Research Article (Open access) |
---|
Int. J. Life. Sci. Scienti.
Res., 3(3): 1100-1105, May
2017
Impact of Biosurfactant
from Kocuria rosea
and Pseudomonas aeruginosa on
Germinating Seedlings of Glycine max, Pisum sativum and Spinacia oleracea
Latika P. Shendre1, Chandrakiran
S. Ukesh2, Sahadeo D. Patil3*
1Research
Scholar, Department of Microbiology and Biotechnology, Shri
Shivaji Science College, Amravati, Maharashtra, India
2Associate
Professor, Department of Microbiology and Biotechnology, Shri
Shivaji Science College, Amravati, Maharashtra, India
3Associate Professor & Head, Department of Microbiology and
Biotechnology, Shri Shivaji
Science College, Amravati, Maharashtra, India
*Address for
Correspondence: Dr. Sahadeo D. Patil,
Associate Professor & Head, Department of Microbiology
and Biotechnology, Shri Shivaji
Science College, Amravati- 444603, Maharashtra, India
ABSTRACT- Biosurfactant
is a structurally diverse group of surface-active molecule, synthesized by
microorganisms. Kocuria rosea
and Pseudomonas aeruginosa strains isolated from pesticide
contaminated soil, which produces biosurfactant were
studied. Curd whey was used as a cheap source of growth medium for biosurfactant production. There was formation of stable
emulsions of biosurfactant containing broth with vegetable
oil and kerosene. These strains produced a clear zone in oil spreading test,
which is an indicative of the good biosurfactant
activity. Both the strains produced extra cellular biosurfactant
in the culture media and showed good foam stability in the culture medium. Biosurfactant was efficiently extracted from the culture
broth by acetone-HCl precipitation. The biosurfactants from the two species, namely Kocuria rosea
and Pseudomonas aeruginosa were found
to have no effects on germinating seedlings of Glycine
max, Pisum sativum and
Spinacia oleracea, when
treated with 25%, 50%, 75% and
100% with the combination of curd whey
in the making of 100ml volume. Curd whey as a control was taken with no
surfactant. Our study suggested an efficient use in surfactant aided
bioremediation in agricultural land.
Key
words- Biosurfactant, Kerosene,
Emulsification, Oil spreading, Kocuria rosea, Pseudomonas aeruginosa, Glycine max, Pisum sativum and Spinacia oleracea
INTRODUCTION-
Surfactants and emulsifiers are indispensable
components of daily life. Biosurfactants are amphiphilic biological compounds produced extracellularly or as part of the cell membrane by a
variety of yeast, bacteria and filamentous fungi [1]. Biosurfactant is extensively used worldwide as it is
preferred to chemical surfactants for it being non toxic and easily
biodegradable [2]. Biosurfactants have
gained importance in the fields of enhanced oil recovery, environment,
bioremediation, food processing and pharmaceuticals [3]. The cost of biosurfactant is much cheaper as compared to chemical
surfactants. They are found to have lower critical micelle concentration value
as compared to chemical surfactant [4]. These compounds find applications in
an extremely wide variety of industrial processes involving emulsification,
foaming, detergency, wetting, dispersing or solubilization [2, 5]. Biosurfactant producing microorganisms
are naturally present in the oil contaminated soil. Oil contaminated environment
contain large amount of hydrocarbons. Hydrocarbons are composed of complex
chemical structure i.e., aliphatic and aromatic hydrocarbons. Microorganisms
exhibit emulsifying activity by producing biosurfactants
and utilize the hydrocarbons as substrate often mineralizing them or converting
them into harmless products [6].
One of the major biosurfactant
applications in environmental protection
is bioremediation. The most cost effective
methods include in-situ bioremediation.
Surface active agents are needed for the hydrophilization
of heavy soil for obtaining good wettability and also
to achieve equal distribution of fertilizers and pesticides in the soils [5]. Most of the pesticides are water soluble and are marketed in
powder or in liquid concentrate form. Before spraying in field, these are
diluted with water and mixed with surface active compound for spontaneous
distribution of the water insoluble pesticides in the aqueous phase as well as
an equal distribution on wetting of the treated areas [4].
Glycine max (Soyabean) and Pisum sativum (Pea) belong to the family fabaceae while Spinacia oleracea (Spinach)
belongs to the family amaranthaceae. The United
States, Brazil and Argentina are the world's largest soybean producers and
represent more than 80% of global soybean production followed by India, and China. China, India, United States of
America, France and Egypt are green pea producing countries. Spinach is grown
mainly in China, United States of America, Japan, Turkey and Indonesia. The seeds are rich in protein,
carbohydrates, fats and vitamins making them a highly nutritious food [7-8].
The present study focused
on the biosurfactant production by Kocuria rosea BS-4 and Pseudomonas aeruginosa BS-14 isolated from pesticide contaminated
soil. The biosurfactant production was screened by
oil spreading technique and emulsification
stability test.
Curd whey was used as a cheap and cost effective growth medium. In this
work we report the effect of biosurfactant from Kocuria rosea and Pseudomonas
aeruginosa on germinating seedlings of Glycine max, Pisum sativum and
Spinacia oleracea.
MATERIALS
AND METHODS- This study was
conducted in the Department of Microbiology and Biotechnology, Shri Shivaji Science College, Amravati
(Maharashtra), India in the duration of 2015-2016. All the chemicals were of
analytical grade and were obtained from Himedia
Laboratories Private Limited, India. Acetone was obtained from Merck, India. The seeds of G.
max, P. sativum
and S. oleracea
were obtained from an authorized dealer.
Enrichment culture and isolation of microbes-
Three pesticide contaminated soil samples were collected at four different
locations from Yawalkar Pesticide Industry, Nagpur
Maharashtra, India. One gram each of the soil samples was added to 100 ml of
mineral salt medium with 2% (v/v) liquid paraffin and incubated at 370C
for 7 days at 180 rpm. The composition of the mineral salt medium was modified
in our laboratory as follows: NaNO3 - 3 g; KH2PO4
- 1.5 g; Na2PO4 - 1.5 g; MgSO4.7H2O
-2 g; CaCl2.2H2O - 0.005 g; FeSO4 - 0.001 g;
ZnSO47H2O - 70 µg; CuSO4.5H2O – 50
µg; H3BO3 - 10 µg; MoO3 - 10 µg per liter; pH
7 ± 0.2. After 7 days 1 ml inoculums were transferred to the same medium and
incubated at 370C for another 7 days. The process was repeated for
three times and 1 ml inoculum from the third
enrichment culture was appropriately serially diluted and 100 µl aliquots from
the last two dilutions were streaked on nutrient agar plate. Isolated pure
colonies were picked up and maintained at 370C on nutrient agar
slant containing hydrocarbon.
Screening
for biosurfactant production- The
isolated pure cultures were transferred separately to culture tubes containing
30 ml mineral salt medium with 2% (v/v) liquid paraffin and incubated in
orbital shaker for 5 days at 180 rpm at 37°C. After the incubation period the
tubes were vortexed for 2 min to record the foaming
and turbidity of the growth medium.
Medium without inoculum served as control.
Culture
supernatant- The mineral
salt medium containing Biosurfactant was centrifuged
at 10,000 rpm for 10 min and supernatant was used for emulsification index and
oil spreading test.
Emulsification
index E24-
Emulsification
index of culture samples were determined by adding 2 ml of oil (kerosene,
vegetable oil, petrol and diesel) to the same amount of culture supernatant,
mixing with a vortex for 2 min, and leaving to stand for 24 hours. The
emulsification index was calculated as percentage of height of emulsified layer
(cm) divided by total height of the liquid column (cm) [9].
Oil Spreading Technique- 50 ml of distilled water was taken in glass Petri
dish with15 cm diameter and then followed by the addition of 20μl of oil
to the surface of water and finally 10μl of culture supernatant was added
to observe the clearance zone [10].
Identification of the selected culture- Best
two isolates (BS-4 and BS-14) screened for biosurfactant
production were identified according to the standard microscopical,
cultural and biochemical tests [11]
and comparing the results with Bergey’ Manual
of Systematic Bacteriology [12]. BS-4 and BS-14 were further
identified to species level using sequencing as Kocuria rosea and
Pseudomonas aeruginosa from Yaazh Xenomics, Mumbai (India).
Curd whey as a low cost growth
medium- The processing of curd whey was done by adjusting the pH
4.1 to 7 by using 5 N NaOH
and heating for 2-3 times. After adjusting the pH, whey was cooled and
centrifuged at 8000 rpm for 12
min to remove casein. Supernatant was adjusted to pH-7.0
again and 30 ml of
processed curd whey was
distributed in a big culture tube and sterilized at 1210c
for 20 minutes.
Sterile whey was separately
inoculated with isolated colonies and incubated
in orbital shaking incubator
for 96 hours (optimum biosurfactant
production time). Fermented whey containing biosurfactant
was centrifuged at 10,000 rpm for 10 min. Supernatants of biosurfactant producing organisms were used for
testing the
effect of biosurfactant on total length proliferation
of germinating seedlings [13].
Extraction of biosurfactant-
Extraction
was done by using acetone and then precipitation by HCI, overnight at 4°C. The
product recovered was dried under vacuum and preserved. The culture broth was
centrifuged (10000 g, 15 min) to remove the cells and thereafter sterilized
with millipore membrane filter. The clear sterile
supernatant served as the source of the crude biosurfactant.
The biosurfactant was recovered from the cell free
culture supernatant by cold acetone precipitation [9].
Effect of biosurfactant on
germinating seedlings- Biosurfactant produced
by Kocuria rosea BS-4
and Pseudomonas aeruginosa BS-14 was evaluated for its effect on
germinating seedlings of Glycine max (Soyabean), Pisum sativum (Pea) and Spinacia oleracea (Spinach). Four different
concentrations of biosurfactant 25%, 50%, 75% and 100% v/v produced
by Kocuria rosea and Pseudomonas aeruginosa
were prepared in curd whey. Curd whey at a concentration of 25%, 50%, 75% and
100% v/v in distilled water was used as a control. Ten healthy seeds of the
above plant species were presoaked in the respective concentration for 24
hours. The soaked seeds were spread on filter paper in petridishes
and the total length (cm) proliferation of the germinating seedlings was
recorded after 4 days.
RESULTS AND DISCUSSION
Isolation of
biosurfactant producing microbes-
The production of
surfactants by microbial cells often results in foaming and
emulsifies hydrophobic substrates [5]. These properties were used in
screening for surfactant producing
strains. A total of 14 isolates were isolated in pure form from three pesticide
contaminated soil samples. All the isolates showed good growth in mineral salt
medium containing 2% (v/v) liquid light paraffin as the sole carbon source. On
the basis of the foaming, turbidity and emulsification index two best isolates
BS-4 and BS-14 were considered for further investigation. Combination
of three tests, foaming, emulsification index and oil spreading tests are
commonly used to identify microbes as potential biosurfactant
producers [14].
Aqueous
solutions of both BS-4 and BS-14 biosurfactants
showed good foaming stability. Total disappearance of the foam was detected
after 2 hours. Stabilization of an oil and water emulsion is commonly used as a
surface activity indicator.
The results of emulsification activity and clearance
zone produced by oil spreading technique are shown in Table 1. All the
hydrocarbons tested served as substrates for emulsification by the biosurfactant. Diesel and kerosene were the best substrates
for both the strains while vegetable cooking oil was less good substrates for
emulsification. Overall BS-4 exhibited higher emulsification activity than
BS-14 with all the hydrocarbons used. Furthermore cell free broth culture of
BS-4 had highest emulsification activity of 72% with kerosene while BS-14 had
highest emulsification activity of 68.85% with diesel.
Table 1: Emulsification activity (E24) and clearance zone of biosurfactant
for different hydrocarbons
E24
value (%)/Clearance zone (mm) |
||||
Strains |
Vegetable
oil |
Kerosene |
Petrol |
Diesel |
BS-4 |
67/10 |
72/46 |
69/48 |
70/51 |
BS-14 |
64/06 |
67/40 |
68/42 |
68.85/48 |
In oil spreading technique, isolate BS-4
and BS-14 produced the maximum clearance zone of 51mm and 48mm respectively in
diesel.
Characterization and identification of the selected
cultures- Strain Kocuria rosea (BS-4)
and Pseudomonas aeruginosa (BS-14) found to be Gram-positive coccus
and Gram-negative rods respectively. Strain Kocuria rosea was found to be non-motile whereas Pseudomonas aeruginosa
proved
to be motile. Both the strains showed negative tests for indole production and MR-VP. Kocuria rosea
was negative for citrate reduction where as Pseudomonas aeruginosa
was positive for citrate reduction. Both were catalase
positive and showed positive test for urea hydrolysis. Kocuria rosea showed growth at temperature of 40C,
100C, 200C, 300C, 370C and 450C.
Growth of Pseudomonas aeruginosa was observed at 200C, 300C,
370C, and 450C. However K. rosea and P. aeruginosa
showed maximum growth at 370C and did not grow at 550C.
The pH range of 6.0 to 11.5 supported the growth of both strains with optimum
growth at pH 7.0.
Curd
whey as a growth medium- Processed curd whey had been used
as a good and cheap substitute for the production of biosurfactant.
Both the strains showed good biosurfactant
productivity when inoculated in processed curd whey. After centrifugation of the curd whey,
supernatants of biosurfactant producing organisms were used for
testing the
effect of biosurfactant on total length proliferation
of germinating seedlings.
Extraction of biosurfactant-
The biosurfactant was separated without loss of its
activity. The yields of biosurfactants of Kocuria rosea and Pseudomonas aeruginosa
were 3.68 g/l and
4.25 g/l, respectively. A maximum yield of around 5.5 g/l was reported by Pruthi and Cameotra[15] by three Pseudomonas species
grown on n-dodecane .
Effect
of biosurfactant in germinating Glycine max, Pisum sativum
and Spinacia oleracea The
total proliferation seedling length was measured on 5th day of
treatment (Table 2-4). Each value represents the mean of three replicates.
Table
2: Effect of curd whey as a control on
germinating seedlings of Glycine max, Pisum
sativum and Spinacia oleracea
Curd Whey |
Seed
type |
No.
of seeds 1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Length of proliferation in centimeters |
|||||||||||
25% |
Glycine
max |
2.1 |
2.9 |
2.6 |
3.1 |
3.2 |
1.9 |
2.5 |
3.4 |
2.8 |
2.0 |
Pisum sativum |
2.4 |
1.6 |
3.7 |
2.9 |
3.1 |
2.9. |
3.8 |
3.3 |
2.5 |
3.8 |
|
Spinacia oleracea |
1.0 |
0.8 |
1.1 |
1.3 |
0.9 |
1.2 |
0.8 |
0.7 |
1.4 |
1.3 |
|
50% |
Glycine
max |
1.9 |
2.7 |
3.1 |
2.8 |
1.7 |
3.0 |
2.2 |
2.7 |
2.9 |
2.1 |
Pisum sativum |
0.9 |
2.2 |
2.4 |
1.8 |
3.0 |
2.3 |
2.4 |
3.1 |
0.9 |
1.9 |
|
Spinacia oleracea |
2.0 |
1.5 |
0.2 |
1.5 |
0.1 |
1.2 |
0.6 |
0.1 |
0.0 |
1.1 |
|
75% |
Glycine max |
1.2 |
1.8 |
0.8 |
1.6 |
2.1 |
2.5 |
0.4 |
0.8 |
1.8 |
2.3 |
Pisum sativum |
2.3 |
1.9 |
2.1 |
1.9 |
0.2 |
0.7 |
0.0 |
2.5 |
3.1 |
2.7 |
|
Spinacia oleracea |
1.4 |
1.6 |
1.1 |
0.2 |
0.0 |
1.4 |
2.1 |
1.7 |
1.0 |
0.8 |
|
100% |
Glycine max |
1.2 |
1.1 |
0.9 |
0.8 |
1.6 |
1.3 |
1.2 |
0.6 |
1.0 |
0.9 |
Pisum sativum |
3.1 |
0.1 |
2.7 |
2.9 |
3.1 |
2.2 |
1.9 |
1.8 |
2.1 |
2.0 |
|
Spinacia oleracea |
0.7 |
1.1 |
0.2 |
0.0 |
1.3 |
0.9 |
1.9 |
2.0 |
0.0 |
0.1 |
Table
3: Effect of Biosurfactants produced by strain Kocuria rosea on
germinating seedlings of Glycine max (Soyabean), Pisum sativum and Spinacia oleracea
Kocuria rosea |
Seed
type |
No.
of seeds 1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Length of proliferation in centimeters |
|||||||||||
25% |
Glycine
max |
1.9 |
2.0 |
2.2 |
1.9 |
2.7 |
1.5 |
3.0 |
3.1 |
2.1 |
1.8 |
Pisum sativum |
1.8 |
3.2 |
2.1 |
2.8 |
2.0 |
3.2 |
3.4 |
3.9 |
2.8 |
3.4 |
|
Spinacia oleracea |
0.5 |
1.4 |
1.6 |
0.8 |
1.4 |
1.5 |
1.3 |
0.9 |
0.4 |
1.7 |
|
50% |
Glycine
max |
2.9 |
1.7 |
3.0 |
2.2 |
3.2 |
2.6 |
1.9 |
2.8 |
2.4 |
1.7 |
Pisum sativum |
3.1 |
2.4 |
3.3 |
2.5 |
2.7 |
1.8 |
2.2 |
3.0 |
2.7 |
3.1 |
|
Spinacia oleracea |
0.0 |
1.2 |
0.9 |
1.4 |
0.7 |
0.4 |
1.6 |
0.4 |
1.3 |
1.5 |
|
75% |
Glycine
max |
2.0 |
1.5 |
1.4 |
2.3 |
2.7 |
0.7 |
2.2 |
1.5 |
2.1 |
2.6 |
Pisum sativum |
3.3 |
0.4 |
2.5 |
2.1 |
0.8 |
1.6 |
0.6 |
3.2 |
2.4 |
3.4 |
|
Spinacia oleracea |
1.6 |
0.1 |
1.3 |
0.0 |
0.9 |
1.7 |
2.3 |
2.4 |
1.2 |
0.7 |
|
100% |
Glycine max |
2.1 |
1.9 |
1.2 |
1.3 |
2.2 |
1.9 |
2.1 |
0.2 |
2.3 |
2.7 |
Pisum sativum |
2.4 |
2.1 |
3.3 |
2.9 |
3.0 |
1.9 |
2.3 |
3.1 |
2.9 |
2.4 |
|
Spinacia oleracea |
0.1 |
1.3 |
1.7 |
0.3 |
1.9 |
2.2 |
2.1 |
2.4 |
0.8 |
0.0 |
Table
4- Effect of Biosurfactants produced by strain
Pseudomonas aeruginosa on germinating seedlings of Glycine
max (Soyabean), Pisum sativum and Spinacia oleracea
Kocuria rosea |
Seed
type |
No.
of seeds- 1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Length of proliferation in
centimeters |
|||||||||||
25% |
Glycine
max |
3.2 |
2.8 |
3.6 |
3.5 |
1.9 |
2.2 |
2.9 |
3.1 |
2.8 |
2.9 |
Pisum sativum |
3.4 |
2.8 |
2.9 |
2.6 |
3.0 |
3.1 |
2.8 |
3.4 |
3.5 |
3.7 |
|
Spinacia oleracea |
1.3 |
2.0 |
0.9 |
1.3 |
0.9 |
1.3 |
1.0 |
1.9 |
1.6 |
1.5 |
|
50% |
Glycine
max |
3.1 |
2.9 |
1.9 |
3.4 |
2.5 |
3.1 |
2.9 |
3.2 |
2.9 |
2.3 |
Pisum sativum |
2.9 |
3.2 |
2.6 |
3.2 |
2.0 |
1.8 |
2.6 |
3.2 |
1.6 |
0.7 |
|
Spinacia oleracea |
1.4 |
2.1 |
0.7 |
1.8 |
2.0 |
0.5 |
0.6 |
0.3 |
0.0 |
1.8 |
|
75% |
Glycine
max |
2.0 |
3.2 |
2.6 |
1.8 |
2.8 |
2.7 |
3.1 |
0.8 |
2.6 |
2.8 |
Pisum sativum |
3.3 |
2.7 |
2.4 |
0.9 |
3.0 |
0.9 |
2.6 |
2.9 |
2.8 |
2.1 |
|
Spinacia oleracea |
2.0 |
1.8 |
0.4 |
0.7 |
0.9 |
0.0 |
2.1 |
1.8 |
0.7 |
0.9 |
|
100% |
Glycine max |
2.3 |
0.8 |
1.7 |
0.7 |
1.4 |
2.3 |
2.1 |
0.8 |
1.3 |
0.9 |
Pisum sativum |
2.9 |
1.8 |
3.2 |
2.7 |
1.1 |
3.1 |
1.4 |
1.8 |
2.3 |
3.0 |
|
Spinacia oleracea |
2.2 |
1.7 |
0.6 |
0.0 |
1.1 |
0.9 |
1.8 |
2.3 |
0.0 |
0.0 |
The biosurfactant produced by Kocuria rosea and
Pseudomonas aeruginosa had no adverse effect
on the total length proliferation on the germinating seedlings of soyabean, peas and spinach.
Biosurfactant producing microorganisms are naturally present in
the pesticide contaminated soils. We successfully isolated bacteria with the
ability to produce biosurfactants from the soil
samples collected from pesticide industry. Further study on the utilization of
curd whey as a growth medium for the large-scale production of biosurfactants was done. The concentration of biosurfactant used in this study ranged from 25%- 100%
which is suggestible for practical application of biosurfactant
in surfactant aided bioremediation of cultivated land. Also, the biosurfactant could possibly be used in hydrophilization
of heavy soil and even distribution of pesticides and fertilizers in
agriculture.
CONCLUSIONS- This
study represented surfactant activity of the bacterial strains isolated from
pesticide contaminated soils from the pesticide industry. The present study is
an attempt to find economically cheaper sources for the large scale production
of microbial biosurfactant. Results obtained in biosurfactant production with curd whey waste suggested the
possibility of industrial production of biosurfactant
using economically cheaper source. We concluded in our result that the biosurfactant from strains Kocuria rosea and Pseudomonas aeruginosa
have no effect on the germination seedlings of Glycine max, Pisum sativum and Spinacia oleracea.
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