 239297 - 107-
16. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 5 in a culture medium; separating the bacteria from the culture medium; and 5 recovery enzyme activity from the culture medium.
17. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 6 in a culture medium; 10 separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium.
18. A method for the preparation of alkali-tolerant enzymes comprising: 15 culturing the bacteria of claim 7 in a culture medium; separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium.
19. A method for the preparation of alkali-tolerant enzymes 20 comprising: culturing the bacteria of claim 8 in a culture medium; separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium. 25
20. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 9 in a culture medium; separating the bacteria from the culture medium; and recovery enzyme activity from the culture medium. 30
21. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 10 in a culture medium; separating the bacteria from the culture medium; and 35 recovery enzyme activity from the culture medium. /' % h V ^ z r, -7 SEP 1993' 239297
22. A method for the preparation of alkali-tolerant enzymes comprising: culturing the bacteria of claim 11 in a culture medium; separating the bacteria from the culture medium; and 5 recovery enzyme activity from the culture medium.
23. A substantially pure preparation of the enzymes of claim 12, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch 10 degrading activities.
24. A substantially pure preparation of the enzymes of claim 13, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch 15 degrading activities.
25. A substantially pure preparation of the enzymes of claim 14, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch 20 degrading activities.
26. A substantially pure preparation of the enzymes of claim 15, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch 25 degrading activities.
27. A substantially pure preparation of the enzymes of claim 16, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch 30 degrading activities.
28. A substantially pure preparation of the enzymes of claim 17, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch 35 degrading activities. • a - 109 - 239297
29. A substantially pure preparation of the enzymes of claim 18, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch degrading activities.
30. A substantially pure preparation of the enzymes of claim 19, wherein the enzymes have lipolytic activity.
31. A substantially pure preparation of the enzymes of claim 10 20, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch degrading activities.
32. A substantially pure preparation of the enzymes of claim 15 21, wherein the enzymes have an activity selected from the group consisting of proteolytic, lipolytic and starch degrading activities.
33. A substantially pure preparation of the enzymes of claim 20 22, wherein the enzymes have an activity selected from the group consisting of proteolytic and starch degrading activities. 5 GIST-BROCADES N.V. By Their Attorneys HENRY HUGHES LTD
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NZ23929791A NZ239297A (en) | 1991-08-07 | 1991-08-07 | Gram negative alkaliphilic bacterial cultures and their use in producing alkali tolerant enzymes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NZ23929791A NZ239297A (en) | 1991-08-07 | 1991-08-07 | Gram negative alkaliphilic bacterial cultures and their use in producing alkali tolerant enzymes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NZ239297A true NZ239297A (en) | 1994-07-26 |
Family
ID=19923692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NZ23929791A NZ239297A (en) | 1991-08-07 | 1991-08-07 | Gram negative alkaliphilic bacterial cultures and their use in producing alkali tolerant enzymes |
Country Status (1)
| Country | Link |
|---|---|
| NZ (1) | NZ239297A (en) |
-
1991
- 1991-08-07 NZ NZ23929791A patent/NZ239297A/en unknown
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