chapter 4 screening, isolation and formulation of...

CHAPTER 4

SCREENING, ISOLATION AND FORMULATION OF

BIOSURFACTANT-PRODUCING MICROBIAL CONSORTIA

4.1 ABSTRACT

Soil samples were collected from various points near a refinery site, petroleum depot,

petrol bunk and automobile workshop for the isolation of biosurfactant-producing bacteria.

Microbial specimens were isolated using the method of serial dilution. The isolated bacteria were

cultured for seven days in MSM containing diesel as the sole carbon source. The isolated

cultures were tested for their ability to produce biosurfactant using the following analytical

methods: oil spreading technique, blood agar haemolysis test, drop collapse test, CTAB agar

plate test, tilted glass slide test and determination of emulsification index and surface activity.

The morphology of the colonies was studied and standard biochemical tests were

performed. 16S rRNA gene sequencing was performed and the sequence was compared with

NCBI GenBank entries by using the BLAST algorithm. One of the main objectives of the

research work was to develop a consortium of microbial isolates with the highest biodegradation

efficiency. Bacillus siamensis was made part of all microbial combinations. Of the two Bacillus

subtilis isolates, the better isolate, in terms of higher reduction of surface tension and better

emulsification index was chosen. This isolate was Bacillus subtilis subsp. inaquosorum. In

similar lines, of the two Pseudomonas aeruginosa isolates, the better was chosen. This isolate

was Pseudomonas aeruginosa. Different combinations of the three isolates were formulated for

development of microbial consortia. The development of microbial consortia was made solely to

study the efficiency of biodegradation of selected polycyclic aromatic hydrocarbons.

The role of plasmid DNA in the production of biosurfactants was verified. The method of

alkaline lysis was used to extract plasmid DNA from each of the bacterial isolates. The samples

were loaded onto an agarose gel and subjected to agarose gel electrophoresis. When the gel was

checked for the presence of bands on the agarose gel, none were observed.

4.2 INTRODUCTION

Soils rich in hydrocarbons like those contaminated with petroleum spillages are a good

source of microbes acclimatized to utilize the hydrocarbons for their metabolism. Many

researchers have isolated a range of novel bacterial specimens that produce extracellular

biosurfactants which in turn can increase the bioavailability of the hydrocarbons.

Seventeen biosurfactant-producing bacterial isolates were isolated from terrestrial and

marine samples contaminated with crude oil or its byproducts in a study by Batista et al., 2006.

All the isolates had produced emulsions with kerosene and twelve exhibited high emulsion-

stabilizing capacity. Eight isolates were capable of reducing the surface tension below 40 mN/m.

Five of these eight exhibited this property in cell-free filtrates. They had concluded that isolation

from petroleum contaminated sites proved to be a rapid and effective manner to identify bacterial

isolates with potential industrial applications.

Two biosurfactant-producing Pseudomonas aeruginosa strains were isolated from

Kuwaiti oil-contaminated soil, which resulted from the Gulf War (Yateem et al., 2002). When

they optimized the environmental conditions that supported the growth and surfactant

production, it was found that the two isolates differed in their biosurfactant-stimulating carbon

source, nitrogen concentration. The biosurfactant produced by one of the strains was found to be

very effective in the emulsification of crude oil.

A total of fifteen oil mixed soil samples were screened and nine bacterial biosurfactant

producing strains were identified (Tambekar and Gadakh, 2012) as Pseudomonas aeruginosa,

Aneurinibacillus miugulanus, Achromobacter insolitus, Bacillus shackletonii,

Ochromobactrum intermedium, Ochromobactrum oryzae by biochemical tests and 16S rRNA

sequencing. Reduction in surface tension was found to be 47–60 mN/m. They could form a

stable emulsion with motor oil.

Biosurfactant production-cum-diesel biodegradation was explored by Moliterni et al.,

2012, using mixed microbial consortia from polluted sites. After designing three microbial

consortia enrichment in diesel, batch experiments were done to study the effects of three

variables (temperature, hydrocarbon concentration and the origin of the consortia) on the diesel

biodegradation and the surface tension evolution. The three enriched consortia contained similar

bacterial genera and degraded diesel with similar efficiencies (approximately 90%). All three

consortia were found to be efficient biosurfactants producers.

Naphthalene degrading bacterial consortium was developed by enrichment culture

technique from sediment collected from the Alang-Sosiya ship breaking yard, Gujarat, India

(Vilas Patel et al., 2012). 16S rRNA sequencing revealed that the consortium consisted of

Achromobacter sp., Pseudomonas sp., Enterobacter sp. and Pseudomonas sp.. The consortium

was able to degrade 1000 ppm of naphthalene in BH medium containing peptone (0.1%) as co-

substrate. The consortium was able to utilize other aromatic and aliphatic hydrocarbons such as

benzene, phenol, carbazole, petroleum oil, diesel fuel, and phenanthrene and 2-methyl

naphthalene as sole carbon source.

Three bacterial isolates capable of utilizing used engine-oil as a carbon source were

isolated from contaminated soils using the enrichment technique (Mandri and Lin, 2007). Three

isolates were identified as Flavobacterium sp., Acinetobacterium calcoaceticum and

Pseudomonas aeruginosa based on biochemical tests and 16S rRNA sequencing. It was found

that A.calcoaceticum and a consortium of the isolates were capable of utilizing 80 and 90% of

used engine oil, respectively, under laboratory conditions in a 4-week period.

Mohammad Abdel-Mawgoud et al., 2008, isolated Bacillus subtilis BS5, from soil which

produced a promising yield of surfactin in mineral salts medium. When they tested for the

presence of plasmid, no plasmid could be detected in the tested isolate, revealing that

biosurfactant production by B. subtilis isolate BS5 is chromosomally mediated but not plasmid-

mediated.

A review by Oluwafemi and Lateef, 2010 presents a deeper analysis on the role of

plasmid in degradation. The degradation of many xenobiotic and hydrocarbon compounds is

known to be mediated by plasmid-encoded enzymes. It has been proven that high level of

plasmid involvement in the degradation of naphthalene and other 2- and 3-ring PAHs. Many of

these are megaplasmids, of linear configuration, encoding part or the whole genes for the

complete pathways.

Padmapriya et al., 2011 worked on Proteus inconstans from which a plasmid with 1.8

kbp was isolated and was cured by acridine orange. Biodegradation and biosurfactant activities

were totally inhibited. Hence, it was determined that the biosurfactant production was purely

plasmid-mediated.

4.3 SCREENING, ISOLATION AND FORMULATION OF BIOSURFACTANT-

PRODUCING MICROBIAL CONSORTIA

For the isolation of biosurfactant-producing bacteria, the samples were collected from

various points near a refinery site, a petroleum depot, a local petrol bunk and an automobile

workshop, as outlined in Section 3.3. The microbes were isolated through serial dilution and

incubated at 25˚C, for 48 hr, as given in Section 3.4. The isolates are then inoculated in MSM

containing 2% (v/v) filtered diesel as the sole carbon source for a duration of 7 days, as described

in Section 3.5. The isolates were screened for the production of biosurfactant using a range of

qualitative and quantitative tests like oil spreading technique, blood agar haemolysis test, drop

collapse test, CTAB agar plate test, tilted glass slide test, emulsification index and determination

of surface activity (Section 3.6).

The morphology of the colonies was studied and standard biochemical tests were

performed. Under the morphological aspects, the colour, shape, size, surface, edge, opacity,

degree of growth and elevation of the colonies were observed and noted. 16S rRNA gene

sequencing was performed at Agharkar Research Institute (Pune, India) to identify the bacterial

strain. PCR was carried out using the universal primers for 1.5 kb fragment amplification for

eubacteria. The PCR product was then processed for cycle sequencing reaction. The sequencing

output was analyzed using the DNA sequence analyzer software. The sequence was compared

with NCBI GenBank entries by using the BLAST algorithm. The consortia are formulated taking

into consideration their genus and specific properties.

Plasmid DNA was extracted from all the isolates using the method of alkaline lysis, as

outlined in Section 3.7. After the samples were subjected to agarose gel electrophoresis, the gel

was observed using a UV transilluminator.

4.4 RESULTS AND DISCUSSION

4.4.1 Isolation of microorganisms from the sample

At the end of the incubation period, a total of twenty three different types of colonies

were selected for further analysis. They were named RT1 through RT23.

4.4.2 Screening for Biosurfactant Production

4.4.2.1 Oil Spreading Technique

Of the twenty three isolates, four samples (RT7, RT9, RT10 and RT21) significantly

displaced the oil layer and started to spread in the water, showing a zone of displacement. The

results of the oil displacement test showed the pattern as given in Figure 4.1.

Figure 4.1 Results of Oil spread test

Morikawa et al. (2000) showed that the area of displacement by a biosurfactant-

containing solution is directly proportional to the concentration of the biosurfactants tested.

Strains secreting a higher concentration of biosurfactant as indicated by the oil spreading test had

low surface tension values. This, plus the fact that the diameter of the clear zone is proportional

to the concentration of a standard biosurfactant, indicated that the oil spreading technique is a

consistent method to detect biosurfactant production. All the images are shown in Appendix 1.

4.4.2.2 Blood Agar Haemolysis Test

Of the twenty three isolates, fifteen strains showed clear zones around the streaks,

confirming the exhibition of partial to considerable hemolytic activity. Five strains (RT3, RT7,

RT10, RT19 and RT21) displayed a high degree of hemolysis. The results of the blood agar

haemolysis test are shown in Figure 4.2. All the images are shown in Appendix 1.

Figure 4.2 Results of Blood agar hemolysis test

However, not all biosurfactants have a hemolytic activity and compounds other than

biosurfactants may cause hemolysis (Youssef et al., 2004). In their study, sixteen percent of the

strains that lysed blood agar tested negative for biosurfactant production with the other two

methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent

of the strains that did not lyse blood agar tested positive for biosurfactant production with the

other two methods and had surface tension values as low as 35 mN/m.

4.4.2.3 Drop Collapse Test

Of the twenty three isolates, six specimens (RT3, RT7, RT9, RT10, RT19 and RT21)

showed appreciable effect on the glass slide. The drop collapsed completely after an hour. The

results of the drop collapse test are depicted in Figure 4.3.

Figure 4.3 Results of Drop collapse test

The use of the drop collapse method as a primary method to detect biosurfactant

producers, followed by the determination of the biosurfactant concentration using the oil

spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant

production (Youssef et al., 2004). Their study had concluded that the drop collapse method may

not be as sensitive as the oil spreading technique in detecting low levels of biosurfactant

production. All the images are shown in Appendix 1.

4.4.2.4 CTAB Agar Plate Test

Of the twenty three isolates, five strains (RT3, RT7, RT9, RT16 and RT21) were

surrounded by considerable bluish-green halos implying good production of extracellular

biosurfactants. The results of the agar plate test are shown in Figure 4.4. All the images are

shown in Appendix 1.

Figure 4.4 Results of CTAB Agar plate test

4.4.2.5 Tilted Glass Slide Test

Of the twenty three isolates, water flowed over the following seven specimens, possibly

indicating the production of an extracellular biosurfactant. The seven isolates are RT3, RT7,

RT9, RT10, RT16, RT19 and RT21. All the images are shown in Appendix 1.

4.4.2.6 Emulsification Index

Of the twenty three isolates, nine strains (RT3, RT7, RT9, RT10, RT11, RT16, RT17,

RT19 and RT21) showed a significant value of emulsification index. The emulsification indices

of the culture supernatants showed the following pattern, as depicted in Figure 4.5. All the

images are shown in Appendix 1.

Figure 4.5 Emulsification Indices of the isolates

4.4.2.7 Determination of Surface Activity

Of the twenty three isolates, seven strains (RT3, RT7, RT9, RT10, RT16, RT19 and

RT21) showed a significant reduction of surface tension values, indicating that the microbe may

be producing an extracellular biosurfactant. The surface tension values of culture supernatants

are shown below in Figure 4.6. The percentage reduction of surface tension has been depicted in

Figure 4.7. All the images are shown in Appendix 1.

Figure 4.6 Surface activity of the isolates

Figure 4.7 Percentage reduction of Surface tension by the isolates

4.4.3 Selection of Microbial Strains

All the tests performed have been used in various studies for preliminary screening of

biosurfactant producers. Though the tests indicate biosurfactant production to various degrees of

sensitivity, the isolates that show consistent results in all the seven tests were chosen. Based on

the results of the seven screening tests including the measurements of surface tension reduction,

the following microbial specimens were chosen for further experimentation: RT3, RT7, RT9,

RT10, RT16, RT19 and RT21.

4.4.4 Morphological Study of Isolates

The basic morphological features of the strains were studied and summarized in Table 4.1.

Table 4.1: Morphological observations of the microbial strains

RT3

RT7

RT9

RT10

RT16

RT19

RT21

Colour Pale yellow Creamy Off

white

Creamy Off

white

Creamy Creamy

Size 1.3 mm 1 mm 2 mm 1.1 mm 1.8 mm 1 mm 1.2 mm

Shape Circular Circular Bead Circular Bead Circular Circular

Microscopic

Observation

(shape)

Rods

&

Cocci

Rods Rods Rods Rods Rods Rods

Surface Shiny &

Smooth

Dull &

Rough Dull Wrinkled Dull

Dull &

Rough

Dull &

Rough

Texture Dry Dry Dry Moist Dry Dry Dry

Edge Undulate Rhizoid Entire Entire Entire Undulate Rhizoid

Elevation Flat Convex Raised

/Plateaux

Convex Raised

/Plateaux

Flat Convex

Opacity Translucent Translucent Opaque Opaque Opaque Translucent Translucent

Degree of

Growth

Scant Profuse Scant Scant Scant Profuse Profuse

4.4.5 Biochemical Characterization of Isolates

The basic biochemical tests were conducted on the strains and the results have been enlisted

below. The images pertaining to the biochemical tests for all the isolates are shown in Appendix

2.

4.4.5.1 Isolate RT3

Gram Staining Reaction: Gram-negative

Motility Test: Non-motile

Starch Hydrolysis Test: Negative (Cannot hydrolyze starch since it cannot produce

amylase & hence, no zone of clearing was observed)

Casein Hydrolysis Test: Negative (Cannot hydrolyze casein since it cannot produce

casesase & hence, no zone of clearing was observed)

Methyl Red – Voges Proskauer Test: MR-negative (Tube turns yellow colour) and VP-

negative (Tube turns copper colour)

Nitrate Reduction Test: Negative (Cannot produce both nitrate reductase and nitrite

reductase and hence, no change of tube colour and no gas collection in Durham tube)

Oxidase Test: Negative (Oxidase cytochrome is absent and hence, oxidase reagent strip

remains colourless)

Catalase Test: Positive (Copious amounts of bubble formation was observed)

Citrate Utilization Test: Positive (Abundant growth was observed on the Simmons Citrate

agar slant and the medium changes from green to blue indicating use of citrate)

Indole production Test: Negative (Upper alcohol layer of the broth turns yellow)

Spore-forming ability Test: Spores were not observed.

4.4.5.2 Isolate RT7

Gram Staining Reaction: Gram-positive

Motility Test: Motile

Starch Hydrolysis Test: Positive (Hydrolyzes starch since it can produce amylase &

hence, a zone of clearing was observed)

Casein Hydrolysis Test: Positive (Hydrolyzes casein since it can produce casesase &



positive (Tube turns red colour)

Nitrate Reduction Test: Positive (Produces both nitrate reductase and nitrite reductase

and hence, tube changes to red colour and gas collection is observed in Durham tube)


remains colourless)




Indole production Test: Negative (The upper alcohol layer of the broth turns yellow

color)

Spore-forming ability Test: Spores were observed near the terminal regions of some cells.

4.4.5.3 Isolate RT9











Oxidase Test: Positive (Oxidase cytochrome is present and hence, oxidase reagent strip

changes from colourless to purple)





color)


4.4.5.4 Isolate RT10


Motility Test: Non-motile



Casein Hydrolysis Test: Negative (Cannot hydrolyze casein since it cannot produce

casesase & hence, no zone of clearing was observed)






remains colourless)


Citrate Utilization Test: Negative (Growth was not observed on the Simmons Citrate agar

slant and the medium remains green indicating non-use of citrate)


color)













Oxidase Test: Positive (Oxidase cytochrome is present and hence, oxidase reagent strip

changes from colourless to purple)





color)














remains colourless)





color)


From the biochemical tests, the likelihood of genus of the isolated microbial specimens is listed

below

RT3: Inconclusive RT16: Pseudomonas

RT7: Bacillus RT19: Bacillus

RT9: Pseudomonas RT21: Bacillus

RT10: Inconclusive

4.4.6 16S rRNA Identification of the Isolates

Microbial isolates were identified through 16S rRNA analysis. The results have been shown in

Table 4.2.

Table 4.2: Results of 16S rRNA identification of isolates

Isolate Genus species

RT 3 Acinetobacter calcoaceticus

RT 7 Bacillus subtilis

RT 9 Pseudomonas aeruginosa

RT 10 Rhodococcus terrae

RT 16 Pseudomonas aeruginosa

RT 19 Bacillus siamensis

RT 21 Bacillus subtilis subsp. inaquosorum

The phylogenetic tree for the isolates RT9, RT19 and RT21 are shown in Fig. 4.8, 4.9 and 4.10,

respectively.

Figure 4.8 Phylogenetic tree of Isolate RT9



4.4.7 Extraction of Plasmid DNA

No bands were observed in all the lanes of the gel. Both high and low molecular weight

plasmids were not observed in the gel. From this observation, it can be safely concluded the

genes responsible for biosurfactant production is not associated with plasmids and must be

located in the chromosomal DNA of the bacterial isolates, tested.

Fleck et al., 2000, when studying biosurfactant production by bacteria isolated from oil-

polluted sites (B.subtilis B1 and P.aeruginosa P1), obtained similar results. Both the strains did

not reveal the presence of plasmids. They had also concluded that the coding genes for

biosurfactant production are located in the chromosomal DNA. Likewise, no plasmids were

observed by Mohammad Abdel-Mawgoud et al., 2008 from Bacillus subtilis BS5 isolated from

the soil.

During scale-up in industrial operations, genetic instability is one of the major setbacks in

hindering a microbial strain from becoming a robust producer of biosurfactant. Thus, the finding

could be perceived as an advantage from industrial viewpoint. As a consequence, the genes

responsible for reduced surface activity are more stable. Hence, at the production scale,

minimum fluctuation would be encountered.

4.4.8 Development of Microbial Consortia

Bacillus siamensis is reported for the first time for the production of biosurfactant.

Hence, this isolate, RT 19, was made part of all microbial combinations. Of the two Bacillus

subtilis isolates (RT7 and RT21), the better isolate, in terms of higher reduction of surface

tension and better emulsification index (from preliminary screening data – Figures 4.5 and 4.7)

was chosen. The better isolate was Bacillus subtilis subsp. inaquosorum (RT 21). In similar lines,

of the two Pseudomonas aeruginosa isolates (RT9 and RT16), the better isolate in terms of

higher reduction of surface tension and better emulsification index (from preliminary screening

data – Figures 4.5 and 4.7) was chosen. The better isolate was Pseudomonas aeruginosa (RT 9).

Another isolate was found to be Rhodococcus terrae (RT 10). It was previously decided

to continue the research work to relate the effect of swarming motility behavior of the isolates on

the action of biosurfactants. Due to the non-motile nature of R.terrae and minor concerns of

possible pathogenicity, the isolate was dropped from the consortium. The last isolate,

Acinetobacter calcoaceticus (RT 3) was also not selected based on similar reasons of non-motile

character.

Different combinations of Bacillus siamensis (RT 19), Bacillus subtilis subsp.

inaquosorum (RT 21) and Pseudomonas aeruginosa (RT 9) were formulated for development of

microbial consortia. The development of microbial consortia was made solely to study the

efficiency of biodegradation of selected polycyclic aromatic hydrocarbons. The following was

the choice of consortia:

• CONS 1: RT 19 + RT 21

• CONS 2: RT 19 + RT 9

• CONS 3: RT 19 + RT 21 + RT 9

4.5 CONCLUSION

The bacteria were isolated from different hydrocarbon-contaminated zones such as

refinery site, petroleum depot, petrol bunk and automobile workshop. Serial dilution and plating

led to isolation of twenty three different bacterial isolates. The isolates were screened for the

production of biosurfactants through a series of qualitative tests that followed after they were

grown in MSM. The tests included oil spreading technique, blood agar haemolysis test, drop

collapse test, CTAB agar plate test, tilted glass slide test, emulsification index and determination

of surface activity. The results of the screening tests indicated that seven of the isolates (RT3,

RT7, RT9, RT10, RT16, RT19, and RT21) showed promise in producing significant quantities of

extracellular biosurfactants. The percentage reduction in surface tension of the cell-free broth

after 24 hr, ranged from 49 % to 62%, among the seven isolates.

Morphological features of the selected strains were observed. The standard biochemical

tests were performed that included Gram Staining Reaction, Motility Test, Starch Hydrolysis

Test, Casein Hydrolysis Test, Methyl Red – Voges Proskauer Test, Nitrate Reduction Test,

Oxidase Test, Catalase Test, Citrate Utilization Test, Indole production Test and Spore Forming

ability Test. From the tests, it was concluded that RT7, RT19 and RT21 were Bacilli. RT9 and

RT16 were Pseudomonas isolates.

Sequencing using 16S rRNA technique helped in the complete identification of the seven

isolates. They were identified as: RT3, RT7, RT9, RT10, RT16, RT19 and RT21 were identified

as Acinetobacter calcoaceticus, Bacillus subtilis, Pseudomonas aeruginosa, Rhodococcus terrae,

Pseudomonas aeruginosa, Bacillus siamensis and Bacillus subtilis subsp. inaquosorum,

respectively. Bacillus siamensis (RT19) has been reported to produce biosurfactant for the first

time, to the best of knowledge. Simlarly, the subsp inaquosorum of B.subtilis is also noted for

the first time, in literature. Different combinations of RT9, RT19 and RT21 were formulated for

development of microbial consortia. The consortium CONS-1 comprised B.siamensis and

B.subtilis subsp. inaquosorum. The consortium CONS-2 was formulated with B.siamensis and

P.aeruginosa. Lastly, the consortium CONS-3 had all the three isolates.

After agarose gel electrophoresis of the samples, the gel, when observed using a UV

transilluminator, did not reveal any bands. It could thus be concluded that the ability of the

bacterial isolates to secrete biosurfactants did not arise from a gene coded on plasmid DNA. In

all probability, the gene responsible for the production of biosurfactant must have been present

on the bulkier chromosomal DNA.

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