hamid paper

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 BakarDepartment of Food Science, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 UPMSerdang, Selangor, Malaysia*Author for correspondence: Tel.: +60-3-894-68386, Fax: +60-3-894-23552, E-mail: [email protected]

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 theSetapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culturesupernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of thebacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highestlipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10were 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 Ralstoniapaucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillusthermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identifiedas Bacillus subtilis.

Introduction

The enormous potential of microbial lipases arises fromthe facts that they are (1) quite stable and active in organicsolvents, (2) do not require cofactors, (3) exhibit a highdegree of enantio- and regioselectivity, and (4) possess awide range of substrate specificity for the conversion ofvarious unnatural substrates (Nakatani et al. 1992;Jaeger & Reetz 1998). More than 50 lipases have beenidentified, purified and characterized to date, whichoriginate from natural sources such as animals, plantsand microorganisms (native or genetically engineered).Lipase-catalysed processes have received great atten-

tion, mainly due to several advantages: (1) they aremore environment-friendly than bulk chemical synthe-ses, (2) they allow manufacture of higher qualityproducts, (3) ease of recovery and re-use of the lipases,and (4) the possibility of use in continuous operations.Ecological concerns have favoured more extensiveapplications of lipases because lipase-catalysed reactionsresemble more closely the pathways designed by naturefor the metabolism of living things. The lipase discrimi-nating ability encompasses features such as stereospeci-ficity, selectivity and substrate specificity, which aremuch higher than those of inorganic catalysts. This

allows the manufacture of high value-added products(Paiva et al. 2000).The investigation and application of thermostable

enzymes from thermophilic microorganisms have re-ceived a lot of attention in academia and industryranging from the petrochemical and waste managementindustries to the food industry. Thermostable lipases arerequired in the food industry for efficient enzymaticprocessing of some lipids.Commercially available thermostable lipases are

mostly produced from mesophilic bacteria and fungi.Due to the possibility of increased stability and resis-tance to high temperature and chemical denaturation,lipases from thermophiles are expected to play asignificant role in the industry. Although many lipasesfrom mesophiles are stable at elevated temperatures,lipases from thermophiles are of interest to furtherenhance thermostability. In addition to higher thermo-stability, proteins from thermophiles often show morestability toward organic solvent and exhibit higheractivity at elevated temperatures.This study will provide a meaningful addition to the

database on bacterial lipase research in the area ofscreening and identification of thermostable lipolyticbacteria. It involves the screening and identification of

World Journal of Microbiology & Biotechnology 19: 961–968, 2003. 961� 2003 Kluwer Academic Publishers. Printed in the Netherlands.

thermostable lipase-producing bacteria from the Setapakhot spring in Malaysia.

Materials and methods

Conventional identification tests

All isolates were initially evaluated by the followingconventional test: Gram stain, growth and morphologiccharacteristics on trypticase soy agar with 5% horseblood and MacConkey agar, catalase, oxidase, triplesugar-iron agar reactions, motility, indole production,gelatin liquefaction, oxidative-fermentative (OF) carbo-hydrate utilization, growth at 42 �C, decarboxylation oflysine, dihydrolase reaction of arginine and ureaseactivity. Additional tests included phenylalanine deami-nation, nitrate reduction, hydrolysis of esculin, starchand DNA and growth in the presence of 6.5 and 7.5%sodium chloride. These tests were considered conven-tional identification methods (Osterhout et al. 1998).

Culture maintenance

The cultures (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST10) that were previously isolated from the Setapak hotspring (Malaysia) were maintained on nutrient agar andstored at 4 �C after incubation at 45 �C for 2 days.

Screening for lipase-producing bacteria

Screening was carried out using Rhodamine B-olive oilagar plate method according to Kouker & Jaeger (1986),modified by Yeap (1998). The growth medium preparedby suspending nutrient agar (28 g) and sodium chloride(4 g) in distilled water (1 l) was adjusted to pH 7.0 withsodium hydroxide and autoclaved at 121 �C for 15 min.10 ml Rhodamine B solution (1 mg/ml) and olive oil(31.25 ml) were added, and the cooled growth mediumwas stirred vigorously and allowed to stand for a fewminutes. Aliquots of 20 ml were poured into each petridish to solidify. Cultures from daughter slants weretransferred and incubated in nutrient agar plates at45 �C for 1 day. Each culture was streaked onto theRhodamine B-olive oil agar plate and incubated at45 �C for 2 days. The lipase-producing bacteria wereindicated by the presence of orange fluorescent halosaround colonies when the plates were irradiated withu.v. light.

Lipolytic assay of bacterial lipase producers

The identified bacterial lipase producers were cultivatedin Tryptone Soya Broth (10 ml) in quadplicate (1control and 3 replicates/samples). After incubation at45 �C for 36 h, the cultures were centrifuged at 5524 · gat 4 �C for 15 min. The crude lipase solution wasobtained by filtering through a Millipore 0.22 lm filtermembrane and concentrated to 2 ml each by ultrafiltra-

tion using Millipore UF membrane with a 10 kDexclusion limit at 4 �C under 2.0 bar of nitrogenpressure.The lipolytic activity was measured according to the

method of Fox & Stepaniak (1983), modified by Yeap(1998). The substrate mixture consisted of olive oil (10%v/v), gum arabic solution (10% w/v in 0.1 M Tris–HClbuffer with a pH of 7.2, 0.5 M NaCl) and CaCl2(20 mM). The reaction mixture consisted of substratemixture (20 ml) and crude lipase (2.0 ml), whichwas added before the reaction started. The lipasesolution for the control was boiled in a water bath for10 min before addition to the reaction mixture. Thereaction mixtures were incubated in a reciprocal shakerwater bath at 125 rev/min at 30 �C for 30 min. 10 ml ofethanol-acetone an mixture (1:1) was added to terminatethe lipase reaction. Phenolphthalein (0.1%) was thenadded and titrated with NaOH (0.02 M) until the endpoint was reached. A unit of lipase activity (U) is definedas the release of 1 lmol of fatty acid per min under theconditions above. The amount of fatty acid liberatedand lipase activity were calculated.

Comparison of thermostability of lipases

In order to determine the thermostability, each enzymewas incubated for 30 min at 40, 50, 60, 70, and 80 �C ina reciprocal water bath with a speed of 125 rev/min. Theresidual activity was then measured as described in thesection above.

Identification of bacteria using the Biolog MicrostationSystem

Gram negative bacteria. Preliminary oxidase and triplesugar ion agar tests were carried out to differentiateGram negative-non-enteric (GN-NENT) bacteria fromGram negative-enteric (GN-ENT) bacteria. The fourunknown cultures were subcultured on Biolog UniversalGrowth (BUG) agar and incubated at 45 �C for 16–24 h. After incubation, the cell suspensions were pre-pared in GN/GP-IF (inoculating fluid) at a cell densityof 52% T (transmittance) ± 3%. The contents werestirred and the turbidity read using the Biolog turbidi-meter. The suspension (150 ll) was dispensed into eachwell of the GN2 MicroPlate using an eight-channelmultipipette. The plates were then incubated at 30 �C(as recommended by the manufacturer) and read usingthe Biolog Microstation at 16 h.Gram positive bacteria. Gram staining was the only

preliminary test required to differentiate Gram positive-rod sporeforming bacteria (GP-ROD SB) from Grampositive-coccus (GP-COCCUS) or Gram positive-rod(GP-ROD) bacteria. The three unknown cultures weresubcultured on BUG + M (0.25% maltose) agar.Before streaking the bacterium, eight drops from athioglycollate ampoule was added into sterile water(3 ml). A sterile swab dipped into the solution was usedto spread a thin film of liquid across the entire surface of

962 N. Sheikh Abdul Hamid et al.

the agar medium and allowed to dry. The bacterium wasinoculated by streaking up and down three times andacross three times to make a narrow ‘+’ (plus) streak torestrain cell growth to two thin lines. The agar plateswere incubated at 45 �C for16–24 h.After incubation, a small amount of cells was scooped

onto the tip of a sterile wooden stick. The active cellsgrowing along the edges of the ‘+’ streak weresuspended in Gram negative/Gram positive-inoculatingfluid (GN/GP-IF) at a cell density of 28% T ± 3%. Thecontents were stirred and the turbidity read in the Biologturbidimeter. The suspension (150 ll) was dispensedinto each well of the GP2 MicroPlate. The plates wereincubated at 45 �C and read using the Biolog Micro-Station at 16 h.

Biochemical tests

Gram negative bacteria. The unknown Gram negativelipase-producing bacteria (ST 1, ST 4, ST 6 and ST 10)were identified using the identification scheme for Gramnegative bacteria according to MacFaddin (1980). Thenine biochemical tests carried out include oxidation-fermentation of glucose, oxidase, motility, urease, ci-trate, gelatin liquefaction, arginine dihydrolase, nitratereduction and growth at 42 �C. Twenty additionalbiochemical tests done according to Osterhout et al.(1998) include antibiotic susceptibility test using agardiffusion method, carbohydrate fermentation (arabi-nose, glucose, 10% lactose, maltose, mannitol andxylose), catalase, deoxyribonuclease (DNase), esculin,indole, lysine decarboxylase, phenylalanine deamina-tion, pigment production, growth in 6.5% sodiumchloride, starch hydrolysis, MacConkey agar, triplesugar ion agar and trypticase soy agar with 5% horseblood, and urease test. The blood agar test was alsodone to determine the haemolysis pattern. All thebiochemical tests were carried out using two incubationtemperatures, (35, 37 or 42 �C depending on thereference and 45 �C). The growth of cultures on nutrientagar plate at incubation temperatures ranging from 30to 65 �C was examined.Gram positive bacteria. The unknown Gram positive

lipase-producing bacteria (ST 7, ST 8 and ST 9) wereidentified using the identification scheme for Grampositive bacteria according to MacFaddin (1980).Biochemical tests performed include catalase, oxida-tion-fermentation glucose, carbohydrate fermentation(arabinose, glucose, 10% lactose, maltose, mannitol andxylose), motility, blood agar, nitrate reduction, ammo-nia, indole, gelatin liquefaction, Simmons citrate, Voges-Proskauer, urease, phenylalanine deamination, 6.5 and7.5% sodium chloride, starch hydrolysis, arginine dihy-drolase and lysine decarboxylase. All the biochemicaltests were carried out at two incubation temperatures,(35, 37 or 42 �C depending on the reference and 45 �C).The growth of cultures on nutrient agar plate atincubation temperatures ranging from 30 to 65 �C wasexamined.

Results and discussion

Comparative lipolytic activity of positive bacterial lipaseproducers

Lipolytic activities of seven lipase-producing bacteriaare shown in Figure 1. All the cultures showed goodlipolytic activities. Strain ST 7 exhibited a high lipolyticactivity of 4.58 U/ml followed by strain ST 6 with anactivity of 3.51 U/ml. Strains ST 10, ST 1 and ST 8 hadmoderate lipolytic activities of 2.62, 2.39 and 2.38 U/mlrespectively. Strains ST 4 and ST 9 showed lowerlipolytic activities of 1.84 and 1.80 U/ml respectively.

Thermostability of crude lipase

The thermostability of crude lipase was determined attemperatures ranging from 40 to 80 �C for 30 min. Theresults for bacterial strains ST 1, ST 6, ST 7, ST 8 andST 10 that had lipolytic activities of more than 2 U/mlare show in Figure 2. Strains ST 7 and ST 8 producedthe most thermostable lipase of the five strains exam-ined, with a recovery of 87 and 88% respectively at80 �C. At 80 �C, strains ST 1, ST 6 and ST 10 showedrecoveries of 36, 61 and 64% respectively.

Colony morphology, Gram reaction and microscopy

Table 1 shows that four of the lipase-producing bacteriawere Gram negative while the rest were Gram positive.All the Gram negative bacteria (ST 1, ST 4, ST 6 and ST10) possessed similar characteristics in terms of colonysize, colony morphology and cellular morphology,giving the possibility that these bacteria evolved fromthe same genus or species of bacteria. The two strains ofGram positive bacteria, ST 7 and ST 9, also showedsimilar characteristics. Strain ST 8 exhibited different

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

ST 1 ST 4 ST 6 ST 7 ST 8 ST 9 ST 10

Lip

olyt

ic A

ctiv

ity

(U)

4.58

3.51

1.84

2.39 2.38

1.80

2.62

Strains

Figure 1. Lipolytic activities of positive lipase-producing bacteria

isolated from the Setapak hot spring. See methods for growth and

assay conditions.

Thermostable bacterial lipase producers 963

characteristics in terms of colony size, colony morphol-ogy and cellular morphology compared to the other sixbacteria.Endospores were observed in all Gram positive

strains. The endospores are impermeable to the Gramstain and were observed as semi-transparent round spotsinside the rods. The endospores in strains ST 7 and ST 9could not be seen clearly. Endospore staining usingmalachite green was carried out to determine theposition of the endospore.

Identification of bacteria using the Biolog MicrostationSystem

Gram negative bacteria. Strains ST 1, ST 4, ST 6 and ST10 were identified as CDC group IVc-2 (Alcaligenes-like)using the Biolog Microstation System. The similarityindex for ST 1, ST 4, ST 6 and ST 10 were 0.739, 0.679,0.634 and 0.734 respectively. Biolog identifications were

reported if the similarity index of the genus or specieswas 0.750 or greater at 4 h, or 0.500 or greater at 24 h.The CDC group IVc-2, now classified as Ralstoniapaucula (Vandamme et al. 1999) is a Gram negativenon-fermentative rod that closely resembles Alcaligenessp. and Bordetella bronchiseptica in terms of biochemicalcharacteristics (Dan et al. 1986; Osterhout et al. 1998).Gram positive bacteria. Biolog identified strains ST 7

and ST 9 as Bacillus thermoglucosidasius, which is nowknown as Geobacillus thermoglucosidasius (Nazina et al.2001) and strain ST 8 as Bacillus subtilis. The similarityindex for ST 7, ST 8 and ST 9 were 0.846, 0.707 and0.704 respectively. Bacillus thermoglucosidasius andBacillus subtilis are aerobic Gram positive sporeformingbacteria. Bacillus thermoglucosidasius is a thermophilicbacteria with an optimum temperature of 61–63 �C(Sneath et al. 1986).The Biolog’s technical literature reported that the

Bacillus species are difficult to identify due to ‘false-positive’ reactions that may be a result of sporulation,utilization of lysed cell material, utilization of storedendogenous substrate or extracellular polysaccharides.Hence, biochemical tests were carried out to confirm theidentity of the bacteria.

Biochemical tests

Gram negative bacteria. Tables 2 and 3 show the resultsof the biochemical tests carried out for the identificationof Gram negative bacteria at two different incubationtemperatures. Incubation temperature was chosen as 35,37 or 42 �C depending on the methods mentioned byMacFaddin (1980) and Osterhout et al. (1998). Theincubation temperature of 45 �C was chosen because thebacteria of interest in this study should be thermophilic.All the four strains showed identical results at bothincubation temperatures with the only difference beingthe rate of reaction. Generally, the strains exhibited ahigher reaction rate at 35 �C as compared to 45 �C. Thismay be due to the strains’ optimum temperature for

30

40

50

60

70

80

90

100

110

40 50 60 70 80

Temperature (ºC)

Rel

ativ

e A

ctiv

ities

(%)

Figure 2. Thermal stability of crude lipases produced by ST 1, ST 6,

ST 7, ST 8 and ST 10. ––¤–– ST 1 ––n–– ST6 ––m–– ST 7 ––·–– ST8 ––+––

ST 10.

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;

smooth surface; beige colour

Negative Short rods



Negative Short rods



Negative Short rods

ST 7 1 Circular; umbonate; undulate edge;


Positive Rods with ellipsoidal endospores

at terminal, swollen cell

ST 8 4 Irregular and spreading; raised margin;

undulate edge; beige colour

Positive Rod with ellipsoidal at central

and subterminal

ST 9 1 Circular; umbonate; undulate edge;


Positive Rods with ellipsoidal endospores

at terminal, swollen cell



Negative Short rods

* 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.


growth that was closer to 35 �C rather than 45 �C.Table 6 shows four of the Gram negative bacteria thatcould grow at temperatures ranging from 30 to 45 �C.They grew weakly at 50 �C and their growth wasinhibited at the temperatures of 55 �C and above.The results obtained from the biochemical tests

tentatively identified strains ST 1 and ST 4, as Alcali-genes sp. (Alcaligenes faecalis or Alcaligenes odorans) atan incubation temperature of 35 �C. Strains ST 6 andST 10 were tentatively identified as B. bronchiseptica atthe same incubation temperature. The results of bio-chemical tests carried out at 45 �C however identifiedstrains ST 1 and ST 10 as belonging to the Alcaligenessp. and strains ST 4 and ST 6 as B. bronchiseptica. Thisdissimilarity was due to the urease test. At 35 �Cincubation temperature, strains ST 6 and ST 10 exhi-bited rapid urease production and were classified as B.bronchiseptica. At 45 �C, ST 4 and ST 6 showed rapidurease production, and were accordingly classified as B.bronchiseptica.

The Biolog System identified all the four cultures asCDC group IVc-2 (Alcaligenes-like), now known asRalstonia paucula (Vandamme et al. 1999). Additionalbiochemical tests were carried out according to Oster-hout et al. (1998) to determine whether the strains wereR. paucula. The results obtained from these biochemicaltests (Tables 2 and 3) were comparable to the resultsmentioned in literature. Except for the lysine decarboxy-lase and rapid urease production tests, the strains wereclassified as R. paucula. Ralstonia paucula exhibited anegative result for lysine decarboxylase test and apositive result for the rapid urease production test(Osterhout et al. 1998). All the four strains showedpositive results for the lysine decarboxylase test. StrainsST 1 and ST 4 exhibited negative result for rapid ureaseproduction at 35 �C. Strains ST 1 and ST 10 showednegative results for this test at 45 �C. Absolute confir-mation that all the unknown Gram negative lipase-producing bacteria were R. paucula was not possible dueto close biochemical resemblance to Alcaligenes spp. andB. bronchiseptica (Dan et al. 1986; Osterhout et al.1998).

Table 2. Results of biochemical tests carried out for Gram negative

bacteria carried out at either 35, 37 or 42 �C.

No. Biochemical tests Code

ST 1 ST 4 ST 6 ST 10

1 Antibiotic susceptibility

(i) amikacin (30 lg) R R R R

(ii) cefuroxime sodium (30 lg) S S S S

(iii) tetracycline (30 lg) S S S S

2 Arginine dehydrolase ) ) ) )3 Blood agar c c c c4 Carbohydrate fermentation

(i) Arabinose ) ) ) )(ii) Glucose ) ) ) )(iii) Lactose (10%) ) ) ) )(iv) Maltose ) ) ) )(v) Mannitol ) ) ) )(vi) Xylose ) ) ) )

5 Catalase + + + +

6 Citrate (Simmons) + + + +

7 Deoxyribonuclease (Dnase) ) ) ) )8 Esculin ) ) ) )9 Gelatin liquefaction ) ) ) )10 Growth at 42 �C + + + +

11 Indole ) ) ) )12 Lysine decarboxylase + + + +

13 MacConkey agar + + + +

14 Motility (37 �C) + + + +

15 Nitrate reduction ) ) ) )16 Oxidase + + + +

17 Oxidation-fermentation glucose ) ) ) )18 Phenylalanine deamination ) ) ) )19 Sodium chloride (6.5%) ) ) ) )20 Starch hydrolysis ) ) ) )21 Triple sugar ion agar ) ) ) )22 Trypticase soy agar with 5%

horse blood (37 �C)+ + + +

23 Urease production + + +R +R

24 Voges–Proskauer ) ) ) )

R – Resistant; S – susceptibility; c – gamma haemolysis (no change

of the medium); ) – negative result; + – positive result; R – rapid

urease (within 4 h).

Table 3. Results of biochemical tests for Gram negative bacteria

carried out at 45 �C.


ST 1 ST 4 ST 6 ST 10

1 Antibiotic susceptibility

(i) Amikacin (30 lg) R R R R

(ii) Cefuroxime sodium (30 lg) S S S S

(iii) Tetracycline (30 lg) S S S S

2 Arginine dehydrolase ) ) ) )3 Blood agar c c c c4 Carbohydrate fermentation

(i) Arabinose ) ) ) )(ii) Glucose ) ) ) )(iii) Lactose (10%) ) ) ) )(iv) Maltose ) ) ) )(v) Mannitol ) ) ) )(vi) Xylose ) ) ) )

5 Citrate (Simmons) + + + +

6 Deoxyribonuclease (Dnase) ) ) ) )7 Esculin ) ) ) )8 Gelatin liquefaction ) ) ) )9 Indole ) ) ) )

10 Lysine decarboxylase + + + +

11 MacConkey agar + + + +

12 Motility + + + +

13 Nitrate reduction ) ) ) )14 Oxidation-fermentation glucose ) ) ) )15 Phenylalanine deamination ) ) ) )16 Pigment production ) ) ) )17 Sodium chloride (6.5%) ) ) ) )18 Starch hydrolysis ) ) ) )19 Triple sugar ion agar ) ) ) )20 Trypticase soy agar with 5%

horse blood (37 �C)+ + + +

21 Urease production + +R +R +

22 Voges–Proskauer ) ) ) )

R – Resistant; S – susceptibility; c – gamma haemolysis (no change

of the medium); ) – negative result; + – positive result; R – rapid

urease (within 4 h).


There was a strong possibility that all the four strainswere R. paucula because Alcaligenes odorans causedgreen discoloration (partial haemolysis or called alphahaemolysis) in blood agar media, was tolerant at 6.5%sodium chloride (NaCl) and showed a negative ureasetest (Gilardi 1978; MacFaddin 1980). Alcaligenes fae-calis also gave a negative urease test and half of thestrains were capable of reducing nitrate to nitrite(Gilardi 1978; MacFaddin 1980). The results shown inTables 2 and 3 show significant differences from theAlcaligenes spp. Nitrate reduction is a conspicuouscharacteristic that differentiates R. paucula from B.bronchiseptica (Osterhout et al. 1998). Bordetella bron-chiseptica was capable of reducing nitrate to nitrite butnot R. paucula (Dan et al. 1986; Osterhout et al. 1998;Vandamme et al. 1999). Besides that, R. paucula wasable to grow in the presence of Tween 80 and exhibitedacid phosphatase activity, phosphoamidase activity andmost importantly, lipase C14 activity (Vandamme et al.1999). Bordetella bronchiseptica did not share thesecharacteristics with Ralstonia paucula.The results based on the biochemical tests carried out

in this study, closely resembled the characteristics ofRalstonia paucula as compared to Alcaligenes spp. andBordetella bronchiseptica. To date R. paucula has notbeen reported as a thermostable lipase producer al-though Vandamme et al. (1999) reported that lipase C14activity was exhibited by R. paucula. However, furtherstudy such as randomly amplified polymorphic DNA(RAPD), cellular fatty acid (CFA) and 16S rRNAanalysis should be carried out for identification confir-mation.Moissenet et al. (1996) concluded that DNA analysis

by the RAPD method proved to be useful for theepidemiological investigation of hospital outbreaks ofRalstonia paucula infection. Osterhout et al. (1998)reported that the combination of CFA and biochemicaldata is generally sufficient for accurate identification ofR. paucula. CFA analysis can be utilized as a rapidscreening method followed by selected biochemical teststo confirm the identification. When phenotypic methodsprove inconclusive, confirmatory genotypic techniquessuch as 16S rRNA gene sequence analysis are beneficial.CFA and rRNAs are two groups of macromolecules thathave recently been made amenable to simplified analysis.Analysis of the 16S rRNA gene sequence has proven tobe most useful in molecular systematics, since it is highlyconserved, universally distributed and contains diagnos-tically significant variable regions. Moissenet et al.(1999) reported that the ID-32-GN identification system(BioMerieux, Marcy-I’Etoile, France), based on 32metabolic assimilations tests, provided a simple, rapidand reliable means of identifying R. paucula with a highdegree of confidence (99.9%).Gram positive bacteria. Tables 4 and 5 show the results

of the biochemical tests carried out for the identificationof Gram positive bacteria. These tests were carried outat two different incubation temperatures, 35 or 37 and45 �C. Three of the strains exhibited higher reaction

rates at 45 �C as compared to 35 �C. This could be dueto the strains’ optimum growth temperature, which werecloser to 45 �C rather than 35 �C. Strains ST 7 and ST 9exhibited similar results at the two different incubationtemperatures. An exception was observed when strainST 9 showed a positive result for the urease test at45 �C. Differences existed in the following biochemicaltests for strain ST 8 at two different incubationtemperatures: (i) arabinose fermentation test, (ii) xylosefermentation test, (iii) Simmons citrate, and (iv) ureaseproduction. For the arabinose fermentation test, strainST 8 exhibited positive results at 35 �C and negativeresults at 45 �C. For the xylose fermentation test, thereaction was delayed when incubated at 35 �C and anegative result was obtained at 45 �C. Strain ST 8exhibited a negative result at 35 �C and a positive resultat 45 �C for Simmons citrate test. For the urease test,strain ST 8 showed a negative result at 35 �C and apositive result at 45 �C. As shown in Table 6, strains ST7 and ST 9 were able to grow at temperatures rangingfrom 30 to 65 �C. They grew weakly at 30 and 35 �C.Strain ST 8 grew at temperatures that ranged from 30 to60 �C but exhibited feeble growth at 55 and 60 �C, andno growth at 65 �C.Strains ST 7, ST 8 and ST 9 were identified as Bacillus

spp. The biochemical test results of strains ST 7 and ST9, showed similar results throughout the study. Bacillus

Table 4. Results of biochemical tests for Gram positive bacteria

carried out at either 35 or 37 �C.


ST 7 ST 8 ST 9

1 Ammonia ) + )2 Arginine dehydrolase ) ) )3 Blood agar b b a4 Carbohydrate fermentation

(i) Arabinose D + D

(ii) Glucose + + +

(iii) Lactose (10%) D ) D

(iv) Maltose + ) +

(v) Mannitol + + +

(vi) Xylose D D D

5 Catalase +w + +w

6 Citrate (Simmons) ) ) )7 Esculin ) + )8 Gelatin liquefaction ) + )9 Indole ) ) )

10 Lysine decarboxylase ) ) )11 Motility + + +

12 Nitrate reduction ) + )13 Oxidation-fermentation glucose F O/F F

14 Phenylalanine deamination ) ) )15 Sodium chloride (6.5%) + + +

16 Sodium chloride (7.5%) + + +

17 Starch hydrolysis ) + )18 Urease production ) ) )19 Voges-Proskauer ) ) )

) – Negative result; + – positive result; D – delayed (no colour

change); F – fermentation; O/F – oxidation and fermentation; b –

beta haemolysis (complete haemolysis); a – alpha haemolysis (partial

haemolysis); w – weak gas production.


stearothermophilus and B. coagulans were the mostpossible candidates for these strains. Strains ST 7 andST 9 had biochemical test results which differed from B.stearothermophilus in terms of O–F glucose, gelatinliquefaction, 7.5% sodium chloride and starch hydro-lysis tests. Strains ST 7 and ST 9 showed differenceswith B. coagulans in terms of 7.5% sodium chlorideand starch hydrolysis tests except for strain ST 9which showed a different result in the urease test.The Biolog system identified strains ST 7 and ST 9 as

B. thermoglucosidasius but the ‘false-positive’ reactionsin which all reaction wells gave positive results including

the control well, do not give credibility to the results.Comparison of the present biochemical test results ofstrains ST 7 and ST 9 with the five characteristics of B.thermoglucosidasius as reported by Nazina et al. (2001),showed that only the esculin test result was identical.Strains ST 7 and ST 9 gave different results for the otherfour tests (arabinose fermentation, gelatin liquefaction,starch hydrolysis and Simmons citrate). As observed inthe Tables 5 and 6, strains ST 7 and ST 9 gave delayed(D) and negative results for a number of tests. Thismight be due to the fact that the optimum temperatureof growth of ST 7 and ST 9 were more than 45 �C. Thereported optimum temperature for B. thermoglucosi-dasius was 61–63 �C (Sneath et al. 1986). Bacillus ther-moglucosidasius was rod-shaped with ellipsoidalendospores at the terminus of the swollen cells. StrainsST 7 and ST 9 possessed the same cellular morphologyas B. thermoglucosidasius. Nevertheless, confirmationtests would have to be performed to identify these twostrains up to the species level. Nazina et al. (2001)proposed the transfer of B. thermoglucosidasius to thenew genus Geobacillus, with Geobacillus thermogluco-sidasius as the type species. An additional characterfound by Kampfer (1994) is that the main cellular fattyacids are iso-15:0, iso-16:0 and iso-17:0, making up morethan 60% of the total fatty acids (Nazina et al. 2001).Thus, cellular fatty acid analysis could be carried outfor further identification.The Biolog system identified strain ST 8 as Bacillus

subtilis but some ‘false-positive’ reactions were evidentnecessitating biochemical tests for further confirmation.Possible matches for strain ST 8, include Bacillussubtilis, B. megaterium, B. licheniformis and B. firmus.The variation in biochemical results between strain ST 8and the four strains mentioned are as follows: (1)differences in the biochemical results of B. subtilis wasobserved in the O–F glucose, Voges–Proskauer, arabi-nose and xylose fermentation tests; (2) differences in thebiochemical results of B. licheniformis was observed inthe O–F glucose, Voges–Proskauer, arabinose and xy-lose fermentation tests; (3) differences in the biochemi-cal results of B. firmus was observed in the O–F glucose,Simmons citrate, urease and phenylalanine deaminase

Table 5. Results of biochemical tests for Gram positive bacteria at

45 �C.


ST 7 ST 8 ST 9

1 Ammonia ) + )2 Arginine dehydrolase ) ) )3 Blood agar b b a4 Carbohydrate fermentation

(i) Arabinose D ) D

(ii) Glucose + + +

(iii) Lactose (10%) D ) D

(iv) Maltose + ) +

(v) Mannitol + + +

(vi) Xylose D ) D

5 Citrate (Simmons) ) + )6 Esculin ) + )7 Gelatin liquefaction ) + )8 Indole ) ) )9 Lysine decarboxylase ) ) )10 Motility + + +

11 Nitrate reduction ) + )12 Oxidation-fermentation glucose F O/F F

13 Phenylalanine deamination ) ) )14 Sodium chloride (6.5%) + + +

15 Sodium chloride (7.5%) + + +

16 Starch hydrolysis ) + )17 Urease production ) + +

18 Voges-Proskauer ) ) )

) – Negative result; + – positive result; D – delayed (no colour

change); F – fermentation; O/F – oxidation and fermentation; b –

beta haemolysis (complete haemolysis); a – alpha haemolysis (partial

haemolysis).

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

1 30 + + + + +w + +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.


tests; (4) differences in the biochemical results of B.megaterium was observed in the motility, O–F glucoseand phenylalanine deaminase tests.Biochemical test results obtained from this study were

similar to the characteristics of Bacillus subtilis. Accord-ing to Sneath et al. (1986), Bacillus subtilis and strainST 8 exhibited negative results for both arginine dihy-drolase and lysine decarboxylase tests. Bacillus licheni-formis showed positive results for the argininedihydrolase test and no results for this test were reportedfor Bacillus firmus. Although Bacillus megaterium ex-hibited same results for both the tests, it could bedifferentiated from Bacillus subtilis because strain ST 8was able to grow at 50 �C. Both Bacillus megateriumand Bacillus firmus were unable to grow at 50 �C(Sneath et al. 1986). Sneath et al. (1986) and Yeap(1998) have also identified Bacillus subtilis as a positivebacterial lipase producer whereas Bacillus megateriumwas not.Due to the lack of discriminating biochemical fea-

tures, more reliable identification systems such as APISystem and 16S rRNA analysis could be carried out forfurther confirmation of the bacterial identity. Logan &Berkeley (1984) reported that the API System tests weremore reproducible than conventional biochemical tests.Ash et al. (1991) and Rainey et al. (1994) reported that16S rRNA gene sequence analysis has revealed highphylogenetic heterogeneity in the genus Bacillus (Nazinaet al. 2001).Although diagnostic keys and tables for Bacillus have

been available for a long time, the identification of theseorganisms is still complicated. Bacillus is an unusuallywide taxon containing mostly aerobic endospore-form-ing rods (Logan & Berkeley 1984). According to Priest(1981), as cited by Logan & Berkeley (1984), in terms ofDNA base ratios it is the equivalent of some bacterialfamilies. Furthermore, some species are ill-defined,existing with closely resembled species as complexes orspectra in which the boundary of a particular species isdifficult or impossible to identify. Even in well estab-lished species, there is considerable variation betweenstrains (Logan & Berkeley 1984). As cited by Nazinaet al. (2001), phylogenetic analysis revealed that thegenus Bacillus and its thermophilic members requireextensive taxonomic revision (Stackebrandt et al. 1987;Ash et al. 1991; Rainey et al. 1994).

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