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World Journal of Microbiology & Biotechnology 19: 961–968, 2003.

961
 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Screening and identification of extracellular lipase-producing thermophilic bacteria


from a Malaysian hot spring

N. Sheikh Abdul Hamid*, Hee B. Zen, Ong B. Tein, Yasin M. Halifah, Nazamid Saari and Fatimah Abu Bakar
Department of Food Science, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
*Author for correspondence: Tel.: +60-3-894-68386, Fax: +60-3-894-23552, E-mail: nazimah@putra.upm.edu.my

Received 7 February 2003; accepted in 22 July 2003

Keywords: Biolog system, hotspring, identification, lipase, screening, thermophilic bacteria

Summary

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the
Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture
supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the
bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest
lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of
3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10
were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia
paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus
thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified
as Bacillus subtilis.

Introduction allows the manufacture of high value-added products


(Paiva et al. 2000).
The enormous potential of microbial lipases arises from The investigation and application of thermostable
the facts that they are (1) quite stable and active in organic enzymes from thermophilic microorganisms have re-
solvents, (2) do not require cofactors, (3) exhibit a high ceived a lot of attention in academia and industry
degree of enantio- and regioselectivity, and (4) possess a ranging from the petrochemical and waste management
wide range of substrate specificity for the conversion of industries to the food industry. Thermostable lipases are
various unnatural substrates (Nakatani et al. 1992; required in the food industry for efficient enzymatic
Jaeger & Reetz 1998). More than 50 lipases have been processing of some lipids.
identified, purified and characterized to date, which Commercially available thermostable lipases are
originate from natural sources such as animals, plants mostly produced from mesophilic bacteria and fungi.
and microorganisms (native or genetically engineered). Due to the possibility of increased stability and resis-
Lipase-catalysed processes have received great atten- tance to high temperature and chemical denaturation,
tion, mainly due to several advantages: (1) they are lipases from thermophiles are expected to play a
more environment-friendly than bulk chemical synthe- significant role in the industry. Although many lipases
ses, (2) they allow manufacture of higher quality from mesophiles are stable at elevated temperatures,
products, (3) ease of recovery and re-use of the lipases, lipases from thermophiles are of interest to further
and (4) the possibility of use in continuous operations. enhance thermostability. In addition to higher thermo-
Ecological concerns have favoured more extensive stability, proteins from thermophiles often show more
applications of lipases because lipase-catalysed reactions stability toward organic solvent and exhibit higher
resemble more closely the pathways designed by nature activity at elevated temperatures.
for the metabolism of living things. The lipase discrimi- This study will provide a meaningful addition to the
nating ability encompasses features such as stereospeci- database on bacterial lipase research in the area of
ficity, selectivity and substrate specificity, which are screening and identification of thermostable lipolytic
much higher than those of inorganic catalysts. This bacteria. It involves the screening and identification of
962 N. Sheikh Abdul Hamid et al.
thermostable lipase-producing bacteria from the Setapak tion using Millipore UF membrane with a 10 kD
hot spring in Malaysia. exclusion limit at 4 C under 2.0 bar of nitrogen
pressure.
The lipolytic activity was measured according to the
Materials and methods method of Fox & Stepaniak (1983), modified by Yeap
(1998). The substrate mixture consisted of olive oil (10%
Conventional identification tests v/v), gum arabic solution (10% w/v in 0.1 M Tris–HCl
buffer with a pH of 7.2, 0.5 M NaCl) and CaCl2
All isolates were initially evaluated by the following (20 mM). The reaction mixture consisted of substrate
conventional test: Gram stain, growth and morphologic mixture (20 ml) and crude lipase (2.0 ml), which
characteristics on trypticase soy agar with 5% horse was added before the reaction started. The lipase
blood and MacConkey agar, catalase, oxidase, triple solution for the control was boiled in a water bath for
sugar-iron agar reactions, motility, indole production, 10 min before addition to the reaction mixture. The
gelatin liquefaction, oxidative-fermentative (OF) carbo- reaction mixtures were incubated in a reciprocal shaker
hydrate utilization, growth at 42 C, decarboxylation of water bath at 125 rev/min at 30 C for 30 min. 10 ml of
lysine, dihydrolase reaction of arginine and urease ethanol-acetone an mixture (1:1) was added to terminate
activity. Additional tests included phenylalanine deami- the lipase reaction. Phenolphthalein (0.1%) was then
nation, nitrate reduction, hydrolysis of esculin, starch added and titrated with NaOH (0.02 M) until the end
and DNA and growth in the presence of 6.5 and 7.5% point was reached. A unit of lipase activity (U) is defined
sodium chloride. These tests were considered conven- as the release of 1 lmol of fatty acid per min under the
tional identification methods (Osterhout et al. 1998). conditions above. The amount of fatty acid liberated
and lipase activity were calculated.
Culture maintenance
Comparison of thermostability of lipases
The cultures (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST
10) that were previously isolated from the Setapak hot In order to determine the thermostability, each enzyme
spring (Malaysia) were maintained on nutrient agar and was incubated for 30 min at 40, 50, 60, 70, and 80 C in
stored at 4 C after incubation at 45 C for 2 days. a reciprocal water bath with a speed of 125 rev/min. The
residual activity was then measured as described in the
Screening for lipase-producing bacteria section above.

Screening was carried out using Rhodamine B-olive oil Identification of bacteria using the Biolog Microstation
agar plate method according to Kouker & Jaeger (1986), System
modified by Yeap (1998). The growth medium prepared
by suspending nutrient agar (28 g) and sodium chloride Gram negative bacteria. Preliminary oxidase and triple
(4 g) in distilled water (1 l) was adjusted to pH 7.0 with sugar ion agar tests were carried out to differentiate
sodium hydroxide and autoclaved at 121 C for 15 min. Gram negative-non-enteric (GN-NENT) bacteria from
10 ml Rhodamine B solution (1 mg/ml) and olive oil Gram negative-enteric (GN-ENT) bacteria. The four
(31.25 ml) were added, and the cooled growth medium unknown cultures were subcultured on Biolog Universal
was stirred vigorously and allowed to stand for a few Growth (BUG) agar and incubated at 45 C for 16–
minutes. Aliquots of 20 ml were poured into each petri 24 h. After incubation, the cell suspensions were pre-
dish to solidify. Cultures from daughter slants were pared in GN/GP-IF (inoculating fluid) at a cell density
transferred and incubated in nutrient agar plates at of 52% T (transmittance) ± 3%. The contents were
45 C for 1 day. Each culture was streaked onto the stirred and the turbidity read using the Biolog turbidi-
Rhodamine B-olive oil agar plate and incubated at meter. The suspension (150 ll) was dispensed into each
45 C for 2 days. The lipase-producing bacteria were well of the GN2 MicroPlate using an eight-channel
indicated by the presence of orange fluorescent halos multipipette. The plates were then incubated at 30 C
around colonies when the plates were irradiated with (as recommended by the manufacturer) and read using
u.v. light. the Biolog Microstation at 16 h.
Gram positive bacteria. Gram staining was the only
Lipolytic assay of bacterial lipase producers preliminary test required to differentiate Gram positive-
rod sporeforming bacteria (GP-ROD SB) from Gram
The identified bacterial lipase producers were cultivated positive-coccus (GP-COCCUS) or Gram positive-rod
in Tryptone Soya Broth (10 ml) in quadplicate (1 (GP-ROD) bacteria. The three unknown cultures were
control and 3 replicates/samples). After incubation at subcultured on BUG + M (0.25% maltose) agar.
45 C for 36 h, the cultures were centrifuged at 5524 · g Before streaking the bacterium, eight drops from a
at 4 C for 15 min. The crude lipase solution was thioglycollate ampoule was added into sterile water
obtained by filtering through a Millipore 0.22 lm filter (3 ml). A sterile swab dipped into the solution was used
membrane and concentrated to 2 ml each by ultrafiltra- to spread a thin film of liquid across the entire surface of
Thermostable bacterial lipase producers 963
the agar medium and allowed to dry. The bacterium was Results and discussion
inoculated by streaking up and down three times and
across three times to make a narrow ‘+’ (plus) streak to Comparative lipolytic activity of positive bacterial lipase
restrain cell growth to two thin lines. The agar plates producers
were incubated at 45 C for16–24 h.
After incubation, a small amount of cells was scooped Lipolytic activities of seven lipase-producing bacteria
onto the tip of a sterile wooden stick. The active cells are shown in Figure 1. All the cultures showed good
growing along the edges of the ‘+’ streak were lipolytic activities. Strain ST 7 exhibited a high lipolytic
suspended in Gram negative/Gram positive-inoculating activity of 4.58 U/ml followed by strain ST 6 with an
fluid (GN/GP-IF) at a cell density of 28% T ± 3%. The activity of 3.51 U/ml. Strains ST 10, ST 1 and ST 8 had
contents were stirred and the turbidity read in the Biolog moderate lipolytic activities of 2.62, 2.39 and 2.38 U/ml
turbidimeter. The suspension (150 ll) was dispensed respectively. Strains ST 4 and ST 9 showed lower
into each well of the GP2 MicroPlate. The plates were lipolytic activities of 1.84 and 1.80 U/ml respectively.
incubated at 45 C and read using the Biolog Micro-
Station at 16 h. Thermostability of crude lipase

Biochemical tests The thermostability of crude lipase was determined at


temperatures ranging from 40 to 80 C for 30 min. The
Gram negative bacteria. The unknown Gram negative results for bacterial strains ST 1, ST 6, ST 7, ST 8 and
lipase-producing bacteria (ST 1, ST 4, ST 6 and ST 10) ST 10 that had lipolytic activities of more than 2 U/ml
were identified using the identification scheme for Gram are show in Figure 2. Strains ST 7 and ST 8 produced
negative bacteria according to MacFaddin (1980). The the most thermostable lipase of the five strains exam-
nine biochemical tests carried out include oxidation- ined, with a recovery of 87 and 88% respectively at
fermentation of glucose, oxidase, motility, urease, ci- 80 C. At 80 C, strains ST 1, ST 6 and ST 10 showed
trate, gelatin liquefaction, arginine dihydrolase, nitrate recoveries of 36, 61 and 64% respectively.
reduction and growth at 42 C. Twenty additional
biochemical tests done according to Osterhout et al. Colony morphology, Gram reaction and microscopy
(1998) include antibiotic susceptibility test using agar
diffusion method, carbohydrate fermentation (arabi- Table 1 shows that four of the lipase-producing bacteria
nose, glucose, 10% lactose, maltose, mannitol and were Gram negative while the rest were Gram positive.
xylose), catalase, deoxyribonuclease (DNase), esculin, All the Gram negative bacteria (ST 1, ST 4, ST 6 and ST
indole, lysine decarboxylase, phenylalanine deamina- 10) possessed similar characteristics in terms of colony
tion, pigment production, growth in 6.5% sodium size, colony morphology and cellular morphology,
chloride, starch hydrolysis, MacConkey agar, triple giving the possibility that these bacteria evolved from
sugar ion agar and trypticase soy agar with 5% horse the same genus or species of bacteria. The two strains of
blood, and urease test. The blood agar test was also Gram positive bacteria, ST 7 and ST 9, also showed
done to determine the haemolysis pattern. All the similar characteristics. Strain ST 8 exhibited different
biochemical tests were carried out using two incubation
temperatures, (35, 37 or 42 C depending on the
reference and 45 C). The growth of cultures on nutrient 5
agar plate at incubation temperatures ranging from 30 4.58
4.5
to 65 C was examined.
Gram positive bacteria. The unknown Gram positive 4
lipase-producing bacteria (ST 7, ST 8 and ST 9) were 3.51
3.5
Lipolytic Activity (U)

identified using the identification scheme for Gram


3
positive bacteria according to MacFaddin (1980). 2.62
2.39 2.38
Biochemical tests performed include catalase, oxida- 2.5
tion-fermentation glucose, carbohydrate fermentation 2 1.84 1.80
(arabinose, glucose, 10% lactose, maltose, mannitol and
1.5
xylose), motility, blood agar, nitrate reduction, ammo-
nia, indole, gelatin liquefaction, Simmons citrate, Voges- 1
Proskauer, urease, phenylalanine deamination, 6.5 and 0.5
7.5% sodium chloride, starch hydrolysis, arginine dihy-
0
drolase and lysine decarboxylase. All the biochemical
ST 1 ST 4 ST 6 ST 7 ST 8 ST 9 ST 10
tests were carried out at two incubation temperatures,
(35, 37 or 42 C depending on the reference and 45 C). Strains
The growth of cultures on nutrient agar plate at Figure 1. Lipolytic activities of positive lipase-producing bacteria
incubation temperatures ranging from 30 to 65 C was isolated from the Setapak hot spring. See methods for growth and
examined. assay conditions.
964 N. Sheikh Abdul Hamid et al.
110 reported if the similarity index of the genus or species
was 0.750 or greater at 4 h, or 0.500 or greater at 24 h.
100
The CDC group IVc-2, now classified as Ralstonia
90
paucula (Vandamme et al. 1999) is a Gram negative
Relative Activities (%)

non-fermentative rod that closely resembles Alcaligenes


80 sp. and Bordetella bronchiseptica in terms of biochemical
characteristics (Dan et al. 1986; Osterhout et al. 1998).
70 Gram positive bacteria. Biolog identified strains ST 7
and ST 9 as Bacillus thermoglucosidasius, which is now
60
known as Geobacillus thermoglucosidasius (Nazina et al.
2001) and strain ST 8 as Bacillus subtilis. The similarity
50
index for ST 7, ST 8 and ST 9 were 0.846, 0.707 and
40 0.704 respectively. Bacillus thermoglucosidasius and
Bacillus subtilis are aerobic Gram positive sporeforming
30 bacteria. Bacillus thermoglucosidasius is a thermophilic
40 50 60 70 80
bacteria with an optimum temperature of 61–63 C
Temperature (ºC) (Sneath et al. 1986).
Figure 2. Thermal stability of crude lipases produced by ST 1, ST 6, The Biolog’s technical literature reported that the
ST 7, ST 8 and ST 10. ––¤–– ST 1 ––n–– ST6 ––m–– ST 7 ––·–– ST8 ––+–– Bacillus species are difficult to identify due to ‘false-
ST 10. positive’ reactions that may be a result of sporulation,
utilization of lysed cell material, utilization of stored
endogenous substrate or extracellular polysaccharides.
characteristics in terms of colony size, colony morphol-
Hence, biochemical tests were carried out to confirm the
ogy and cellular morphology compared to the other six
identity of the bacteria.
bacteria.
Endospores were observed in all Gram positive
strains. The endospores are impermeable to the Gram Biochemical tests
stain and were observed as semi-transparent round spots
inside the rods. The endospores in strains ST 7 and ST 9 Gram negative bacteria. Tables 2 and 3 show the results
could not be seen clearly. Endospore staining using of the biochemical tests carried out for the identification
malachite green was carried out to determine the of Gram negative bacteria at two different incubation
position of the endospore. temperatures. Incubation temperature was chosen as 35,
37 or 42 C depending on the methods mentioned by
Identification of bacteria using the Biolog Microstation MacFaddin (1980) and Osterhout et al. (1998). The
System incubation temperature of 45 C was chosen because the
bacteria of interest in this study should be thermophilic.
Gram negative bacteria. Strains ST 1, ST 4, ST 6 and ST All the four strains showed identical results at both
10 were identified as CDC group IVc-2 (Alcaligenes-like) incubation temperatures with the only difference being
using the Biolog Microstation System. The similarity the rate of reaction. Generally, the strains exhibited a
index for ST 1, ST 4, ST 6 and ST 10 were 0.739, 0.679, higher reaction rate at 35 C as compared to 45 C. This
0.634 and 0.734 respectively. Biolog identifications were may be due to the strains’ optimum temperature for

Table 1. Bacterial morphology and Gram reaction of lipase producers.

Code Colony size (mm) Colony morphology Gram reaction Cellular morphology

ST 1 1 Circular; convex; entire edge; Negative Short rods


smooth surface; beige colour
ST 4 1 Circular; convex; entire edge; Negative Short rods
smooth surface; beige colour
ST 6 1 Circular; convex; entire edge; Negative Short rods
smooth surface; beige colour
ST 7 1 Circular; umbonate; undulate edge; Positive Rods with ellipsoidal endospores
smooth surface; beige colour at terminal, swollen cell
ST 8 4 Irregular and spreading; raised margin; Positive Rod with ellipsoidal at central
undulate edge; beige colour and subterminal
ST 9 1 Circular; umbonate; undulate edge; Positive Rods with ellipsoidal endospores
smooth surface; beige colour at terminal, swollen cell
ST 10 1 Circular; convex; entire edge; Negative Short rods
smooth surface; beige colour

* Cellular morphology was studied using bright field microscopy of a Gram-stained preparation. When observed under the microscope, Gram
positive cells were purple or blue in colour while Gram negative cells were pink or red in colour.
Thermostable bacterial lipase producers 965
Table 2. Results of biochemical tests carried out for Gram negative The Biolog System identified all the four cultures as
bacteria carried out at either 35, 37 or 42 C. CDC group IVc-2 (Alcaligenes-like), now known as
No. Biochemical tests Code Ralstonia paucula (Vandamme et al. 1999). Additional
biochemical tests were carried out according to Oster-
ST 1 ST 4 ST 6 ST 10 hout et al. (1998) to determine whether the strains were
1 Antibiotic susceptibility R. paucula. The results obtained from these biochemical
(i) amikacin (30 lg) R R R R tests (Tables 2 and 3) were comparable to the results
(ii) cefuroxime sodium (30 lg) S S S S mentioned in literature. Except for the lysine decarboxy-
(iii) tetracycline (30 lg) S S S S lase and rapid urease production tests, the strains were
2 Arginine dehydrolase ) ) ) )
3 Blood agar c c c c
classified as R. paucula. Ralstonia paucula exhibited a
4 Carbohydrate fermentation negative result for lysine decarboxylase test and a
(i) Arabinose ) ) ) ) positive result for the rapid urease production test
(ii) Glucose ) ) ) ) (Osterhout et al. 1998). All the four strains showed
(iii) Lactose (10%) ) ) ) ) positive results for the lysine decarboxylase test. Strains
(iv) Maltose ) ) ) )
(v) Mannitol ) ) ) )
ST 1 and ST 4 exhibited negative result for rapid urease
(vi) Xylose ) ) ) ) production at 35 C. Strains ST 1 and ST 10 showed
5 Catalase + + + + negative results for this test at 45 C. Absolute confir-
6 Citrate (Simmons) + + + + mation that all the unknown Gram negative lipase-
7 Deoxyribonuclease (Dnase) ) ) ) ) producing bacteria were R. paucula was not possible due
8 Esculin ) ) ) )
9 Gelatin liquefaction ) ) ) )
to close biochemical resemblance to Alcaligenes spp. and
10 Growth at 42 C + + + + B. bronchiseptica (Dan et al. 1986; Osterhout et al.
11 Indole ) ) ) ) 1998).
12 Lysine decarboxylase + + + +
13 MacConkey agar + + + +
14 Motility (37 C) + + + + Table 3. Results of biochemical tests for Gram negative bacteria
15 Nitrate reduction ) ) ) ) carried out at 45 C.
16 Oxidase + + + +
17 Oxidation-fermentation glucose ) ) ) ) No. Biochemical tests Code
18 Phenylalanine deamination ) ) ) )
ST 1 ST 4 ST 6 ST 10
19 Sodium chloride (6.5%) ) ) ) )
20 Starch hydrolysis ) ) ) ) 1 Antibiotic susceptibility
21 Triple sugar ion agar ) ) ) ) (i) Amikacin (30 lg) R R R R
22 Trypticase soy agar with 5% + + + + (ii) Cefuroxime sodium (30 lg) S S S S
horse blood (37 C) (iii) Tetracycline (30 lg) S S S S
23 Urease production + + +R +R 2 Arginine dehydrolase ) ) ) )
24 Voges–Proskauer ) ) ) ) 3 Blood agar c c c c
4 Carbohydrate fermentation
R – Resistant; S – susceptibility; c – gamma haemolysis (no change (i) Arabinose ) ) ) )
of the medium); ) – negative result; + – positive result; R – rapid (ii) Glucose ) ) ) )
urease (within 4 h). (iii) Lactose (10%) ) ) ) )
(iv) Maltose ) ) ) )
(v) Mannitol ) ) ) )
growth that was closer to 35 C rather than 45 C. (vi) Xylose ) ) ) )
Table 6 shows four of the Gram negative bacteria that 5 Citrate (Simmons) + + + +
6 Deoxyribonuclease (Dnase) ) ) ) )
could grow at temperatures ranging from 30 to 45 C. 7 Esculin ) ) ) )
They grew weakly at 50 C and their growth was 8 Gelatin liquefaction ) ) ) )
inhibited at the temperatures of 55 C and above. 9 Indole ) ) ) )
The results obtained from the biochemical tests 10 Lysine decarboxylase + + + +
tentatively identified strains ST 1 and ST 4, as Alcali- 11 MacConkey agar + + + +
12 Motility + + + +
genes sp. (Alcaligenes faecalis or Alcaligenes odorans) at 13 Nitrate reduction ) ) ) )
an incubation temperature of 35 C. Strains ST 6 and 14 Oxidation-fermentation glucose ) ) ) )
ST 10 were tentatively identified as B. bronchiseptica at 15 Phenylalanine deamination ) ) ) )
the same incubation temperature. The results of bio- 16 Pigment production ) ) ) )
chemical tests carried out at 45 C however identified 17 Sodium chloride (6.5%) ) ) ) )
18 Starch hydrolysis ) ) ) )
strains ST 1 and ST 10 as belonging to the Alcaligenes 19 Triple sugar ion agar ) ) ) )
sp. and strains ST 4 and ST 6 as B. bronchiseptica. This 20 Trypticase soy agar with 5% + + + +
dissimilarity was due to the urease test. At 35 C horse blood (37 C)
incubation temperature, strains ST 6 and ST 10 exhi- 21 Urease production + +R +R +
bited rapid urease production and were classified as B. 22 Voges–Proskauer ) ) ) )
bronchiseptica. At 45 C, ST 4 and ST 6 showed rapid R – Resistant; S – susceptibility; c – gamma haemolysis (no change
urease production, and were accordingly classified as B. of the medium); ) – negative result; + – positive result; R – rapid
bronchiseptica. urease (within 4 h).
966 N. Sheikh Abdul Hamid et al.
There was a strong possibility that all the four strains Table 4. Results of biochemical tests for Gram positive bacteria
were R. paucula because Alcaligenes odorans caused carried out at either 35 or 37 C.
green discoloration (partial haemolysis or called alpha No. Biochemical tests Code
haemolysis) in blood agar media, was tolerant at 6.5%
sodium chloride (NaCl) and showed a negative urease ST 7 ST 8 ST 9
test (Gilardi 1978; MacFaddin 1980). Alcaligenes fae- 1 Ammonia ) + )
calis also gave a negative urease test and half of the 2 Arginine dehydrolase ) ) )
strains were capable of reducing nitrate to nitrite 3 Blood agar b b a
(Gilardi 1978; MacFaddin 1980). The results shown in 4 Carbohydrate fermentation
(i) Arabinose D + D
Tables 2 and 3 show significant differences from the (ii) Glucose + + +
Alcaligenes spp. Nitrate reduction is a conspicuous (iii) Lactose (10%) D ) D
characteristic that differentiates R. paucula from B. (iv) Maltose + ) +
bronchiseptica (Osterhout et al. 1998). Bordetella bron- (v) Mannitol + + +
chiseptica was capable of reducing nitrate to nitrite but (vi) Xylose D D D
5 Catalase +w + +w
not R. paucula (Dan et al. 1986; Osterhout et al. 1998; 6 Citrate (Simmons) ) ) )
Vandamme et al. 1999). Besides that, R. paucula was 7 Esculin ) + )
able to grow in the presence of Tween 80 and exhibited 8 Gelatin liquefaction ) + )
acid phosphatase activity, phosphoamidase activity and 9 Indole ) ) )
most importantly, lipase C14 activity (Vandamme et al. 10 Lysine decarboxylase ) ) )
11 Motility + + +
1999). Bordetella bronchiseptica did not share these 12 Nitrate reduction ) + )
characteristics with Ralstonia paucula. 13 Oxidation-fermentation glucose F O/F F
The results based on the biochemical tests carried out 14 Phenylalanine deamination ) ) )
in this study, closely resembled the characteristics of 15 Sodium chloride (6.5%) + + +
Ralstonia paucula as compared to Alcaligenes spp. and 16 Sodium chloride (7.5%) + + +
17 Starch hydrolysis ) + )
Bordetella bronchiseptica. To date R. paucula has not 18 Urease production ) ) )
been reported as a thermostable lipase producer al- 19 Voges-Proskauer ) ) )
though Vandamme et al. (1999) reported that lipase C14
activity was exhibited by R. paucula. However, further ) – Negative result; + – positive result; D – delayed (no colour
study such as randomly amplified polymorphic DNA change); F – fermentation; O/F – oxidation and fermentation; b –
beta haemolysis (complete haemolysis); a – alpha haemolysis (partial
(RAPD), cellular fatty acid (CFA) and 16S rRNA haemolysis); w – weak gas production.
analysis should be carried out for identification confir-
mation.
Moissenet et al. (1996) concluded that DNA analysis rates at 45 C as compared to 35 C. This could be due
by the RAPD method proved to be useful for the to the strains’ optimum growth temperature, which were
epidemiological investigation of hospital outbreaks of closer to 45 C rather than 35 C. Strains ST 7 and ST 9
Ralstonia paucula infection. Osterhout et al. (1998) exhibited similar results at the two different incubation
reported that the combination of CFA and biochemical temperatures. An exception was observed when strain
data is generally sufficient for accurate identification of ST 9 showed a positive result for the urease test at
R. paucula. CFA analysis can be utilized as a rapid 45 C. Differences existed in the following biochemical
screening method followed by selected biochemical tests tests for strain ST 8 at two different incubation
to confirm the identification. When phenotypic methods temperatures: (i) arabinose fermentation test, (ii) xylose
prove inconclusive, confirmatory genotypic techniques fermentation test, (iii) Simmons citrate, and (iv) urease
such as 16S rRNA gene sequence analysis are beneficial. production. For the arabinose fermentation test, strain
CFA and rRNAs are two groups of macromolecules that ST 8 exhibited positive results at 35 C and negative
have recently been made amenable to simplified analysis. results at 45 C. For the xylose fermentation test, the
Analysis of the 16S rRNA gene sequence has proven to reaction was delayed when incubated at 35 C and a
be most useful in molecular systematics, since it is highly negative result was obtained at 45 C. Strain ST 8
conserved, universally distributed and contains diagnos- exhibited a negative result at 35 C and a positive result
tically significant variable regions. Moissenet et al. at 45 C for Simmons citrate test. For the urease test,
(1999) reported that the ID-32-GN identification system strain ST 8 showed a negative result at 35 C and a
(BioMerieux, Marcy-I’Etoile, France), based on 32 positive result at 45 C. As shown in Table 6, strains ST
metabolic assimilations tests, provided a simple, rapid 7 and ST 9 were able to grow at temperatures ranging
and reliable means of identifying R. paucula with a high from 30 to 65 C. They grew weakly at 30 and 35 C.
degree of confidence (99.9%). Strain ST 8 grew at temperatures that ranged from 30 to
Gram positive bacteria. Tables 4 and 5 show the results 60 C but exhibited feeble growth at 55 and 60 C, and
of the biochemical tests carried out for the identification no growth at 65 C.
of Gram positive bacteria. These tests were carried out Strains ST 7, ST 8 and ST 9 were identified as Bacillus
at two different incubation temperatures, 35 or 37 and spp. The biochemical test results of strains ST 7 and ST
45 C. Three of the strains exhibited higher reaction 9, showed similar results throughout the study. Bacillus
Thermostable bacterial lipase producers 967
Table 5. Results of biochemical tests for Gram positive bacteria at the control well, do not give credibility to the results.
45 C. Comparison of the present biochemical test results of
No. Biochemical tests Code strains ST 7 and ST 9 with the five characteristics of B.
thermoglucosidasius as reported by Nazina et al. (2001),
ST 7 ST 8 ST 9 showed that only the esculin test result was identical.
1 Ammonia ) + ) Strains ST 7 and ST 9 gave different results for the other
2 Arginine dehydrolase ) ) ) four tests (arabinose fermentation, gelatin liquefaction,
3 Blood agar b b a starch hydrolysis and Simmons citrate). As observed in
4 Carbohydrate fermentation the Tables 5 and 6, strains ST 7 and ST 9 gave delayed
(i) Arabinose D ) D
(ii) Glucose + + +
(D) and negative results for a number of tests. This
(iii) Lactose (10%) D ) D might be due to the fact that the optimum temperature
(iv) Maltose + ) + of growth of ST 7 and ST 9 were more than 45 C. The
(v) Mannitol + + + reported optimum temperature for B. thermoglucosi-
(vi) Xylose D ) D dasius was 61–63 C (Sneath et al. 1986). Bacillus ther-
5 Citrate (Simmons) ) + )
6 Esculin ) + )
moglucosidasius was rod-shaped with ellipsoidal
7 Gelatin liquefaction ) + ) endospores at the terminus of the swollen cells. Strains
8 Indole ) ) ) ST 7 and ST 9 possessed the same cellular morphology
9 Lysine decarboxylase ) ) ) as B. thermoglucosidasius. Nevertheless, confirmation
10 Motility + + + tests would have to be performed to identify these two
11 Nitrate reduction ) + )
12 Oxidation-fermentation glucose F O/F F
strains up to the species level. Nazina et al. (2001)
13 Phenylalanine deamination ) ) ) proposed the transfer of B. thermoglucosidasius to the
14 Sodium chloride (6.5%) + + + new genus Geobacillus, with Geobacillus thermogluco-
15 Sodium chloride (7.5%) + + + sidasius as the type species. An additional character
16 Starch hydrolysis ) + ) found by Kampfer (1994) is that the main cellular fatty
17 Urease production ) + +
18 Voges-Proskauer ) ) )
acids are iso-15:0, iso-16:0 and iso-17:0, making up more
than 60% of the total fatty acids (Nazina et al. 2001).
) – Negative result; + – positive result; D – delayed (no colour Thus, cellular fatty acid analysis could be carried out
change); F – fermentation; O/F – oxidation and fermentation; b – for further identification.
beta haemolysis (complete haemolysis); a – alpha haemolysis (partial The Biolog system identified strain ST 8 as Bacillus
haemolysis).
subtilis but some ‘false-positive’ reactions were evident
necessitating biochemical tests for further confirmation.
stearothermophilus and B. coagulans were the most Possible matches for strain ST 8, include Bacillus
possible candidates for these strains. Strains ST 7 and subtilis, B. megaterium, B. licheniformis and B. firmus.
ST 9 had biochemical test results which differed from B. The variation in biochemical results between strain ST 8
stearothermophilus in terms of O–F glucose, gelatin and the four strains mentioned are as follows: (1)
liquefaction, 7.5% sodium chloride and starch hydro- differences in the biochemical results of B. subtilis was
lysis tests. Strains ST 7 and ST 9 showed differences observed in the O–F glucose, Voges–Proskauer, arabi-
with B. coagulans in terms of 7.5% sodium chloride nose and xylose fermentation tests; (2) differences in the
and starch hydrolysis tests except for strain ST 9 biochemical results of B. licheniformis was observed in
which showed a different result in the urease test. the O–F glucose, Voges–Proskauer, arabinose and xy-
The Biolog system identified strains ST 7 and ST 9 as lose fermentation tests; (3) differences in the biochemi-
B. thermoglucosidasius but the ‘false-positive’ reactions cal results of B. firmus was observed in the O–F glucose,
in which all reaction wells gave positive results including Simmons citrate, urease and phenylalanine deaminase

Table 6. Growth of lipase-producing bacteria at different temperatures.

No. Temperature (C) Code


Gram negative bacteria Gram positive bacteria

ST 1 ST 4 ST 6 ST 10 ST 7 ST 8 ST 9
w
1 30 + + + + + + +w
2 35 + + + + +w + +w
3 40 + + + + + + +
4 45 + + + + + + +
5 50 +w +w +w +w + + +
6 55 ) ) ) ) + +w +
7 60 ) ) ) ) + +w +
8 65 ) ) ) ) + ) +

w
– weak.
968 N. Sheikh Abdul Hamid et al.
tests; (4) differences in the biochemical results of B. Gilardi, G.L. 1978 Glucose Nonfermenting Gram Negative Bacteria in
megaterium was observed in the motility, O–F glucose Clinical Microbiology. New York: CRC Press, Inc. ISBN 0-
84935331-9.
and phenylalanine deaminase tests. Jaeger, K.E. & Reetz, M.T. 1998 Microbial lipases form versatile tools
Biochemical test results obtained from this study were for biotechnology. Trends in Biotechnology 16, 396–403.
similar to the characteristics of Bacillus subtilis. Accord- Kampfer, P. 1994 Limits and possibilities of total fatty acid analysis
ing to Sneath et al. (1986), Bacillus subtilis and strain for classification and identification of Bacillus species. Systematic
ST 8 exhibited negative results for both arginine dihy- and Applied Microbiology 17, 86–89.
Kouker, G. & Jaeger, K.E. 1986 Specific and sensitive plate assay for
drolase and lysine decarboxylase tests. Bacillus licheni- bacterial lipases. Applied and Environmental Microbiology 52, 211–
formis showed positive results for the arginine 213.
dihydrolase test and no results for this test were reported Logan, N.A. & Berkeley, R.C.W. 1984 Identification of Bacillus strains
for Bacillus firmus. Although Bacillus megaterium ex- using the API system. Journal of General Microbiology 130, 1871–
hibited same results for both the tests, it could be 1882.
MacFaddin, J.F. 1980 Biochemical Tests for Identification of Medical
differentiated from Bacillus subtilis because strain ST 8 Bacteria. 2nd ed. USA: Waverly Press, Inc. ISBN 0-68305315-9.
was able to grow at 50 C. Both Bacillus megaterium Moissenet, D., Goujon, C.P., Antonie, G.C. & Hoang, V.T. 1999 CDC
and Bacillus firmus were unable to grow at 50 C Group IVc2: a New Ralstonia Species Close to Ralstonia eutropha.
(Sneath et al. 1986). Sneath et al. (1986) and Yeap Journal of Clinical Microbiology 37, 1777–1781.
(1998) have also identified Bacillus subtilis as a positive Moissenet, O., Tabone, M.D., Girardet, J.P., Laverger, G., Garbarg-
Chenon, A. & Vu-Thien, H. 1996 Nosocomial CDC group IV c-2
bacterial lipase producer whereas Bacillus megaterium bacteremia: epidemiological investigation by randomly amplified
was not. polymorphic DNA analysis. Journal of Clinical Microbiology 34,
Due to the lack of discriminating biochemical fea- 1264–1266.
tures, more reliable identification systems such as API Nakatani, T., Hiratake, J., Yoshikawa, K., Nishioka, T. & ODA, J.
System and 16S rRNA analysis could be carried out for 1992 Chemical inactivation of lipase in organic solvent: A lipase
from Pseudomonas aeruginosa TE3285 is more like a typical serine
further confirmation of the bacterial identity. Logan & enzyme in an organic solvent than in aqueous media. Bioscience
Berkeley (1984) reported that the API System tests were Biotechnology and Biochemistry 56, 1118–1123.
more reproducible than conventional biochemical tests. Nazina, T.N., Tourova, T.P., Poltaraus, A.B., Novikova, E.V.,
Ash et al. (1991) and Rainey et al. (1994) reported that Grigoryan, A.A., Ivanova, A.E., Lysenko, A.M., Petrunyaka,
16S rRNA gene sequence analysis has revealed high V.V., Osipov, G.A. & Ivanov, M.V. 2001 Taxonomic study of
aerobic thermophilic bacilli: descriptions of Geobacillus subterran-
phylogenetic heterogeneity in the genus Bacillus (Nazina eus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from
et al. 2001). petroleum reservoirs and transfer of Bacillus stearothermophilus,
Although diagnostic keys and tables for Bacillus have Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus
been available for a long time, the identification of these kaustophilus, Bacillus thermoglucosidasius and Bacillus thermode-
organisms is still complicated. Bacillus is an unusually nitrificans to Geobacillus as the new combinations G. stearothermo-
philus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus,
wide taxon containing mostly aerobic endospore-form- G. thermoglucosidasius and G. thermodenitrificans. International
ing rods (Logan & Berkeley 1984). According to Priest Journal of Systematic and Evolutionary Microbiology 51, 433–446.
(1981), as cited by Logan & Berkeley (1984), in terms of Osterhout, G.J., Valentine, J.L. & Dick, J.D. 1998 Phenotypic and
DNA base ratios it is the equivalent of some bacterial Genotypic Characterization of Clinical Strains of CDC Group
families. Furthermore, some species are ill-defined, IVc-2. Journal of Clinical Microbiology 36, 2618–2622.
Paiva, A.L., Balcao, V.M. & Malcata, F.X. 2000 Kinetics and
existing with closely resembled species as complexes or mechanisms of reactions catalyzed by immobilized lipases. Enzyme
spectra in which the boundary of a particular species is and Microbial Technology 27, 187–204.
difficult or impossible to identify. Even in well estab- Priest, F.G. 1981 DNA homology in the genus Bacillus. In The Aerobic
lished species, there is considerable variation between Endospore Forming bacteria: Classification and identification. eds.
strains (Logan & Berkeley 1984). As cited by Nazina Berkeley, R.C.W. & Goodfellow, M., pp. 33–57. London: Academic
Press. ISBN 0-12091250-3.
et al. (2001), phylogenetic analysis revealed that the Rainey, F.A., Fritze, D. & Stackebrandt, E. 1994 The phylogenetic
genus Bacillus and its thermophilic members require diversity of thermophilic members of the genus Bacillus as revealed
extensive taxonomic revision (Stackebrandt et al. 1987; by 16S rDNA analysis. FEMS Microbiology Letter 115, 205–211.
Ash et al. 1991; Rainey et al. 1994). Sneath, P.H.A., Mair, N.S., Sharpe, M.E. & Holt, J.G. 1986 Bergey’s
Manual of Systematic Bacteriology. vol. 2. USA: Waverly Press,
Inc. ISBN 0-71670621-0.
Stackebrandt, E., Ludwig, W., Weizenegger, M., Dorn, S., McGill,
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