a new enrichment method for isolation of bacillus thuringiensis from diverse sample types

A New Enrichment Method for Isolation of Bacillusthuringiensis from Diverse Sample Types

Ketan D. Patel & Forum C. Bhanshali &Avani V. Chaudhary & Sanjay S. Ingle

Received: 4 February 2012 /Accepted: 18 February 2013# Springer Science+Business Media New York 2013

Abstract New or more efficient methodologies having different principles are needed, asone method could not be suitable for isolation of organisms from samples of diverse typesand from various environments. In present investigation, growth kinetics study revealed ahigher germination rate, a higher growth rate, and maximum sporulation of Bacillusthuringiensis (Bt) compared to other Bacillus species. Considering these facts, a simpleand efficient enrichment method was devised which allowed propagation of spores andvegetative cells of Bt and thereby increased Bt cell population proportionately. The newenrichment method yielded Bt from 44 out of 58 samples. Contrarily, Bt was isolated onlyfrom 16 and 18 samples by sodium acetate selection and dry heat pretreatment methods,respectively. Moreover, the percentages of Bt colonies isolated by the enrichment methodwere higher comparatively. Vegetative whole cell protein profile analysis indicated isolationof diverse population of Bt from various samples. Bt strains isolated by the enrichmentmethod represented novel serovars and possibly new cry2 gene.

Keywords Bacillus . Spore . Enrichment . Growth kinetics

AbbreviationBt Bacillus thuringiensis

Introduction

Bacillus thuringiensis is a Gram-positive soil bacterium known to produce larvicidal Cry,Cyt, and Vip toxins. It occurs naturally in diverse habitats like aquatic environments,phylloplanes, brackish sediments, mountain soils, etc. [1–6]. It is the most valuable andwidely used environment-friendly biopesticide [7], which occupies more than 90 % of theworld biopesticide market [8]. The main advantages of B. thuringiensis (Bt) biopesticides aretarget-specific action, lack of toxicity to humans, and ease of commercial production.

Appl Biochem BiotechnolDOI 10.1007/s12010-013-0145-y

K. D. Patel (*) : F. C. Bhanshali : A. V. Chaudhary : S. S. IngleGround Floor Laboratory, Department of Microbiology and Biotechnology Center,The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, Indiae-mail: [email protected]

However, few problems have been encountered with Cry toxins of Bt which demandssearch for novel toxins. The most recent is the emergence of secondary (sap sucking) pestswhich pose a great threat to the use of Cry toxins. The sap-sucking pests such as cottonaphids (Aphis gossypii), mealybug (Phenacoccus species), whitefly (Bemisia tabaci), andmirid (Creontiades species) are not susceptible to Bt toxins [9]. Resistance development inHelicoverpa armigera, Plutella xylostella, and Trichoplusia ni insects is another majorconcern and necessitates the use of novel toxins with no cross-resistance [10–12]. Theinability of Bt toxins to target more than one insect needs to be addressed by a search fortoxins with broader spectrum of activity. Thus, continuous isolation and screening ofBt strains with novel toxins are needs of the time to meet these challenges.

A variety of methods for isolation of Bt from different types of samples have beenreported [13, 14]. The sodium acetate selection method as described by Travers et al. [15]is used extensively for Bt isolation [16, 17]. Other methods involve heat treatment to soilsuspension or antibiotic treatments to eliminate bacteria other than Bt [18, 19]. Santana et al.[20] found dry heat pretreatment to effectively reduce non-Bt spores from soil samples.However, during present investigation, successful isolation of Bt was not achieved frommost of the soil samples by sodium acetate selection, dry heat pretreatment, and antibioticselection. Thus, an efficient and simple isolation of Bt from diverse sample types wasdevised.

Materials and Methods

Cultures Used

Standard strains of B. thuringiensis subsp. kurstaki HD1, B. thuringiensis subp. kurstakiHD73, Bacillus cereus ATCC 14579, Bacillus megaterium QMB1551, Bacillus sphaericusWHO 2297, and Bacillus subtilis 1012M15 were obtained from the Bacillus Genetic StockCentre (BGSC), Ohio State University, USA. Bacillus licheniformis K125 was obtainedfrom our departmental culture collection [21].

Growth Kinetics in GYS Sporulation Medium

In order to perform growth kinetic study for seven different Bacillus species as mentionedearlier, spore suspension with equal spore concentration (107 spores per ml) was prepared tobe used as an inoculum. An equal number of spores (250 μL aliquot from 107 spores/ml ofsuspension), for each of the seven standard Bacillus species, were inoculated separately into25 ml glucose yeast extract salt (GYS) medium prepared in 250-ml flasks. Inoculated GYSmedium was incubated at 30 °C with 200 rpm shaking condition for 2 days. Aliquots werewithdrawn at regular intervals to monitor the growth by viable count.

Bt Isolation Methods

Soil samples were collected in sterile plastic collection vials and stored at 4 °C until use. Inthe enrichment method, 1 g of soil sample was used as an inoculum and added aseptically to20 ml of sterile GYS sporulation medium prepared in 250 ml flask (GYS per liter: 1 gglucose, 2 g yeast extract powder, 2 g NH4(SO4)2, 0.06 g MnSO4, 0.4 g MgSO4·7H2O,0.08 g CaCl2, 5 g K2HPO4). This mixture was incubated at 30 °C for 2 days on a rotaryshaker at 200 rpm. An aliquot of 1 ml was withdrawn and gently centrifuged at 1,000 rpm

Appl Biochem Biotechnol

for 1 min to settle down soil particles. Heat treatment was given to the supernatant at 80 °Cfor 3 min and serially diluted up to 10−8. An aliquot of 200 μL from 10−6 to 10−8 dilutionswas spread on Luria-Bertani broth (LB) agar plates. Bt-like colonies were observed afterincubation of plates at 30 °C for 2 days. When low percentages of Bt like colonies wereobserved, plating was repeated on 120 mm Petri plates using 500 μL aliquot. In case ofphylloplane samples, ∼2 cm2 sections were cut from three different leaves of the same sampleand added aseptically in 20 ml GYS medium. For sewage sample, 2 ml of mixed liquid wasadded directly to 20ml GYSmedium in aseptic condition. Rest of themethod of Bt isolation forphylloplane and sewage sample was performed in same manner as for soil sample.

The sodium acetate selection and dry heat pretreatment methods were performed asdescribed by Travers et al. [15] and Santana et al. [20], respectively. From each isolationmethod, Polymyxin B sulfate selection was performed by plating 200 μL of inoculum fromthree methods on plates containing 50 U/ml of the antibiotic.

Statistical Analysis of Bt Isolation

In order to perform statistical analysis, isolation of Bt from ten of the 53 soil samples wascarried out in triplicates by all three methods. Soil samples 1 and 2 were from Jaspur,samples 3, 4, and 5 from Raika, sample 6 from Baroda, and sample 8 from Sarangpur area ofGujarat State, India. Each sample from Jaspur and Raika was from different sites. Sample 7was from Kutch, sample 9 from Khordung La, Jammu State, and sample 10 from Bhopal,Madhya Pradesh State, India. Screening for Bt isolates was done by phase contrast micros-copy followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)and confirmation by PCR analysis [22]. Comparison of the percentage of Bt colonies to othercolonies on the isolation plate was done. Statistical analysis was performed by one-wayANOVA test using Fisher’s LSD in SigmaState 3.5 software to derive significant differencein the percentage of Bt colonies isolated by the three methods.

Vegetative Whole Cell Protein Profile

A single Bt colony was inoculated in 5 ml LB broth medium and grown until OD at 600 nmreaches ∼0.8 absorbance unit. Two-milliliter culture was pelleted down, washed twice with0.85 % NaCl, resuspended in 100 μL of 50 mM sodium hydroxide, and incubated for 10 minat RT. Immediately, 20 μL of 6× loading dye was added to 100 μL suspension, boiled for2 min, and 30 μL was loaded onto 10 % SDS-PAGE. Gels were stained with CoomassieBrilliant Blue R250 staining dye.

Comparative Results for Three Methods

In order to compare the characteristics of Bt strains isolated by three methods,isolation from ten soil samples was done by three methods (Table 2). Sample 1 wasfrom Chandoli District, Uttar Pradesh, India and samples 2 to 10 were from differentregions of Gujarat State, India. Sodium acetate selection, dry heat pretreatment, andenrichment methods yielded 12, 11, and 19 different strains of Bt, respectively, fromthe ten soil samples. Sample preparation for phase contrast microscopy of crystalinclusions, Cry protein analysis by SDS-PAGE, and for detection of cry genes byPCR analysis were done as described in our previous studies [22]. Frequency andpercent observation of various types of crystal inclusions, Cry protein bands, andcry/cyt genes were compared for ten soil samples (Table 2).


Results

Growth Kinetics of Bacillus Species in GYS Medium

Differences in the rate of spore germination, growth rate, and spore formation were observedin growth kinetic studies of different Bacillus species with Bt having the highest growth rate(Fig. 1). Germination of Bt and B. cereus spores was found most rapid compared to otherBacillus species used in the study, while that of B. licheniformis and B. megaterium was theslowest. Bt and B. cereus, which are considered alike species, showed higher growth rates inthe exponential phase as compared to other Bacillus species. Maximum sporulation wasobserved for Bt and B. cereus followed by B. licheniformis and B. subtilis after 48 h ofgrowth. Though B. sphaericus had growth rate higher than B. licheniformis, the extent ofspore formation was found to be comparatively less.

Bt Isolation by Different Methods

When 53 soil samples from diverse geographical regions were screened for the presence ofBt, we could isolate Bt only from 16 and 18 soil samples by sodium acetate selection methodand dry heat pretreatment method, respectively (Table 1). Polymyxin-sensitive strains of Btwere found to be present in many soil samples. In order to improve isolation of Bt from thesesamples, soil samples were added aseptically to GYS medium and after incubationproceeded for Bt isolation. Surprisingly, Bt strains were isolated from 39 soil samples bythe enrichment method. Furthermore, Bt strains were isolated from two sewage samples andthree phylloplane samples only by enrichment method.

A significant difference was observed in the percentages of Bt colonies obtained by theenrichment method (Fig. 2). Higher percentages of Bt colonies (55–75 %) as compared toother methods (5–15 %) were observed from soil samples 1, 2, 3, 4, and 10 by theenrichment method. Bt colonies were obtained only by the enrichment method from soil

Fig. 1 Growth kinetics of different Bacillus species in GYS sporulation medium


samples 5, 6, 8, and 9 though at low percentages (10–30%). These samples showed Bt colonieswith background of B. cereus-like or mixed Bacillus colonies. The total number of differentstrains isolated by enrichment method was higher comparatively (Table 2). Bt strains isolates byenrichment method showed higher frequency of crystal inclusions, cry/cyt genes, and Cryprotein bands, while the percentage of observation was higher or comparable.

Vegetative Whole Cell Protein Profile Analysis

In order to exclude sibling species, vegetative whole cell protein profile analysis wasperformed [23]. Thereby, the number of different Bt strains present in a soil sample wasdetermined. Bt colonies obtained by the enrichment method showed diverse population of Btstrains in most of the soil samples. Bt strains isolated by enrichment method from soilsamples 1 and 3 showed three and four different banding patterns, respectively (Fig. 3). Itthus indicated the presence of three and four different Bt strains in samples 1 and 3,respectively. Bt strains from samples 2, 5, 6, and 10 showed two distinct patterns. In contrast,

Table 1 Comparison of Bt isolation by different methods

Isolation method Total samples examineda Bt-positive samplesb Bt index range

Enrichment method 58 44 (75.8) 0.8–0.08

Sodium acetate selection 58 16 (27.5) 0.12–0.01

Dry heat pretreatment 55 18 (33.7) 0.17–0.03

Polymyxin selection 28 4 (14.2) ND

Numbers in brackets indicate percentagesa Number of total samples examined which include soil, phylloplane, and sewage samplesb Percentage of samples with at least one Bt isolate

ND Not Determined

Fig. 2 Statistical analysis of Bt isolation by sodium acetate selection, dry heat pretreatment, and enrichmentmethod from soil samples 1–10. Note: n=3; a, b, and c represent significant differences in percentage of Btcolonies obtained by different methods (p value<0.05). Percent Bt colonies were calculated as the percentageof Bt colonies to other colonies. Bt colonies were identified by microscopy, SDS-PAGE, and or PCR analysis


Table 2 Comparative results of frequency and percent observation for crystal inclusions, cry/cyt genes, andCry protein bands in Bt strains isolated from ten samples by three methods

Sodium acetateselection method (12)

Dry heat pretreatmentmethod (11)

Enrichmentmethod (19)

Crystal inclusions

Spherical 5 [42] 4 [36] 7 [37]

Bipyramidal 6 [50] 6 [55] 12 [63]

Cuboidal 5 [42] 6 [55] 12 [63]

Amorphous 0 0 2 [10]

cry and cyt genes

cry1 6 [50] 6 [55] 12 [63]

cry2 6 [50] 6 [55] 11 [58]

cry3,7,8 0 0 2 [10]

cry11 2 [17] 2 [18] 4 [21]

cyt 0 0 3 [16]

Cry protein sizesa

130–135 6 [50] 6 [55] 10 [53]

90–97 12 [100] 11 [100] 19 [100]

70–85 4 [33] 4 [36] 7 [37]

60–66 9 [75] 9 [82] 13 [68]

50–55 9 [75] 8 [73] 14 [74]

45 4 [33] 3 [27] 5 [26]

27–29 1 [8] 1 [9] 2 [10]

Note: Figure in round brackets indicate number of Bt strains isolated by the respective method; figures insquare brackets indicate percent observations in isolatesa Size of band in SDS-PAGE analysis for Cry proteins

Fig. 3 Vegetative whole cell protein profile of Bt isolates from samples 1–10 isolated by sodium acetateselection, dry heat pretreatment and enrichment method. Note: SA, Bt strains isolated by sodium acetateselection method; EN, Bt strains isolated by enrichment method; DH, Bt strains isolated by dry heatpretreatment method; numbers in brackets are soil sample numbers as given in Fig. 2. Lane M Proteinmolecular weight marker


Bt strains isolated by sodium acetate selection and dry heat pretreatment methods from samples1, 2, and 3 showed a single pattern, except sample 4 which showed two patterns.

Characterization of Bt Isolates

As a result of isolation by new enrichment method from soils, Bt isolates with novel types ofcrystal inclusion formations were obtained in previous studies [22]. Novel serovars withunknown H3abce and H3ab type were found which could not be classified under any knownserotype [24]. Partial cry2 gene sequence from isolate Bt LI93 (NCBI accession no.FJ416494) showed only 95 % nucleotide sequence identity to cry2Ab and cry2Ah genesand thus could be a novel cry2 gene [25]. Few of the isolates showed RFLP digestionpatterns which did not match to any of the known cry1A genes [25]. SDS-PAGE analysis forCry protein content of Bt isolates showed protein bands of 140, 130, 120, 95, 80, 75, 70, 66,55, 35, and 29 kDa, and PCR analysis revealed presence of various cry genes [22, 24–26].

Discussion

Bt isolation was not successful from many of the soil samples using sodium acetate selectionas reported by several authors [4, 20, 27]. Similarly, isolation using dry heat pretreatmentmethod did not yield Bt colonies from more samples. This could be due to low density of Btin soil. Henceforth, a new method for isolation of Bt was investigated which would allowpropagation of Bt cells from samples with low density of spores and vegetative cells of Bt.

During initial study of Bt isolation, larger size of Bt colony was observed compared toother Bacillus species which indicated a higher growth rate of Bt comparatively. Thisobservation prompted us to compare growth kinetics of different Bacillus species. Growthkinetic study revealed a higher rate of germination of Bt along with B. cereus and had ahigher growth rate compared to other Bacillus species (Fig. 1). Shnayderman et al. [28]similarly observed the growth rate of Bt to be higher than B. subtilis and E. coli in LBmedium at 30 °C. Also, Bt showed maximum sporulation at the end of 48 h of incubation.Considering these observations, a new method was devised on the basis of growth rate of Bt.

In the enrichment method, a sample is added to the GYS medium which contains only 0.1 %glucose and 0.2 % yeast extract. Initially, it allows germination of Bacillus spores and growth ofvegetative cells. But with the depletion of nutrients, spore-forming bacteria like Bt initiatesporulation, while the non-spore formers get killed or their growth is prevented. Bt and otherBacillus species of B. cereus group with rapid germination, higher growth rate, and maximumsporulation increase in number proportionately among survivors. Incubation until 48 h and heattreatment in a subsequent step completely eliminate remaining vegetative cells without affectingspores in the suspension. The enrichmentmethod allows propagation of both spores and vegetativecells of Bt initially and thus increases Bt spore population proportionately at the end of 48 h ofincubation, thereby improving the chances of Bt isolation from samples with low density of Bt.

Isolation of Bt by the enrichment method yielded higher percentages of Bt colonies(55–75 %) in many soil samples (Fig. 2) and higher frequency of crystal inclusions, Cryprotein bands, and cry/cyt genes (Table 2). Some samples showed Bt isolation only by theenrichment method at low percentage (10–30 %) with background of B. cereus-like colonies,which could be due to the propagation of Bt. It indicated isolation of Bt, even from sampleswith higher proportion of other Bacillus species and low density of Bt. Polymyxin-sensitivestrains of Bt were found to be present in many samples. Similarly, polymyxin-sensitiveBt strains were isolated from India [29] and from other parts of the world [15, 29].


Since the enrichment method allows propagation of Bt cells, dominance by one strain of Btcould occur. However, vegetative whole cell protein profile analysis showed isolation ofdifferent Bt strains from same soil samples (Fig. 3). Johnson and Bishop [31] observed diversityin Bt strains, isolated after three rounds of propagation in the penicillin cycling method. Thus,the possibility of domination by one strain of Bt can be ruled out unless one strain being presentin very high proportion. On the other hand, Bt strains with slow growth rate, if present, could beexcluded during isolation by the enrichment method. To our experience, all Bt strains isolatedby the three methods exhibited similar growth rate during vegetative whole cell protein profilestudy. Similarly, Travers et al. [15] found 40 strains of Bt having similar growth pattern. Hence,if found to be present, there would be less number of Bt strains with slow growth rate.

Since one method could not suit for isolation from diverse samples, there is a continuingneed to explore the benefits of new methodologies for the detection of Bt from theenvironment. The enrichment method with its efficiency of isolating Bt from samples havinglow density of Bt and without any selection pressure will be useful for isolation with easefrom diverse types of samples.

Acknowledgments KDP would like to thank Gujarat State Biotechnology Mission, Gandhinagar, Gujarat,India for the research fellowship and grant for the project. All authors are very thankful to Dr. Daniel R.Zeigler, BGSC, Ohio State University, Ohio, USA for kindly providing Bacillus cultures. All authors wouldlike to thank Prof. Anjana Desai for providing and developing excellent department facilities.

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a new enrichment method for isolation of bacillus thuringiensis from diverse sample types

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