24 interactions among bacilli

Ayesha Patela and Anthony Ouelletteb, 1

a Department of Chemistry and

b Department of Biology & Marine Science,

Jacksonville University, Jacksonville, Fl, USA

This paper describes Ayesha Patel’s senior research project carried

out spring 2010, in partial fulfillment for the Bachelor of Science

in Biochemistry

Resubmitted on: April 15, 2011

1 Corresponding author: 2800 University Blvd N, Jacksonville, FL 32211. Office #: 904-256-7299.

Fax #: 904-256-7573. E-mail address: aouelle@ju.edu.

Abstract

Seven bacterial samples were isolated from upstate New York water bodies. Bacterial

strains 101, 105 and 106 were isolated from Rice creek whereas 203, 205, 206 and 207

were isolated from Rice pond in Rice Creek Field station at State University of New

York, Oswego, NY. To determine whether there were chemical interactions between

bacteria, six different bacteria were grown in bottom agar whereas a seventh bacterium

was mixed with agar and poured on top of bottom agar. Potential interactions were

observed in order to determine if any of the six bacteria inhibited the growth of the

seventh bacterium. There were three triplicates for each plate in each of the three

conditions summing up to 63 plates. Zones of inhibition were measured and are reported

in percentages. Bacillus pumilus (106) inhibited all other six strains whereas Bacillus

pumilus (206) inhibited only Bacillus pumilus (105) and Bacillus licheniformis (205).

Bacillus cereus inhibited Bacillus pumilus and Bacillus licheniformis. Bacterial strains

were identified using Enterotubes II and metabolic tests. Bacterium strain 101 was

Bacillus cereus whereas 203 and 205 were Bacillus licheniformis. Bacterial strains 105,

106, 206 and 207 were different strains of Bacillus pumilus. These results displayed the

intraspecific and interspecific competition among the bacteria which may be

representative of interactions in natural environment.

Introduction

Bacillus cereus, Bacillus pumilus, and Bacillus licheniformis are three organisms

identified in this paper. Bacillus cereus can be usually found in soil, groundwater, and

other dried food [11]. Bacillus licheniformis is a toxic and saphrophytic organism,

commonly found in soil and in aquatic species. It can be found in food such as poultry,

meat, stews and curries [4]. Bacillus pumilus can be also found in soil, water and air [9].

Bacteria compete for the limited resources to survive in an ecosystem. There can

be several types of competition. Two types of competition applicable to this paper are

intraspecific and interspecific competition. Intraspecific competition is between members

(bacteria) of the same species whereas interspecific is between members (bacteria) of

different species. Intraspecific competion may result in logistic population growth over

time. An example of intrasepecific competition would be between two different strains of

Bacillus pumilus. An example of intraspecific competition would be between Bacillus

cereus and Bacillus pumilus. Two possible outcomes can occur: either they balance by

sharing the resources or out-compete each other [1].

There is another type of effect that an organism can have on another: Allelopathy.

The word Allelopathy derives from “allelon” and “pathos” which means “each other” and

“to suffer,” respectively. In allelopathy, an organism releases a chemical substance, an

allelochemical, into an environment which acts as growth inhibitor to another organism.

Allelochemicals can be divided into three categories: kariomones, allomones and

synomones. Kariomones benefit the receiving organism but harm the producing

organism whereas allomones benefit the producer and do not affect the receiver.

Synomones benefit both producer and receiving organism

[http://csip.cornell.edu/Projects/CEIRP/AR/Allelopathy.htm].

Bacteriocins, peptide-based chemicals, are a type of antibacterial allelochemical.

There are three types of bacteriocins: lanthibiotics (which contains modified amino acids

lanthionine), cystibiotics (containing one or more disulfide bonds important for their

activity) and thiolbiotics (which contains an active-SH group) [12]. Bacteriocins can kill

nearby cells by destroying cellular DNA, preventing synthesis of proteins or by creating

channels in cytoplasmic membranes [10]. They can be used as food-preservatives and

also as therapeutic agents [2].

Bacteriocin-producing organisms can be identified using several methods such as

metabolic tests, staining methods, protein analysis such as electrophoresis, serological

methods, flow cytometry, gene analysis and southern blotting, phage typing etc

[http://www.mansfield.ohio-state.edu/~sabedon/ biol3010.htm#protein_analysis].

Metabolic tests, Enterotubes II and staining methods such as gram staining and endospore

staining are used to identify bacteria in this paper. Metabolic tests such as fermentation

tubes, oxidative-fermentative metabolism, starch hydrolysis, citrate, urease, gelatin

hydrolysis, indole, phenylalanine deamination, hydrogen sulfide production, nitrate

reduction, oxidase and catalase tests were also used. Fermentation tests such as adonitol,

lactose, glucose, arabinose, sorbitol, mannitol, and dulcitol were used to detect acid and

gas production from these carbohydrates. Starch hydrolysis is used to detect the presence

of starch by adding Gram’s iodine. Citrate tests identify whether the bacteria can ferment

citrate, which will turn the media blue. Phenylalanine deamination removes an amino

group from L-phenylalanine, which produces phenylpyruvic acid and ammonia. Catalase

is detected by adding hydrogen peroxide to the bacteria which forms bubbles [6].

The objectives of this paper are (1) to determine whether there are bacterial

interactions between seven samples and if one bacterium inhibits the growth of other

bacteria in different conditions by measuring zones of inhibition and (2) to identify the

interacting bacteria. First, six bacteria (potential producer strains) were stabbed in media

and incubated under certain condition. On the next day, the seventh bacterium (indicator

strain) was mixed with agar and poured on already grown bacteria and incubated at the

same condition. The zones of inhibition were then measured and recorded. At last, the

interacting bacteria were identified using metabolic tests.

Materials and Methods

Bacterial strains and culture conditions

Bacterial strains 101, 105 and 106 were isolated from Rice Creek whereas 203,

205, 206 and 207 were isolated from Rice Pond at Rice Creek field station located at the

State University of New York College, Oswego, NY by John F. Heagerty (Figure 1 & 2)

[Personal communication]. These samples were frozen in glycerol stocks at -70ºC in

Ultra-low freezer (SO-Low). They were streaked twice and incubated at 37º C in Tryptic

Soy Agar medium (TSA, Fischer Scientific). Then, each bacterial strain was grown in

Tryptic Soy Broth (TSB) at 37º C while shaking.

Stab plate assay and pour plate technique

Six bacteria (potential producer strains) from TSB culture tubes were stabbed

vertically once in a TSA plate. On the same day, the seventh bacterium (indicator strain)

was inoculated in TSB tube and grown at 37º C in an incubator shaker. On the next day, 4

mL of the indicator strain was mixed with 20 mL of TSA in an Erlenmeyer flask. Then,

this mixture was poured on top of the six grown bacteria (in bottom agar) as much as

needed to cover the producer strains. The plates were incubated at three different

conditions (Figure 3). After stabbing six bacteria, the plates were either incubated at 30º

C for 24 hours, room temperature (~ 20º C) for 24 hours, or at room temperature for 48

hours. After the seventh bacterium (indicator strain) was poured on top of the six grown

bacteria (producer strains in bottom agar), the plates were incubated at the same condition

as the stabbed plates (Figure 3).

Measurement of inhibition zone:

After the poured plates were incubated, they were observed using a colony

counter (by Leica Quebec Darkfield) to measure the zone of inhibition with a ruler. The

zone of inhibition was determined for each bacterium by measuring the diameter of outer

circle (area of clear edge) minus diameter of inner circle (growth of producer strain).

Three triplicates for each plate and each condition were carried out. Therefore, inhibition

zones were reported in percentages which state how many times out of three plates a

bacterial strain inhibits the growth of other strain.

Morphology of bacteria (Staining methods):

Gram staining was performed on all bacterial strains to determine the Gram

reaction, shape and size of the bacteria from both semi-solid and liquid culture.

Micrographs were taken and the sizes of bacteria were measured. Five measurements

were obtained and averaged together to get the length and width of each bacterium using

Swift cam Imaging II. Endospore stains were also performed on one-week old cultures.

Identification of bacteria (using metabolic tests and Enterotubes II):

All seven bacterial strains were inoculated in Enterotubes II (BD BBL by Beckton

Dickinson). In addition, nitrate reduction, fermentation of mannitol, starch hydrolysis,

catalase and oxidase tests were performed on all bacterial strains. Strains 203 and 205

were also identified using additional tests such as fermentation of sucrose, gelatin

hydrolysis, ornithine decarboxylase, sulfide production, indole production, motility,

phenylanine deamination and oxidation-fermentation metabolism tests [6]. Bacterial

strains 101, 105, 106, 206 and 207 were previously identified using 16S rRNA gene

analysis by Ouellette [data not shown]. Therefore, metabolic tests and enterotubes were

used as support tests to support the identified bacteria [6].

Results

Identification of bacteria (using metabolic tests and Enterotubes II):

Based on the results of enterotubes II, other metabolic tests and Bergey’s Manual

[11], the identification of bacteria was determined for 203 and 205 whereas results of

101, 105, 106, 206 and 207 were used as confirmation (as they were previously identified

using 16S rRNA gene analysis) (Table 1 & 2 ). Bacterium strain 101 is B. cereus whereas

203 and 205 are B. licheniformis (Table 1 & 2). Strains 101, 105, 106, 206 and 207 were

B. pumilus; although they are different strains (Table 1). Nitrate reduction, fermentation

of mannitol and arabinose, and starch hydrolysis tests were used to support and

differentiate the identities of 101, 105, 106, 206 and 207 by comparing with metabolic

results of B.s pumilus and B. cereus (Table 1) [11].

Morphology of bacteria (using staining methods):

All seven bacterial strains were Gram-positive and bacilli in shape (Figure 4a &

4b; Appendix), and formed endospores. B. cereus (101) had central endospores whereas

B. pumilus (106) had central, terminal and free endospores. Endospores of B.pumilus

(206) were mostly seen as free endospores (Figure 5a & 5b, Appendix). B.cereus (101)

was largest whereas B.pumilus (206) was smallest among seven strains. B.pumilus (105,

106, and 207) and B.licheniformis (203 & 205) were similar in sizes (Table 1 & 2).

Bacterial interactions among Bacilli

Each plate was inoculated with six potential producer strains (potential to produce

bacteriocins) and a seventh bacterium strain as an indicator strain (to see if it is inhibited

by any of the producer strain). Each bacterial strain was used as an indicator strain

against the other six bacteria. Therefore, seven differently stabbed plates were used for

the seven different bacterial strains. There was a total of 21 plates (7 bacterial strains 3

trials/triplicates) for each condition summing up to 63 plates (21 plates 3 conditions).

Zones of inhibition were measured to determine whether one bacterium inhibits

the growth of the other bacteria, and if temperature and/or time has an influence. An

example of one of the plates with zone of inhibition is shown in Figure 6. Zones of

Inhibition differed in all three conditions: 30º C for 24 hours, room temperature for 24

hours and room temperature for 48 hours. Zones of Inhibition were reported in

percentages to determine how many indicator strains were inhibited by producer strains

out of three plates in each condition (Table 3).

At 30º C for 24 hours, strain B.cereus (101) was inhibited only by B.pumilus

(106) whereas B.licheniformis (205) was inhibited by B.cereus (101), B.pumilus (105,

106) and B.licheniformis (203). Bacterial strains B.pumilus (206 and 207) did not inhibit

any strains at all (Table 3). At room temperature for 24 hours, B.cereus (101) and

B.pumilus (106) was not inhibited by other strains at all whereas B.licheniformis (205)

was inhibited by all other strains (Table 3). At room temperature for 48 hours, B.cereus

(101) was inhibited by B.pumilus (207) but not any other strains whereas 205 was

inhibited by only B.pumilus (106). Bacterium strain 206 (B.pumilus) was inhibited by

B.licheniformis (203 & 205) and B.pumilus (106 & 207) (Table 3).

When all three conditions were considered, there were total 24 bacterial

interactions out of potential 42 interactions among seven bacteria. Some of the important

trends seen across all three conditions were as follows: B.pumilus (106) inhibited all other

six strains whereas B.pumilus (206) inhibited only B.pumilus (105) and B.licheniformis

(205). B.cereus (101) inhibited B.pumilus (105 &206) and B.licheniformis (203 & 205)

whereas 203 (B.licheniformis) inhibited B.pumilus (105, 106, & 206) and B.licheniformis

(206). The bacterial inhibitions differed among three conditions showing that one

bacterium could interact differently with other bacteria depending on the different

conditions. These were notable differences among three conditions: At 30º C for 24

hours, B.cereus (101) was inhibited only by B.pumilus (106) whereas B.licheniformis

(205) was inhibited by B.cereus (101), B.pumilus (105 and106) and B.licheniformis (203)

but at room temperature for 24 hours, B.cereus (101) was not inhibited by other strains at

all whereas B.licheniformis (205) was inhibited by all other strains. At room temperature

for 48 hours, B.cereus (101) was inhibited by B.pumilus (207) but not any other strains

whereas B.licheniformis (205) was inhibited by only B.pumilus (106) (Table 3).

Discussion

Bacterial strains 203 and 205 were identified as Bacillus licheniformis whereas

bacterial strains 101, 105, 106, 206 and 207 were confirmed using metabolic tests,

Entertotubes II and Bergey’s manual [11]. Strains 203 and 205 are perhaps two different

strains of Bacillus licheniformis because 205 is a weak fermentor of sucrose and mannitol

whereas 203 is a good fermentor of sucrose and mannitol. These fermentations are the

only observed differences in the results of metabolic tests and Enterotubes II. When 205

was inoculated into sucrose and mannitol tube, it changed from red to peach-orange color

whereas 203 changed from red to yellow color meaning the pH was 6.8 or below. Both

strains of Bacillus licheniformis (203 and 205) ferments glucose well, producing acids of

pH 6.8 or below. Bacterial strains 203 and 205 (Bacillus licheniformis) also use citrate as

a carbon source and ammonium as a nitrogen source (Table 2). Both strains hydrolyze

gelatin to produce amino acids which in turn are used by bacteria as energy sources when

there is no supply of carbohydrates. They also produce the enzyme catalase, which breaks

down hydrogen peroxide produced by aerobic respiration, which is deadly for the cells of

bacteria (Table 2).

Bacillus licheniformis (203 and 205) cannot convert tryptophan (an amino acid)

into indole. They are also unable to remove amino group from phenylanine and therefore

unable to convert it into ammonia, nor can they hydrolyze urea to form ammonia and

carbon dioxide. Bacillus licheniformis cannot decarboxylate (remove carbon dioxide)

from lysine (Table 2). Strains 203 and 205 did not produce amylase (as assessed by the

iodine test) but Bacillus licheniformis should be able to hydrolyze the starch [11].

Perhaps mutations that inactivated the enzyme have occurred.

As stated earlier, strains 101, 105, 106, 206 and 207 were previously identified

using 16S rRNA gene analysis. The results from 16S rRNA gene analysis suggested that

the two possible identifications are Bacillus pumilus and Bacillus cereus. Therefore,

metabolic tests such as nitrate reduction, starch hydrolysis, fermentation of mannitol and

arabinose producing acid were used as support tests to distinguish between Bacillus

pumilus and Bacillus cereus for bacterial strains 101, 105, 106, 206 and 207. Bacillus

cereus (101) reduced nitrates to nitrite and hydrolyzed starch (assessed by adding iodine

to a culture of bacteria grown on a starch plate). However, it could not ferment arabinose

and mannitol and therefore was unable to produce acids (which would decrease the pH of

broth and turn it yellow). These results match the metabolic tests results for Bacillus

cereus. Bacterial strains 105, 106, 206 and 207 are Bacillus pumilus. Bacterium strain

106 is a different strain from the others because it could not ferment mannitol &

arabinose and produce acids. However, the other three strains could ferment mannitol and

arabinose. All four strains of Bacillus pumilus did not reduce nitrates to nitrite or

hydrolyze the starch (Table 1).

Characteristics of Bacilli

Bacillus cereus (101) is commonly found in soil, groundwater, milk, cereals and

dried foods [8 & 11]. Their endospores are widely spread and can result in rapid cell

division in foods, which may lead to food poisoning. This can cause gastric flu resulting

in diarrhea, nausea, and vomiting and interestingly also used for probiotics for animals

[8]. Bacillus licheniformis (203 and 205) is a saphrophytic organism, a bacterium that can

live on other dead organisms, commonly found in soil and in aquatic species which may

endure severe heat treatment and can be used to produce amylases, proteases and other

antibiotics [4]. However, it is also resistant to erythromycin and chloramphenicol [11].

Bacillus pumilus (105, 106, 206 and 207) is found in soil, water and air and requires

biotin and other amino acids [11] that can endure extreme environmental conditions such

as little or no nutrient, desiccation, presence of hydrogen peroxide and chemical

disinfections but is sensitive to penicillin and resistant to cyanide. B. pumilus can cause

decay of plant tissue, skin infections or life-threatening bacteremia (presence of bacteria

in the blood) to individuals whose immune system is weak and some products made by

Bacillus pumilus may be deadly to mice and eukaryotic cells in humans [9].

Bacterial Interactions (inhibitions) among Bacilli

Three different conditions were used to determine whether incubation time and

temperature plays a role in bacterial inhibitions or interactions. Three conditions for

bacterial interactions were 30º C for 24 hours, room temperature for 24 hours and room

temperature for 48 hours. Across all three conditions, it was determined that 106

(Bacillus pumilus) inhibited all other six strains whereas 206 (Bacillus pumilus) inhibited

only 105 (Bacillus pumilus) and 205 (Bacillus licheniformis). Bacterial strains 101

(Bacillus cereus) and 203 (Bacillus pumilus) inhibited 4 different strains. 101 (Bacillus

cereus) inhibited 105 (Bacillus pumilus), 203 & 205 (Bacillus licheniformis) and Bacillus

pumilus (206) whereas 203 (Bacillus licheniformis) inhibited 105, 206 &106 (Bacillus

pumilus) and 205 (Bacillus licheniformis) (Table 3 and Figure 7). These data show that

some of Bacillus pumilus strains inhibited other strains of Bacillus pumilus, Bacillus

cereus, and Bacillus licheniformis. In addition, Bacillus licheniformis inhibited different

strains of Bacillus pumilus but not any strains of Bacillus cereus. However, Bacillus

cereus inhibited both Bacillus pumilus and Bacillus licheniformis.

Possible causes of Inhibition

The inhibitions might be caused by bacteriocins [5], which are peptide-like

substances made ribsomally by bacteria to destroy or inhibit the growth of other

organisms. Bacillus cereus produces trypsin-sensitive cerein that inhibits Gram-positive

bacteria. Its activity can be detected before the accumulation of heat-resistant endospores.

Cerein has a unique amino-terminal sequence which is not similar to any other proteins or

peptides. The six-amino acids sequence is Gly-Trp-Gly-Asp-Val-Leu. Bacillus subtilis,

Bacillus pumilus, some strains of Clostridium and Listeria are sensitive to cerein and

Micrococcus luteus is most sensitive among all. Cerein can be used as antimicrobial

molecule for some Gram-positive bacteria [8].

Bacillus licheniformis produces four types of bacteriocins [4]. The first

bacteriocin discovered was Bacitracin. Other bacteriocins produced by different strains of

Bacillus licehniformis are Lichenin, Bacillocin 490 and P40 which have different masses,

antangonistic specificities and heat-resistances. When the B. licheniformis ZJU12 is

treated with trypsin and proteinase K, the antagonistic activity vanishes indicating that it

is sensitive to them. It does not inhibit Gram-negative bacteria but does inhibit some

Gram-positive bacteria such as Bacillus subtilis, Bacillus pumilus, Micrococcus flaveus,

staphylococcus aureus, staphylococcus epidermidis and some fungi such as Alternaria

brassicae, Pyricularia grisea, and Fusarium oxysporum and therefore can be used for

controlling plant diseases and medicines for humans [4].

Bacillus pumilus produces pumilicin which inhibits the growth of other Gram-

positive bacteria such as other strains of Bacillus pumilus, Bacillus licheniformis,

Bacillus cereus, Vancomycin Resistant E. faecalis (VRE) and Methicillin-Resistant

Staphlococcus aureus (MRSA). VRE & MRSA are major concerns in public health

because they can cause fatal infections such as skin infections and pneumonia [2].

Pumilicin, one of the components in Biosubtyl, a probiotic for humans [2], is a 1.99 kDa

peptide that is soluble in ethanol and methanol indicating that it is a hydrophobic

polypeptide.

It was hypothesized that the incubation temperature and time will be positively

related to the inhibition zones of bacteria. However, based on the results, it is found that

the incubation conditions did not correlate with the inhibition zones of bacteria. There are

several reasons why some of the replicates produced zones of inhibition, whereas in other

replicates they did not. First, bacterial strains inoculated in triplicates and in three

conditions would not have been exact. Second, the producer strains might have not grown

at the same rate in incubation shaker. Third, all of the tryptic soy agar plates were not

made at same time. Therefore, there might have been minor differences in concentration.

Finally, bacteriocin-producing organisms’ incubation conditions also affect the

production of active bacteriocin. Conditions such as composition of growth medium,

temperature and time of incubation, aeration, and pH can have great impact on

production of active bacteriocin which directly affects the bacterial interactions [12]. For

example, cerein can be produced maximally at initial pH between 6.5 and 9.0,

temperature between 22 and 34 °C and soybean protein concentration higher than 20 g l−1

[3].

Bacterial strains 101 (Bacillus cereus), 105 and 106 (Bacillus pumilus) were

isolated from Rice Creek whereas 203 and 205 (Bacillus licheniformis), 206 and 207

(Bacillus pumilus) were isolated from Rice Pond. Rice creek feeds into Rice pond which

then leads water stream to Lake Ontario, NY. Twenty-four bacterial interactions were

observed out of potential 42 interactions among three conditions. Although these are

limited data, they show how bacteria inhibit each other perhaps to compete for resources.

Some species inhibit their own type as seen with Bacillus pumilus (106) inhibiting

Bacillus pumilus (105).

Acknowledgements

A.P. wants to thank Dr. Sonnenberg, Professor and Director of Millar Wilson

Laboratory for her valuable advice and support throughout my research project. A.P.

would like to especially thank Gabriela Block, a good friend, lab mate and classmate who

in addition to providing insight in certain areas, was also encouraging me and supportive

during the research.

Figure 1: Map of Upstate

New York (Google Map)

Figure 2: Map of Rice

Creek Field Station

(http://www.oswego.edu/academics/opportunities/rice_creek_field_station/facilities/rice_

crk_trail_090909_fnt.pdf)

203, 205,

206 and 207

101, 105 and 106

SUNY OSWEGO

Rice Creek field

station

Figure 3. Flowchart for Stab plate and Pour Plate at three different conditions.

A: Conditon 1 B: Condition 2 C: Condition 3

1: Stabbed plates- Six bacterial trains (producer strains) stabbed in bottom agar

2: Poured plates- seventh bacterium (indicator strains) poured on top of six grown

bacteria

3: Zones of inhibition measured (diameter of brown circle- diameter of white circle)

Incubated @ 30 º

C for 24 hrs

Incubated @ RT

for 24 hrs

Incubated @ RT

for 48 hrs

Incubated @ 30 º

C for 24 hrs

Incubated @ RT

for 24 hrs

Incubated @ RT

for 24 hrs

1

-

1

-

1

-

2

-

3

-

3

-

3

-

2

-

2

-

A C B

ENTEROTUBES II and Metabolic tests Results

Metabolic

Tests

Bacterial Strains

101 B.

cereus*

106 105 206 207 *B.

pumilus

Glucose + + + (O) + + + +

Gas - - - - - - -

Lysine - - - - - - -

Ornithine - NA - - - - NA

H2S - NA - - - - NA

Indole - - - - - - -

Acid from

Adonitol

- NA - - - - NA

Lactose - - - - - - NA

Arabinose - - - + (P) + (P) +(L O) +

Sorbitol - - - - - NA

Vogues -

Paskeur

+ + - + + + -

Dulcitol - - - - - - NA

Phenylalanine

deamination

- - - - - - -

Urea + (P) d + - - - -

Citrate - + - (G/B) - + - +

Nitrate

Reduction

+ + - - - - -

Acid from

Mannitol

- - - + + + +

Starch

Hydrolysis

+ + - - - - -

Acid from

Arabinose

- - - + (P) + (P) +

(L O)

+

Size of

bacteria (lw)

(µm)

3.48±0.36

1.14±0.26

3-5

(length)

1-1.2

(width)

2.52±0.59

0.98±0.29

2.84±0.86

0.76±0.13

1.84±0.78

0.6±0.16

2.76±0.25

0.64±0.17

2-3

(length)

0.6-0.7

(width)

Endospores C + T, C + + + +

Table 1: Comparing results of metabolic tests for five strains with B. pumilus and B. cereus. (+) :

positive result ; (-) negative result for the particular test. (P): peach color, (O): Orange color,

(Y/O): yellow/orange color, (L O): light orange color and (G/B): green/blue color. C: central, T:

Terminal endospores ; NA: not available in Bergey’s Manual. d: 11-89% are positive; l: length,

w: width.* : Bacilus pumilus and Bacillus cereus- data from Bergey’s Manual

Note: Nitrate Reduction, Acid from Mannitol, Starch Hydrolysis, Acid from Arabinose tests were

the only tests used to compare and contrast the results with Bacillus pumilus and Bacillus cereus

Results of Enterotubes II and Metabolic tests for 203 and 205 (and

Bacillus licheniformis) Metabolic tests Bacterial Strains

203 205 Bacillus

licheniformis*

Glucose + + +

Gas - - (-) p

Lysine - - -

Ornithine - - NA

H2S - - NA

Indole - - -

Adonitol - - NA

Lactose - - NA

Arabinose + (Y/O) + (P) +

Sorbitol - - NA

Vogues-Paskeur + + +

Dulcitol - - NA

Phenylalanine - - -

Urea - - -

Citrate + + +

Starch Hydrolysis - - +

Acid from Sucrose + (Peach/orange) + (Yellow) NA

Mannitol + (Peach/orange) + (Yellow) +

Gelatin Hydrolysis + + +

Nitrate reduction - - +

Oxidase - - d

Catalase + + +

Ornthine

Decarboxylase

+ + NA

Sulfide Production - - NA

Indole Production - - -

Motility + + NA

Phenylanine

deamination

- - -

Size of bacteria

(lw)

(µm)

2.68 ± 0.81

0.74 ± 0.21

2.46 ± 0.50

0.78 ± 0.15

1.5-3 (length)

0.6-0.8(width)

Endospres + + +

Table 2: Identification of 203 and 205 as Bacillus licheniformis. The results are

compared to metabolic tests for Bacillus licheniformis. (+) : positive result ; (-) negative

result for the particular test. (P): peach color, (Y/O): yellow/orange color; (-) p

: few

bubbles may be formed; NA: result for the test is Not Available in Bergey’s Manual [9].

l: length, w: width, * data from Bergey’s Manual.

A.

B.

Figure 4: Gram stain. Gram-positive and bacillus in shape (a) 203 (b) 207

A.

B.

Figure 5. Endospore stains (a) 106- central, terminal and free endospores (b) 206 - free

endospores (shown above as green).

Terminal &

central

endospores

Figure 6. Inhibition zone. 101, 105, 106,

203, 205 and 206 were inoculated as

potential producer strains while 207 was

poured on top as indicator strain. Shows 207

was inhibited by 106 but not any other

strains.

Inhibition zone

Inhibitions at different conditions (%) – (Producer strains)

Conditions 101(B.c) 105(B.p) 106(B.p) 203(B.l) 205(B.l) 206(B.p) 207(B.p)

101

B.cereus

(B.c)

24 hrs at

30ºC

-

0 33 0 0 0 0

24 hrs at

RT

-

0 0 0 0 0 0

48 hrs at

RT

-

0 0 0 0 0 33

105

B.pumilus

(B.p)

24 hrs at

30ºC

33 -

66 66 100 0 0

24 hrs at

RT

33 -

0 33 66 33 0

48 hrs at

RT

0 -

0 100 0 0 0

106

B.pumilus

(B.p)

24 hrs at

30ºC

0 0 - 33 0 0 0

24 hrs at

RT

0 0 - 0 0 0 0

48 hrs at

RT

0 0 - 0 0 0 0

203

B.licheniformis

(B.l)

24 hrs at

30ºC

66 0 33 - 0 0 0

24 hrs at

RT

0 0 33 - 0 0 0

48 hrs at

RT

0 0 100 - 0 0 0

205

B.licheniformis

(B.l)

24 hrs at

30ºC 33 100 33 66

- 0 0

24 hrs at

RT 33 100 33 66

- 66 100

48 hrs at

RT 0 0 100 0

- 0 0

206

B.pumilus

(B.p)

24 hrs at

30ºC 33 0 0 33 100

- 0

24 hrs at

RT 0 100 33 66 66

- 100

48 hrs at

RT 0 0 100 33 66

- 33

207

B.pumilus

(B.p)

24 hrs at

30ºC 0 0 66 0 0 0

-

24 hrs at

RT 0 0 33 0 33 0

-

48 hrs at

RT 0 0 100 0 0 0

-

Table 3. Inhibtions at three conditions: 30ºC for 24 hours, room temperature (~ 22 ºC)

for 24 hours and room temperature for 48 hours. The percentage indicates how many

times out of three plates a producer strain inhibited indicator strain across three

conditions. (-): the bacterium was not tested against itself; (33): producer strain inhibited

indicator strain in 1 out of 3 plates; (66): producer strain inhibited indicator strain in 2 out

of 3 plates and (100): producer strain inhibited indicator strain in 3 out of 3 plates.

Figure 7. Twenty-four interactions (inhibitions) among Bacilli

Producer organism (inhibitor) inhibiting other organism (inhibited)

105

B.pumilus

206

B.pumilus

106

B.pumilus

101

B.cereus 203

B.licheniformis

205

B.licheniformis

207

B.pumilus

References

1. A.G. Fredrickson, G. Stephanopoulos. 1981. Abstr..Microibal competition.

Science. 213: 972-979

2. Aunpad, R., and Na-Bangchang K. 2007. Pumilicin 4, a novel bacteriocin with

anti-MRSA and anti-VRE activity produced by newly isolated bacteria Bacillus

pumilus strain WAPB4. Curr Microbiol. 55: 308-213.

3. Dominguez, A.P.M., Bizani , D., Cladera-Olivera, F., and Brandelli, A. 2007.

Cerein 8A production in soybean protein using response surface methodology.

Biochem. Eng. J. 35: 238-243.

4. He, L., Chen, W., and Liu,Y. 2006. Production and partial characterization of

bacteriocin-like pepitdes by Bacillus licheniformis ZJU12. Microbiol Res.

161:321-326.

5. Jack, R.W, Tagg J.R., and Ray B. 1995. Bacteriocins of gram-positive bacteria.

Microbiol Rev.59:171-200.

6. Johnson, T.R., and Case, C.L. 2007. In L.Berriman, B.Robbins, W.Earl (ed.),

Laboratory Experiments in Microbiology, 8th

ed. Pearson Benjamin Cummings.

San Francisco, CA.

7. Kekessy, D.A. and Piguet, J.D. 1970. New Method for Detecting Bacteriocin

Production. Appl Microbiol. 20:282-283.

8. Oscáriz, J.C., Lasa, I., and Pisabarro, A.G. 1999. Detection and

characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a

broad spectrum of activity. FEMS Microbiol Lett. 178:337-341.

9. Parvithi,A., Krishna, K.,Jose, J., Joseph, N., Nair, S. 2009.Biochemical and

molecular characterization of Bacillus pumilus isolated from coastal environment

in Cochin, India. Braz. J. Microbiol. 40:269-275.

10. Riley, M.A., and Gordon, D.M.1999. The ecological role of bacteriocins in

bacterial competition. Trends Microbiol.7:129-133.

11. Sneath, P.H.A, Mair, N.S., Sharpe, M.E., and Holt, J.G. 1986. Endospore-

forming Rods and Cocci.,p1105-1138. Bergey’s Manual of Systematic

Bacteriology, 2nd

ed.,vol.2. Williams & Wikins. Baltimore, MD.

12. Tagg J.R., Dajani A.S., and Wannamaker L.W. 1976. Bacteriocins of gram-

positive bacteria. Bacteriol Rev.40:722-756.

APPENDIX

Figure A.1: Gram-staining

Fig A.1a: Gram stain for 101 (Bacillus

cereus)

Fig A.1b: Gram stain for 105 (Bacillus

pumilus)

Fig A.1c: Gram stain for 106 (Bacillus

pumilus)

Fig A.1d: Gram stain for 203 (Bacillus

licheniformis)

Fig A.1e: Gram stain for 205 (Bacillus

licheniformis)

Fig A.1f: Gram stain for 206 (Bacillus

pumilus)

Fig A.1g Gram stain for 207(Bacillus

pumilus)

Figure A.2 Endospore-staining

Fig A.2a: Endospore stain for 101

(Bacillus cereus)

Fig A.2b: Endospore stain for 105

(Bacillus pumilus)

Fig 2c: Endospore stain for 106

(Bacillus pumilus)

Fig A.2d: Endospore stain for 203

(Bacillus licheniformis)

Fig A.2e: Endospore stain for 205

(Bacillus licheniformis)

Fig 2f: Endospore stain for 206

(Bacillus pumilus)

Fig.A.2g: Endospore stain for 207

(Bacillus pumilus)

Figure A.3: Some Metabolic tests

Fig.A.3a: Controls for Mannitol test

Fig.A.3b: Controls for Sucrose test

Fig.A.3c: Controls for Hydrogen sulfide

production test

Figure 4: Enterotubes II for controls

Fig. 4a: Escherichia coli’s result for Enterotubes II used as control (shown above).

Fig. A.4b: Pseudomonas aerogenes’ result for Enterotubes II used as control (shown

above).

Fig. A.4c: Proteus vulgaris’ result for Enterotubes II used as control (shown above).

Figure 5: Two examples of plates showing zones of inhibition.

Fig.A.5a: 105 (Bacillus pumilus

inhibited by 106: Bacillus pumilus) at RT

for 48 hours.

Fig.A.5b: 101, 105, 106, 203, 205

and 207 are producer strains. 206 is

inhibited by 106, 203, 205 and 207.

* These are just two examples from many pictures of plates.

Table A.1: Size of bacteria in Microns

101 B.cereus

105 B.pumilus

106 B.pumilus

Number of bacteria Length Width Length Width Length Width 1 3.8 0.9 3.7 0.9 2.8 1.1

2 2.9 0.9 2.5 0.6 2.3 1

3 3.7 1.1 2.6 0.7 3.3 1.3

4 3.4 1.5 3.7 0.9 1.7 0.5

5 3.6 1.3 1.7 0.7 2.5 1

Average 3.48 1.14 2.84 0.76 2.52 0.98 Standard deviation 0.36 0.26 0.86 0.13 0.59 0.29

Table A.1a: Size of bacteria (101- Bacillus cereus, 105 and 106 - Bacillus pumilus) in

microns with their averages and standard deviation.(shown above)

203 B.licheniformis

205 B.licheniformis

206 B.pumilus

207 B.pumilus

# of bacteria Length Width Length Width Length Width Length Width

1 1.6 0.7 1.9 0.7 1.1 0.5 2.6 0.6

2 3.3 0.6 2.5 0.8 1.1 0.6 2.4 0.4

3 2.7 0.5 2.9 0.6 1.7 0.4 2.9 0.6

4 2.2 1 3 1 2.7 0.7 3 0.8

5 3.6 0.9 2 0.8 2.6 0.8 2.9 0.8

Average 2.68 0.74 2.46 0.78 1.84 0.6 2.76 0.64 Standard deviation 0.81 0.21 0.50 0.15 0.78 0.16 0.25 0.17

Table A.1b: Size of bacteria (203 and 205- Bacillus licheniformis, 206 and 207 -

Bacillus pumilus) in microns with their averages and standard deviation (shown above)

Table A.2: Inhibition zones at different conditions.

Inhibitions at different conditions Producer Strains (mm)

Average and Standard deviation (mm)

Conditions 101 105 106 203 205 206 207

101 24 hrs at

30ºC

-

0 ± 0 1 ±1 .73 0 ± 0 0 ± 0 0 ± 0 0 ± 0

24 hrs at

RT

- 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0

48 hrs at

RT

-

0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.33±4.04

105 24 hrs at

30ºC

1.33±2.31

-

2.67±3.06 1±1 2.33±1.53 0.33±0.58 0±0

24 hrs at

RT

1±1.53

-

0±0 0.33±0.58 1±0.58 0.33±0.58 0±0

48 hrs at

RT

0 ±0

-

0 ±0

7±5.2

0±0

0±0

0±0

106 24 hrs at

30ºC

0±0

0±0

-

0.67±1.15

0±0

0±0

0±0

24 hrs at

RT

0±0

0±0

-

0±0

0±0

0±0

0±0

48 hrs at

RT

0 ± 0 0 ± 0 - 0 ± 0 0 ± 0 0 ± 0 0 ± 0

203 24 hrs at

30ºC

2 ± 2

0 ± 0

0.33 ± 0

-

0 ± 0

0 ± 0

0 ± 0

24 hrs at

RT

0 ± 0

0.67 ± .58

1 ± 1.73

-

0 ± 0

0 ± 0

0 ± 0

48 hrs at

RT

0 ± 0

0 ± 0

6.67±3.06

-

0 ± 0

0 ± 0

0 ± 0

205 24 hrs at

30ºC 1±1.73 1.67±0.58 0.33±0.58 1.33±1.15

-

0 ± 0

0 ± 0

24 hrs at

RT 1.33±2.31 3.67±1.53 0.33±0.58 3±3.61

-

1.33±1.53

1.33±0.58

48 hrs at

RT

0±0

0±0

4.33±2.52

0±0

-

0±0

0±0

206 24 hrs at

30ºC 1.33±2.31 0±0 0.67±0.58 0.33±0.58 1.67±1.15

-

0±0

24 hrs at

RT 0±0 3±1 2±3.46 1.67±1.53 1.67±1.53

-

3±2

48 hrs at

RT 0±0 0±0 2.33±0.58 1.33±2.31 1.33±1.15

-

0.33±0.58

207 24 hrs at

30ºC 0±0 0±0 1.33±1.15 0±0 0±0 0±0

-

24 hrs at 0±0 0.33±0.58 2.33±4.04 0±0 0.33±0.58 0±0 -

RT

48 hrs at

RT

0±0

0±0

5.33±0.58

0±0

0±0

0±0

-

* 101: Bacillus cereus; 105, 106, 206 and 207: Bacillus pumilus and 203 and 205:

Bacillus licheniformis.

Table A.3: Enterotubes II results for Controls

ENTEROTUBES II

Metabolic

Tests

Bacteria

Escherichia coli Proteus vulgaris Pseudomonas

aerogenes

Glucose + + +

Gas - - -

Lysine + - -

Ornithine + - -

H2S - + -

Indole + + -

Adonitol - - -

Lactose + - -

Arabinose + - +

Sorbitol + - -

VP

Dulcitol - -

Phenylalanine - +

Urea - +

Citrate - + +

Table A.4: Oxidative-Fermentation Metabolism test results

Oxidation- Fermentation Metabolism

Condition 203- Bacillus

licheniformis

205- Bacillus

licheniformis

Growth Color Growth Color

Aerobic (without

oil)

- Green - Green

Anaerobic (with

oil)

+ Yellow + Yellow

Metabolism Fermentative Fermentative

Inhibitons at 30ºC for 24 hours

Inhibition in Percentages (%)

Producer Strains

101

B.

cereus

105

B.

pumilus

106

B.

pumilus

203

B.

licheniformis

205

B.

licheniformis

206

B.

pumilus

207

B.

pumilus

101

B. cereus

-

0 33 0 0 0 0

105

B. pumilus

33 -

66 66 100 0 0

106

B. pumilus

0 0 - 33 0 0 0

203

B.

licheniformis

66 0 33 - 0 0 0

205

B.

licheniformis

33 100 33 66 - 0 0

206

B. pumilus 33 0 0 33 100

- 0

207

B. pumilus 0 0 66 0 0 0

-

Table A.5. the percentage a bacterium inhibited other bacteria (from three plates). (-): the

bacterium was not tested against itself; (33): producer strain inhibited indicator strain in 1

out of 3 plates; (66): producer strain inhibited indicator strain in 2 out of 3 plates and

(100): producer strain inhibited indicator strain in 3 out of 3 plates.

Inhibitions at RT for 24 hours

Inhibition in Percentages (%)

Producer Strains

101

B.

cereus

105

B.

pumilus

106

B.

pumilus

203

B.

licheniformis

205

B.

licheniformis

206

B.

pumilus

207

B.

pumilus

101

B. cereus

-

0 0 0 0 0 0

105

B. pumilus

33 -

0 33 66 33 0

106

B. pumilus

0 0 - 0 0 0 0

203

B.

licheniformis

0 0 33 - 0 0 0

205

B.

licheniformis 33 100 33 66

- 66 100

206

B. pumilus 0 100 33 66 66

- 100

207

B. pumilus 0 0 33 0 33 0

-

Table A.6. The percentage a bacterium inhibited other bacteria (from three plates). (-):

the bacterium was not tested against itself; (33): producer strain inhibited indicator strain

in 1 out of 3 plates; (66): producer strain inhibited indicator strain in 2 out of 3 plates and

(100): producer strain inhibited indicator strain in 3 out of 3 plates.

Inhibitions at RT for 48 hours

Inhibition in Percentages (%)

Producer Strains

101

B.

cereus

105

B.

pumilus

106

B.

pumilus

203

B.

licheniformis

205

B.

licheniformis

206

B.

pumilus

207

B.

pumilus

101

B. cereus

-

0 0 0 0 0 33

105

B. pumilus

0 -

0 100 0 0 0

106

B. pumilus

0 0 - 0 0 0 0

203

B.

licheniformis

0 0 100 - 0 0 0

205

B.

licheniformis 0 0 100 0

- 0 0

206

B. pumilus 0 0 100 33 66

- 33

207

B. pumilus 0 0 100 0 0 0

-

Table A.7. The percentage a bacterium inhibited other bacteria (from three plates). (-):

the bacterium was not tested against itself; (33): producer strain inhibited indicator strain

in 1 out of 3 plates; (66): producer strain inhibited indicator strain in 2 out of 3 plates and

(100): producer strain inhibited indicator strain in 3 out of 3 plates.

Size of Bacterial strains (µm)

Average Standard deviation

Bacteria Length Width Length Width

101

B. cereus

3.48 1.14 0.36 0.26

105

B. pumilus

2.84 0.76 0.86 0.13

106

B. pumilus

2.52 0.98 0.59 0.29

203

B. licheniformis

2.68 0.74 0.81 0.21

205

B. licheniformis

2.46 0.78 0.50 0.15

206

B. pumilus

1.84 0.6 0.78 0.16

207

B. pumilus

2.76 0.64 0.25 0.17

Table A.8. Five measurements were used to determine the average size of bacterium.