4.1 isolation and screening of serratia...
TRANSCRIPT
97
RESULTS AND DISCUSSION
4.1 Isolation and Screening of Serratia marcescens
4.1.1. Soil sample
Microbiological studies were conducted and revealed the presence of bacteria
in soil samples obtained from the different sites of the Allahabad region of Uttar Pradesh,
India. Total viable bacterial counts and total potential Serratia marcescens counts
from the soil samples are summarized in Table 4.1. The total number of bacterial
colonies resembling Serratia marcescens was counted by colony counting method.
Bacterial colonies producing the distinct red pigment on Luria Bertani agar at 27±2˚C
were isolated from 60 soil samples.
4.1.2 Water sample
Microbiological studies of different water samples from the vicinity of the
Sangam region of Allahabad as well as different households showed the presence of a
vast variety of microorganisms that have the ability to thrive in various different types
of water sources. Total viable bacterial counts and total potential Serratia marcescens
counts from the water samples are summarized in Table 4.2. Bacterial colonies
producing the distinct red pigment on Luria Bertani agar at 27±2˚C were isolated
from 40 water samples.
The result thus obtained suggests that the soil and water of the Allahabad region of
Uttar Pradesh, India especially in and around the river banks is a thriving source for
innumerable varieties of bacteria with potential Serratia marcescens bacteria present
in moderate proportions. High density of such different varieties of bacteria including
Serratia may be due to the rise in human activities by pilgrims who visit in great
numbers annually which has a profound impact on the substantial rise in
environmental pollution and bacterial resistance to a wide number of diseases.
The specific study of these potential Serratia isolates from simple environmental
sources and its metabolites and biological products will be definitely useful to find
means to create an antimicrobial agent that can be targeted against potentially disease
causing bacteria and may also find applications in other industries.
98
Table 4.1 Isolation of Serratia marcescens from soil samples of Allahabad, Uttar Pradesh
Sample
no.
Sampling site Total Viable
Count
(cfu x106/g)
No. of
presumptive
Serratia
marcescens
Sangam region
2 10cm depth at river bank (RS 2) 2
feet away from RS 1
7.1 2
10 10cm depth at river bank (RS 10) 2
feet away from RS 9
10.3 1
18 10cm depth 3 ft. away from RS 5
towards higher ground (RS18)
0.64 2
Saraswati Ghat
27 10cm depth 3 ft. from RS 21 towards
higher ground (RS 27)
42 2
34 10cm depth 2 feet from RS 25
towards higher ground (RS 34)
0.105 1
Balua Ghat:
38 10cm depth at river bank (RS 38) 2
feet away from RS 37
0.7 2
44 10cm depth 2 feet from RS 39
towards higher ground (RS 44)
0.47 2
Company Garden soil, Allahabad
55 10cm depth Park soil PS 55 4.5 1 ND – not detected
99
Table 4.2 Isolation of Serratia marcescens from water samples of Allahabad,
Uttar Pradesh
Sample
no.
Sampling site Total Viable
Count
(cfu x106/ml)
No. of potential
Serratia
marcescens
isolates
Sangam region
65 Site 5: 10-15cm depth River water
near river bank (RW 65)
0.079 1
68 Site 8: 10-15cm depth River water
near river bank (RW 68)
7.6 1
Balua Ghat
77 Site 1: 10-15cm depth River water
near river bank (RW 77)
7.7 2
84 Site 8: 10-15cm depth River water
near river bank (RW 84)
0.067 1
Rainwater logging at Parade Ground, Allahabad
100 Site 2: (PG 100) 0.76 1 ND – not detected
4.2. Screening and selection of Serratia marcescens from soil and water samples
Out of the hundred samples collected, sixty and forty samples comprised of soil and
water respectively. All samples were collected from in and around Allahabad city of
Uttar Pradesh, India. The soil and water samples were serially diluted to minimize
overcrowding by other bacterial species. Out of the 60 soil samples, 13 potential
isolates of Serratia marcescens were obtained which produced a red to orange
coloured pigment (Table 4.1.1). 6 potential Serratia marcescens isolates were
obtained from 40 water samples which also produced red coloured pigment (Table
4.1.2). In order to confirm that the 19 isolates obtained after sampling were that of the
100
desired organism, morphological and biochemical tests were conducted to establish
this fact.
Identification and characterization of potential isolates of Serratia
marcescens
4.3. Identification of potential isolates
The isolates were identified by studying morphological and biochemical
characteristics according to Bergey’s Manual of Determinative Bacteriology (Holt et
al., 1989). The procedures for the various tests were performed in accordance with
Benson’s Microbiological Applications, Laboratory Manual in General Microbiology
(Brown, 2007). Detailed morphological and biochemical tests of the isolates are
given in Tables of 4.3. and 4.4. respectively. Two or more isolates from the same
source and dilution have been named as ‘a, b’ respectively.
On the basis of the morphological tests, isolates RW65 and PG 100 were eliminated
since they were identified as gram positive cocci. The remaining 17 isolates were
subjected to biochemical tests for final identification and confirmation.
Biochemical tests indicated that isolates 18a and 18b respectively produced acid from
lactose which is not in accordance with the identification of Serratia marcescens but
holds true for another species of the same genus (Tables 4.4) Hence the above
mentioned strains were eliminated from the final list of isolates.
The final group of 15 isolates were renamed as ‘Sm’ strains and were serially
numbered with ‘S’ and ‘m’ denoting Serratia and marcescens respectively (Table
4.5).
101
PG100
RW
84
RW
77b
RW77a
RW
68
RW
65
PS55
RS44b
RS44a
RS38b
RS38a
RS34
RS27b
RS27a
RS18b
RS18a
RS10
RS2b
RS2a
Isolate no.
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Round
Configuration
Colony m
orphology
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Entire
Margin
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Convex
Elevation
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Surface
Pink
Orange red
Red
Red
Orange red
Pink
Pink red
Orange red
Orange red
Pink red
Pink red
Red
Pink red
Pink red
Orange red
Orange red
Pink red
Red
Red
Pigment
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opaque
Opacity
+ - - - - + - - - - - - - - - - - - -
Gram
’s staining
Cocci
Bacilli
Bacilli
Bacilli
Bacilli
Cocci
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Bacilli
Cell shape
Tetrads
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Tetrads
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Singly & groups
Arrangem
ent
- - - - - - - - - - - - - - - - - - -
Spores
+ + + + + + + + + + + + + + + + + + +
Motility
Table 4.3: Morphological characteristics of Serratia m
arcescens isolates
102
RW
84
RW
77b
RW77a
RW
68
PS55
RS44b
RS44a
RS38b
RS38a
RS34
RS27b
RS27a
RS18b
RS18a
RS10
RS2b
Isolate no.
- - - - - - - - - - - + + - - -
Lactose
Sugar Fermentations
+ + + + + + + + + + + + + + + +
Glucose
+ + + + + + + + + + + + + + + +
Mannitol
+ + + + + + + + + + + + + + + +
Catalase
- - - - - - - - - - - - - - - -
Oxidase
- - - - - - - - - - - - - - - -
Indole
- - - - - - - - - - - - - - - -
Methyl
Red
+ + + + + + + + + + + + + + + +
Voges
Proska-uer
+ + + + + + + + + + + + + + + +
Citr-
ate
- - - - - - - - - - - - - - - -
H2 S
produc-tion
+ + + + + + + + + + + + + + + +
Gas
production from
glucose
+ + + + + + + + + + + + + + + +
Lip-
ase T
est
- - - - - - - - - - - - - - - -
Star-ch
Hydro
lysis
- - - - - - - - - - - - - - - -
Gelatin
Liquefact
-ion
Table 4.4: Biochemical characteristics of Serratia m
arcescens isolates
103
Table 4.5: Naming of the isolates after identification as Serratia marcescens
Serial
No.
Previous naming of potential
Serratia marcescens isolates
Final naming of Serratia
marcescens isolates after
identification
1 RS2a Sm1
2 RS2b Sm2
3 RS10 Sm3
4 RS27a Sm4
5 RS27b Sm5
6 RS34 Sm6
7 RS38a Sm7
8 RS38b Sm8
9 RS44a Sm9
10 RS44b Sm10
11 PS55 Sm11
12 RW68 Sm12
13 RW77a Sm13
14 RW77b Sm14
15 RW84 Sm15
4.4 Characterization of identified isolates
4.4.1. Heavy metal tolerance capacity
The minimal inhibitory concentration (MIC) of heavy metals for all Serratia
marcescens isolates have been shown in Table 4.6. It was observed that all the strains
were inhibited by Cadmium even at concentrations as low as 25µg/ml. Most of the
strains were tolerant towards relatively high concentrations of Lead followed by
Copper. Sm1, Sm6, Sm9 and Sm13 were able to tolerate 100 µg/ml of lead with Sm4,
Sm5, Sm10 and Sm12 growing at an even higher concentration of 200µg/ml
respectively. The isolates showed moderate tolerance towards Nickel with strains
Sm1 and Sm9 showing considerably high tolerance. All the isolates showed low
tolerance towards Chromium and Cobalt. Maximum tolerance towards heavy metals
104
was seen in the case of Sm1 and Sm9 towards Chromium, Copper, Cobalt, Lead and
Nickel respectively. Minimum inhibitions towards heavy metals were observed for
Sm2, Sm3, Sm7, Sm11, Sm14 and Sm15 respectively.
Table 4.6 Minimum Inhibitory Concentration (MIC) of heavy metals of
Serratia marcescens isolates
Serratia
marcescens
strains
Heavy metal (ìg ml-1)
Cadmium
(Cd)
Chromium
(Cr)
Copper
(Cu)
Cobalt
(Co)
Lead
(Pb)
Nickel
(Ni)
Sm1 < 25 25 100 25 100 400
Sm2 < 25 < 25 < 25 < 25 < 25 50
Sm3 < 25 < 25 < 25 < 25 50 25
Sm4 < 25 < 25 50 < 25 200 50
Sm5 < 25 < 25 50 < 25 200 50
Sm6 < 25 25 100 25 100 50
Sm7 < 25 < 25 < 25 < 25 < 25 25
Sm8 < 25 < 25 50 25 50 50
Sm9 < 25 25 100 < 25 100 400
Sm10 < 25 < 25 50 < 25 200 50
Sm11 < 25 < 25 < 25 < 25 50 25
Sm12 < 25 < 25 50 < 25 200 50
Sm13 < 25 25 100 25 100 50
Sm14 < 25 < 25 50 < 25 < 25 25
Sm15 < 25 < 25 < 25 25 < 25 50 * < - less than
4.4.2. Antibiotic susceptibility
The results for antibiotic susceptibility are given in Table 4.7. All Serratia
isolates were subjected to antibiotic susceptibility tests using 22 different antibiotics
which are routinely used clinically. All the isolates were completely susceptible in the
presence of Amikacin, Ceftriaxone, Cefuroxime, Cephotaxime, Chloramphenicol,
Ciprofloxacin, Cloxacillin, Erythromycin, Gentamicin, Neomycin sulfate, Netilmicin,
Polymyxin, Tetracycline and Vancomycin antibiotics respectively. All the isolates
105
were resistant towards Cefepime, Ceftazidime, Nalidixic acid and Trimethoprim
respectively. Certain isolates like Sm1, Sm3, Sm4, Sm5 and Sm15 showed resistance
towards Ampicillin whereas the rest of the isolates were sensitive towards drug.
Except for Sm3, Sm5, Sm8, Sm10, Sm12, Sm13 and Sm15, all the remaining strains
grew in the presence of Cephalexin. Sm1, Sm6, Sm9, Sm13 and Sm15 were sensitive
towards Streptomycin while all the other isolates grew in its presence. The interesting
fact was that strains Sm5, Sm9 and Sm13 were resistant towards Imipenem (with
cilastatin), a fourth generation carbapenem which is a drug of choice to treat severe
Gram negative bacterial infections.
Table 4.7 Antibiotic susceptibility pattern in Serratia isolates
Antibiotics Potency (mcg/ units)
Resistant (+) / Sensitive (-) Sm strains 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Amikacin (AMK) 30 - - - - - - - - - - - - - - -
Ampicillin (AMP) 10 + - + + + - - - - - - - - - +
Cefepime (FEP) 30 + + + + + + + + + + + + + + +
Ceftazidime (CAZ) 30 + + + + + + + + + + + + + + +
Ceftriaxone (CRO) 30 - - - - - - - - - - - - - - -
Cefuroxime (CXM) 30 - - - - - - - - - - - - - - -
Cephalexin (LEX) 30 + + - + - + + - + - + - - + -
Cephotaxime (CTX)
30 - - - - - - - - - - - - - - -
Chloramphenicol (CHL)
30 - - - - - - - - - - - - - - -
Ciprofloxacin (CIP)
5 - - - - - - - - - - - - - - -
Cloxacillin (CLX) 1 - - - - - - - - - - - - - - -
Erythromycin (ERY)
15 - - - - - - - - - - - - - - -
Gentamicin (GEN) 10 - - - - - - - - - - - - - - -
Imipenem (IPM) 10 - - - - + - - - + - - - + - -
Nalidixic acid (NAL)
30 + + + + + + + + + + + + + + +
Neomycin 30 - - - - - - - - - - - - - - -
106
sulphate (NMS)
Netilmicin (NET) 30 - - - - - - - - - - - - - - -
Polymyxin (PMB) 300 units - - - - - - - - - - - - - - -
Streptomycin (STR)
10 - + + + + - + + - + + + - + -
Tetracycline (TET) 30 - - - - - - - - - - - - - - -
Trimethoprim (TMP)
23.75
units
+ + + + + + + + + + + + + + +
Vancomycin (VAN)
30 - - - - - - - - - - - - - - -
4.4.3 Plasmid Curing
Plasmid curing experiment was conducted using acridine orange
(20µg/ml) as the mutagenic agent, which gets intercalated between the bases of DNA
thereby causing a mutation. This leads to changes in the concerned DNA bases and
inhibits plasmid replication without altering or inhibiting normal chromosomal
replication of the host cell DNA. Such inhibition can lead to loss of plasmid and if the
gene for antibiotic resistance or metal tolerance is present on such a plasmid, then
after treatment the host cell will lose this property of resistance or tolerance.
All of the 15 isolates of Serratia marcescens from different sources were
tested on Luria Bertani agar Petri plates incorporated with metals and antibiotics
towards which the organisms showed resistance (Table 4.8). Plasmid curing studies
showed variable results for all the strains of Serratia marcescens revealing that they
behaved differently in their plasmid borne resistant gene patterns.
Sm1 showed about 90% curing for nickel alone. Sm3 showed about 69%
curing for lead. Sm6 revealed 76% curing for copper, 66% curing for chromium and
83% curing for lead. Sm9 showed about 72% curing for only nickel. Some of the
remaining isolates did show curing but the results were not significant and were
recorded at below 50%. The rest of the isolates did not give significant curing results
(Table 4.9 and Figure 4.1(i)).
Sm1 showed moderate levels of curing for ampicillin and ceftazidime and
absolute curing for cefepime and cephalexin. Sm2 showed significantly high levels of
curing for ceftazidime, cefepime, cephalexin and streptomycin and 60% curing for
107
trimethoprim. Sm3 showed moderate levels of curing for ampicillin, ceftazidime, and
nalidixic acid and 90% for streptomycin. Sm4 showed very high levels of curing for
nearly all antibiotics. Results obtained showed 100% curing for ampicillin,
cephalexin, nalidixic acid and streptomycin. It also showed 87% curing for
ceftazidime, 91% for cefepime and 76% for trimethoprim. Sm5 showed very high
levels of curing for ampicillin, ceftazidime, cefepime, nalidixic acid, streptomycin and
trimethoprim and moderate levels of curing for imipenem. Sm6 showed reasonable
levels of curing for ceftazidime and cefepime and 100% curing for cephalexin. Sm7
depicted moderate levels of curing for ceftazidime and cefepime and high levels of
curing for cephalexin. Sm8 showed reasonable levels of curing for only streptomycin
and trimethoprim. Sm9 showed absolute curing for cefepime and nalidixic acid with
moderate curing for imipenem and ceftazidime recorded at 52.5% and 65.8%
respectively. It however showed low levels of curing for cephalexin and trimethoprim
with values of curing calculated at 34% and 47% respectively. 100% curing for
trimethoprim was seen in the case of Sm10 and moderate curing for ceftazidime and
streptomycin. It showed low curing levels for cefepime and nalidixic acid. Sm11
however, did not show significant levels of curing for any of the antibiotics that were
used for the plasmid curing experiment except for ceftazidime where it showed
moderate levels of curing. The rest of the values were very low and zero curing was
seen for streptomycin. Sm12 showed moderate levels of curing for ceftazidime,
cefepime and trimethoprim and low levels were seen in the case of streptomycin and
the lowest for nalidixic acid. Complete curing for cefepime was observed for Sm13
with moderate curing of about 62% for imipenem and low levels of curing for
ceftazidime, nalidixic acid and trimethoprim. Nearly complete curing was observed
for cefepime in the case of Sm14. The isolate recorded moderate level of curing for
trimethoprim and low levels for ceftazidime, cephalexin, nalidixic acid and
streptomycin. Sm15 showed moderate curing for ampicillin and trimethoprim and
high level of 85% for cefepime. However, it showed low level of curing for
ceftazidime and poor curing level for nalidixic acid. All the above results are shown
in Table 4.10 and Figure 4.1(ii). All plasmid curing results greater than or equal to
50% were considered as successful and have been depicted in Table 4.11 for each
Serratia marcescens isolate.
108
Table 4.8 Selection of Serratia marcescens strains for plasmid curing of
specific antibiotics and heavy metals
Isolate No. Antibiotics Heavy Metals
Sm1 AMP, CAZ, FEP, LEX, NAL, TMP Co, Cr, Cu, Ni, Pb
Sm2 CAZ, FEP, LEX, NAL, STR, TMP Ni
Sm3 AMP, CAZ, FEP, NAL, STR, TMP Ni, Pb
Sm4 AMP, CAZ, FEP, LEX, NAL, STR, TMP Cu, Ni, Pb
Sm5 AMP, CAZ, FEP, IPM, NAL, STR, TMP Cu, Ni, Pb
Sm6 CAZ, FEP, LEX, NAL, TMP Co, Cr, Cu, Ni, Pb
Sm7 CAZ, FEP, LEX, NAL, STR, TMP Ni
Sm8 CAZ, FEP, NAL, STR, TMP Co, Cu, Ni, Pb
Sm9 CAZ, FEP, IPM, LEX, NAL, TMP Cr, Cu, Ni, Pb
Sm10 CAZ, FEP, NAL, STR, TMP Cu, Ni, Pb
Sm11 CAZ, FEP, LEX, NAL, STR, TMP Ni, Pb
Sm12 CAZ, FEP, NAL, STR, TMP Cu, Ni, Pb
Sm13 CAZ, FEP, IPM, NAL, TMP Co, Cr, Cu, Ni, Pb
Sm 14 CAZ, FEP, LEX, NAL, STR, TMP Cu, Ni
Sm 15 AMP, CAZ, FEP, NAL, TMP Co, Ni *Abbreviations for heavy metals and antibiotics given in Tables 4.4.1 and 4.4.2 respectively
Table 4.9 Plasmid curing of heavy metal tolerance capacity (≥ 50%) in
Serratia isolates
Isolate No. Heavy Metal No. of
colonies
tested
No. of
colonies
cured
Percentage
curing (%)
Sm1 Ni 120 107 89.16
Sm3 Pb 120 83 69.16
Sm6
Cr 120 79 65.8
Cu 120 91 75.8
Pb 120 99 82.5
Sm8 Pb 120 60 50
Sm9 Ni 120 86 71.66
109
Sm12 Cu 120 60 50
Pb 120 69 57.5 *Abbreviations for heavy metals given in Table 4.4.1
Figure 4.1(i): Comparison of Serratia isolates towards plasmid curing of heavy metal tolerance capacity
Table 4.10 Plasmid curing of antibiotic susceptibility (≥ 50%) in Serratia isolates
Isolate
No.
Antibiotic No. of colonies
tested
No. of colonies
cured
Percentage curing
(%)
Sm1
AMP 120 80 66.66
CAZ 120 95 79.16
FEP 120 120 100
LEX 120 120 100
Sm2
CAZ 120 99 82.5
FEP 120 101 84.16
LEX 120 117 97.5
110
STR 120 114 95
TMP 120 73 60.83
Sm3
AMP 120 81 67.5
CAZ 120 77 64.16
FEP 120 66 55
NAL 120 95 79.16
STR 120 109 90.83
TMP 120 63 52.5
Sm4
AMP 120 120 100
CAZ 120 105 87.5
FEP 120 110 91.66
LEX 120 120 100
NAL 120 120 100
STR 120 120 100
TMP 120 91 75.8
Sm5
AMP 120 120 100
CAZ 120 111 92.5
FEP 120 101 84.16
IPM 120 75 62.5
NAL 120 101 84.16
STR 120 105 87.5
TMP 120 98 81.6
Sm6
CAZ 120 74 61.66
FEP 120 86 71.6
LEX 120 120 100
111
NAL 120 64 53.3
Sm7
CAZ 120 78 65
FEP 120 72 60
LEX 120 109 90.8
STR 120 63 52.5
TMP 120 64 53.3
Sm8
CAZ 120 63 52.5
FEP 120 66 55
STR 120 91 75.8
TMP 120 91 75.8
Sm9
IMP 120 63 52.5
CAZ 120 79 65.8
FEP 120 120 100
NAL 120 120 100
Sm10
CAZ 120 65 54.1
STR 120 74 61.66
TMP 120 120 100
Sm11 CAZ 120 63 52.5
Sm12
CAZ 120 72 60
FEP 120 71 59.16
TMP 120 66 55
Sm13 IMP 120 74 61.66
FEP 120 120 100
Sm14 FEP 120 117 97.5
TMP 120 66 55
112
Sm15
AMP 120 74 61.66
FEP 120 102 85
TMP 120 64 53.33
*Abbreviations for antibiotics given in Table 4.4.2
Figure 4.1(ii): Comparison of Serratia isolates towards plasmid curing of antibiotic resistance
113
Table 4.11: Comparison of successful plasmid curing (≤50%) of heavy metal tolerance and antibiotic susceptibility in Serratia isolates
Isolate
No.
Successful curing of Antibiotics
(≤50%)
Successful curing
Heavy Metals (≤ 50%)
Sm1 AMP, CAZ, FEP, LEX Ni Sm2 CAZ, FEP, LEX, STR, TMP -
Sm3 AMP, CAZ, FEP, NAL, STR, TMP Pb
Sm4 AMP, CAZ, FEP, LEX, NAL, STR, TMP -
Sm5 AMP, CAZ, FEP, IMP, LEX, NAL, STR, TMP
-
Sm6 CAZ, FEP, LEX, NAL Cr, Cu, Pb
Sm7 CAZ, FEP, LEX, STR, TMP -
Sm8 CAZ, FEP, STR, TMP Pb Sm9 IPM, CAZ, FEP, NAL Ni
Sm10 CAZ, STR, TMP - Sm11 CAZ - Sm12 CAZ, FEP, TMP Cu, Pb Sm13 IPM, FEP - Sm14 FEP, TMP - Sm15 AMP, FEP, TMP -
4.5. Optimization of physiological parameters for maximum
production of prodigiosin
Mekhael and Yousif (2008) studied optimization of prodigiosin production
by using a specific formula to estimate the amount of prodigiosin produced for each
parameter tested. Since a UV-spectrophotometer was used by them and a colorimeter
has been used in this case, the wavelengths had to be altered to the closest possible
wavelengths to the reference UV- spectrophotometer. The standard graph obtained
after changing wavelengths and estimating the values at both 490nm and 610nm was
prepared. The value for the new constant was calculated after taking the average
values of the ratio of cell density at 490nm and 610nm at three vital stages of cell
growth. The final constant was obtained and was substituted in place of the existing
114
constant ‘1.381’ and the modified formula was then used for the optimization of
prodigiosin production using several parameters.
4.5.1. Effect of Incubation time on growth and prodigiosin production
The pattern of growth for all isolates of Serratia marcescens showed a steady
increase and prodigiosin production was also enhanced with increasing incubation
time at 30˚C at pH 7.0 grown on Luria-Bertani media. The isolates were incubated
separately at 24, 48, 72, 96, 120 and 144 hours respectively. Sm1 and Sm8 grew
rapidly in the first 48 hours. The rest of the isolates showed rapid growth which
reached maximum levels at 72 hours. The pigment production was initially negligible
in case of all isolates since it is a secondary metabolite. In the case of Sm1, maximum
cell growth occurred at 72 hours and pigment production was also highest at this level
(79.55units/cell). After this, pigment production reduced to 48.54units/cell at 96
hours. Beyond this, there was a decline in cell density and pigment production. In
case of Sm2, although cell growth occurred rapidly until 72 hours, pigment
production was very low and occurred after 120 hours of incubation. It was found to
be only 57.57units/cell. Sm3 showed produced 202.63units/cell of pigment after 72
hours of incubation. Sm4 also showed reasonably high levels of pigment production
of 200units/cell. Sm5 produced very low amounts of the pigment (20units/cell) after
96 hours and maximum cell density at 72 hours. Sm6 produced the highest amount of
pigment when compared to the rest of the isolates which was found to be
381.66units/cell at 96 hours which was considered to be highly significant based on
statistical analysis (F test) as seen in Appendix 4 Anova Table 1c. Sm7, Sm9 and
Sm15 produced modest amounts of the pigment calculated to be 145units/cell,
105.26units/cell and 156units/cell respectively. Sm8, Sm13, Sm14 produced moderate
quantities of 50units/cell, 61.19units/cell and 77.77units/cell respectively. However,
Sm10, Sm11 and Sm12 produced very low levels of prodigiosin such as
6.94units/cell, 7.56units/cell and 19.46units/cell respectively. Most strains showed
highest production after 72 hours of incubation which is in accordance with Samrot
et al. (2011) which was also found to be significant for most isolates as shown in
Appendix 4 Anova Table 1b. This also corresponded with the late exponential phase
of cell growth. The effect of incubation time on each individual isolate has been
demonstrated between Figures 4.2(i) and 4.2(xv). The comparison of incubation time
of all isolates for prodigiosin production has been shown in Figure 4.2(xvi).
115
Figure 4.2(i): Effect of incubation time on Sm1
Figure 4.2(ii): Effect of incubation time on Sm2
Figure 4.2(iii): Effect of incubation time on Sm3
116
Figure 4.2(iv): Effect of incubation time on Sm4
Figure 4.2(v): Effect of incubation time on Sm5
Figure 4.2(vi): Effect of incubation time on Sm6 (***highest production)
117
Figure 4.2(vii): Effect of incubation time on Sm7
Figure 4.2(viii): Effect of incubation time on Sm8
Figure 4.2(ix): Effect of incubation time on Sm9
118
Figure 4.2(x): Effect of incubation time on Sm10
Figure 4.2(xi): Effect of incubation time on Sm11
Figure 4.2(xii): Effect of incubation time on Sm12
119
Figure 4.2(xiii): Effect of incubation time on Sm13
Figure 4.2(xiv): Effect of incubation time on Sm14
Figure 4.2(xv): Effect of incubation time on Sm15
120
Figure 4.2(xvi): Comparison of effect of incubation time on prodigiosin production from Serratia isolates (Highly significant ***F cal17086.25 > Ftab2.085 at 5% with 44 df at 72 hours incubation time)
4.5.2. Effect of Incubation temperature
The biosynthesis of a pigment is significantly affected by the physiological
parameter, temperature (Hejazi and Falkiner, 1997). In order to determine the effect
of temperature on the production of prodigiosin by the different strains of Serratia,
the cells were incubated at 25, 30, 35, 40 and 45˚C respectively. The incubation time
was kept at the optimum for each isolate from the above data obtained from the above
parameter. Incubation temperature was found to be a critical parameter as all the
isolates were affected by changes in incubation temperature.
Figure 4 illustrates the optimum temperature required for pigment production
for the 15 strains of Serratia. Six of the strains produced optimum quantities of the
pigment at 25˚C. Sm1, Sm4, Sm6, Sm7, Sm13 and Sm15 produced 147.02, 248,
257.14, 196.15, 96.29 and 169.38units/cell of pigment respectively. The remaining
isolates showed optimum prodigiosin production at 30˚C. Sm2, Sm3, Sm5, Sm8,
Sm9, Sm10 and Sm12 produced high quantities of 196.15, 285.2, 182.61, 139.13,
144.44, 142.86, and 120.88units/cell of prodigiosin respectively. Sm11 and Sm14
synthesized lower quantities of 50units/cell and 96.26units/cell of pigment
121
respectively. The effect of incubation temperature of individual isolates on
prodigiosin production has been depicted between Figures 4.3(i) and 4.3(xv).
All pigment production was inhibited completely at 45˚C. Pigment production
was shown to decrease with increasing temperature. 30˚C was selected to be the
optimum temperature for pigment production based on the results obtained and
showed significant results of pigment production as seen by statistical analysis in
Appendix 4 Anova Table 2b. 35˚C showed reduced levels of pigment production
though cell density was the highest at this point. Khanafari et al. (2006) and Giri et
al. (2004) showed similar results with optimum pigment production temperature
recorded to be 28˚C and 30˚C respectively. Hence, it can be concluded that lower
temperature definitely favours prodigiosin formation though optimal growth
temperature of bacterial cells is 37˚C. Comparison of incubation of all isolates has
been shown in Figure 4.3(xvi).
Figure 4.3(i): Effect of incubation temperature on Sm1
Figure 4.3(ii): Effect of incubation temperature on Sm2
122
Figure 4.3(iii): Effect of incubation temperature on Sm3 (***highest production)
Figure 4.3(iv): Effect of incubation temperature on Sm4
Figure 4.3(v): Effect of incubation temperature on Sm5
123
Figure 4.3(vi): Effect of incubation temperature on Sm6
Figure 4.3(vii): Effect of incubation temperature on Sm7
Figure 4.3(viii): Effect of incubation temperature on Sm8
124
Figure 4.3(ix): Effect of incubation temperature on Sm9
Figure 4.3(x): Effect of incubation temperature on Sm10
Figure 4.3(xi): Effect of incubation temperature on Sm11
125
Figure 4.3(xii): Effect of incubation temperature on Sm12
Figure 4.3(xiii): Effect of incubation temperature on Sm13
Figure 4.3(xiv): Effect of incubation temperature on Sm14
126
Figure 4.3(xv): Effect of incubation temperature on Sm15
Figure 4.3(xvi): Effect of incubation temperature on prodigiosin production from Serratia isolates (Fcal17638.29>Ftab 2.085 at 5% with 44df at 30°C incubation temperature)
4.5.3. Effect of pH
The growth and type of pigment produced by an organism is affected largely
by the pH of the medium in which the microorganism is grown. Slight changes in pH
can also alter the shade of colour produced (Joshi et al., 2003). The influence of pH
on the production of prodigiosin was studied at different pH values ranging from 6 to
10. The effect of pH on prodigiosin production by individual isolates has been
recorded between Figures 4.4(i) and 4.4(xv). The graphical representation shows that
127
increased production of prodigiosin occurred at mainly pH 8 and pH 9. For all
isolates, pH 6 and 10 showed the lowest synthesis of the pigment. At pH 7, low to
moderate quantity of the pigment was produced for all the isolates.
Five isolates showed their optimum pH to be 9. Sm1, Sm6, and Sm8 produced
considerably high yield of the pigment which amounted to 151.85, 226.08, and
233.33units/cell respectively. Sm10 and Sm14 produced low to moderate amounts of
62.5 and 100units/cell respectively. Khanafari et al. (2006) reported that pH 9 was
the optimum pH for cell growth of Serratia. Nine of the Serratia isolates produced
maximum amounts of the pigment at pH 8. Sm2, Sm3, Sm4, Sm5, Sm7, Sm9 and
Sm15 generated increased levels of 234.61, 232.35, 210.81, 208, 202.94, 157.14 and
172.05units/cell of prodigiosin. Sm12 and Sm13 synthesized relatively moderate
quantities of prodigiosin (77.77 and 67.5units/cell) respectively. According to
Mekhael and Yousif (2008) the optimum pH was observed to be 8 which
substantiated the find. The amount of prodigiosin produced was found to be highly
significant at this pH as seen by statistical analysis in Appendix 4 Anova Table 3c
with Sm2 producing the highest amount of the pigment.
Only Sm11 showed optimum pigment production amounting to very low
amounts at pH 7 which was found to be 16.94units/cell. Enhanced pigment production
at pH 7 has been reported by Samrot et al. (2011) and Tao et al. (2005). The
comparative effect of pH on all isolates has been depicted in the Figure 4.4(xvi).
Hence, it can be concluded that most of the pigment producers showed
optimum production at pH 8 hence tending towards slight to moderate alkaline
environment except for Sm11 which showed optimum pigment production at the
neutral pH of 7.
128
Figure 4.4(i): Effect of pH on Sm1
Figure 4.4(ii): Effect of pH on Sm2 (***highest production)
Figure 4.4(iii): Effect of pH on Sm3
129
Figure 4.4(iv): Effect of pH time on Sm4
Figure 4.4(v): Effect of pH on Sm5
Figure 4.4(vi): Effect of pH on Sm6
130
Figure 4.4(vii): Effect of pH on Sm7
Figure 4.4(viii): Effect of pH on Sm8
Figure 4.4(ix): Effect of pH on Sm9
131
Figure 4.4(x): Effect of pH on Sm10
Figure 4.4(xi): Effect of pH on Sm11
Figure 4.4(xii): Effect of pH on Sm12
132
Figure 4.4(xiii): Effect of pH on Sm13
Figure 4.4(xiv): Effect of pH on Sm14
Figure 4.4(xv): Effect of pH on Sm15
133
Figure 4.4(xvi): Effect of pH on prodigiosin production from Serratia isolates (Highly significant***Fcal9854>Ftab2.085 at 5% with 44df at pH 8)
4.5.4. Effect of different carbon sources
The use of alternate and inexpensive sources of carbon is important for the
synthesis of the pigment is of vital importance since it can not only reduce the overall
cost of production but can also enhance the production. Various carbon sources such
as glucose, glycerol, sesame seed and peanut seed extracts were evaluated for their
contribution towards pigment enhancement by maintaining all the above physical
parameters to a constant level.
When glucose was used in the media, the level of prodigiosin produced by
most strains was relatively reduced when compared to previous levels of production
(Figure 4.5(xvii)). Sm1, Sm3, Sm8, Sm10, Sm11, Sm12, Sm13 and Sm15 produced
generated very low yield of the pigment calculated to be 57.1, 75.67, 59.1, 33.33, 20,
18.42, 57.57 and 54.38units/cell respectively when compared to previous results.
Sm14 however, did not produce any pigment in the presence of glucose showing
suppression of pigment producing mechanism. Sm2, Sm4, Sm5, Sm6 and Sm7
produced moderate levels of the pigment calculated to be 146.42, 123.68, 188.88,
218.75 and 96.77units/cell respectively. According to Haddix and Werner (2000),
glucose reduced pigment production to nearly half when compared to other carbon
sources and sugars. Glucose addition causes catabolic repression resulting in reduced
134
prodigiosin production (Khanafari et al., 2006). Figures 4.5(i) to 4.5(xv) show the
effect of carbon source on individual Serratia isolates for prodigiosin production.
The use of glycerol as a source of carbon showed substantial increase
production of pigment when compared to glucose in almost all cases (Figure
4.5(xviii)). Sm1, Sm2, Sm3, Sm4, Sm5, Sm6, Sm7, Sm8, Sm9, Sm10, Sm12, Sm13,
Sm14 and Sm15 produced 166.66, 218.75, 204.76, 202.94, 193.54, 217.85, 167.5,
178.94, 150, 50, 29.78, 63.41, 109.52 and 157.69units/cell of pigment respectively, all
except in the case of Sm11 where there was no synthesis.
Fatty acid containing substances like sesame seed and peanut seed contain
very high amounts of saturated fats. Peanut contains a higher concentration of
saturated fat than sesame thereby giving a higher yield of prodigiosin (Khanafari et
al., 2006) which was also established by the results obtained for all the strains as well
as greater cell density. Overall, peanut seed extract produced the maximum quantity
of prodigiosin when compared to other carbon sources as shown in Figure 4.5(xvi).
For Sm2, Sm3 and Sm4 sesame seed seemed to give rise to greater pigment
production than peanut seed extract estimated to be 234.14, 225.8, 182.14units/cell
respectively when compared to 210, 90.63, 144.44units/cell respectively. This showed
the ability of the above mentioned isolates to prefer sesame seed over peanut seed as a
carbon source. Sm5 and Sm15 showed a very marginal increase in pigment
production in peanut seed extract when compared to sesame seed medium. The
former gave values of 255.26units/cell and 183.63unit/cell respectively for Sm5 and
Sm15 while in the case of the latter the values were estimated to be 251.42units/cell
and 182.35units/cell respectively. In the case of Sm1, Sm6, Sm7, Sm8, Sm9, Sm10,
Sm11, Sm12, Sm13 and Sm14 peanut seed extract showed pigment production
calculated to be 260.97, 277.8, 233.33, 269, 182.6, 105.5, 61.53, 92.72, 118.18 and
143.14units/cell respectively. This was a moderate increase when compared to
isolates grown in sesame seed extract from which the calculated values obtained were
142.85, 242.5, 201.96, 240.9, 157.14, 64.51, 40.74, 76.92, 90.62 and 114.89units/cell
respectively. In case of Sm10, Sm11, Sm12 and Sm13 the production of pigment
though moderate in quantity, was the maximum when compared to the amount
produced using any other carbon source. Hence peanut seed extract as carbon source
135
showed the maximum production of prodigiosin which was considered to be highly
significant based on statistical analysis shown in Appendix 4 Anova Table 4d.
Figure 4.5(i): Effect of carbon source on Sm1
Figure 4.5(ii): Effect of carbon source on Sm2
136
Figure 4.5(iii): Effect of carbon source on Sm3
Figure 4.5(iv): Effect of carbon source on Sm4
Figure 4.5(v): Effect of carbon source on Sm5
137
Figure 4.5(vi): Effect of carbon source on Sm6 (*** highest production)
Figure 4.5(vii): Effect of carbon source on Sm7
Figure 4.5(viii): Effect of carbon source on Sm8
138
Figure 4.5(ix): Effect of carbon source on Sm9
Figure 4.5(x): Effect of carbon source on Sm10
Figure 4.5(xi): Effect of carbon source on Sm11
139
Figure 4.5(xii): Effect of carbon source on Sm12
Figure 4.5(xiii): Effect of carbon source on Sm13
Figure 4.5(xiv): Effect of carbon source on Sm14
140
Figure 4.5(xv): Effect of carbon source on Sm15
Figure 4.5(xvi): Effect of carbon source on prodigiosin production from Serratia isolates (Fcal26479.55>Ftab2.085 at 5% with 44df using peanut seed as carbon source)
141
Figure 4.5(xvii): Comparison of glucose and commercial LB media
Figure 4.5(xviii): Comparison of glucose with glycerol as sole carbon
source for pigment production
4.5.5. Effect of different nitrogen sources
The synthesis of secondary metabolites by microorganisms has been reported
to be greatly influenced by many physical factors. Nutritional sources, especially
142
nitrogen sources have always been of great interest to researchers in the industry for
low cost media design. Hence, keeping the economical aspect in perspective, the
search for inexpensive sources of nutrients has always been of great importance by
industries (Juwon and Emmanuel, 2012).
Three different nitrogen sources were used to study their effect on prodigiosin
production. Yeast extract, peptone and ammonium sulfate were used as nitrogen
sources in the absence of other sources of nitrogen in each media preparation. When
only yeast extract was used in the absence of tryptone or peptone in the medium, the
amount of prodigiosin produced was much lower when compared to the absence of
yeast extract in the medium. Sm1, Sm2, Sm3, Sm4, Sm5, Sm6, Sm7, Sm8, Sm9,
Sm10, Sm11, Sm12, Sm13, Sm14 and Sm15 produced 76.66, 150, 75, 150, 161.29,
166.66, 105.55, 80, 66.66, 42.5, 32.25, 48.78, 88, 25.64 and 63.33units/cell of
prodigiosin respectively with yeast extract as nitrogen source which was much lower
compared to peptone which gave values such as 126.32, 157.14, 100, 200, 165.71,
193.55, 133.33, 125, 96.77, 66.66, 34.48, 62.5, 66.66, 51.42 and 109.52 units/cell of
prodigiosin respectively. Wei and Chen (2005) showed similar results when they
compared prodigiosin-like-pigment production by their mutant strains of Serratia.
The last nitrogen source used was ammonium sulfate which gave low to
negligible quantities of prodigiosin. Sm1, Sm2, Sm3, Sm4, Sm5, Sm6, Sm8, Sm9 and
Sm13 gave reduced levels of pigment estimated as 35.09, 73.68, 42.5, 85.29, 67.5,
88.23, 45.2, 22.64, 8.51 and 24.39units/cell respectively. The effect of nitrogen
sources on individual isolates have been shown between Figures 4.6(i) and 4.6(xv).
However, Sm10, Sm11, Sm12, Sm14 and Sm15 did not produce any pigment since it
was not measurable by the colorimeter. Figure 4.6(xvi) depicts comparative graph
showing the effect of each selected nitrogen source on all isolates. Ammonium sulfate
is an inorganic source of nitrogen lacking in the rich supplements of vitamins,
minerals and other proteinaceous nutrients present in peptone and yeast extract.
Highest production of prodigiosin was seen when peptone was used as a
nitrogen source and the statistical results reported were highly significant for the same
as shown in Appendix 4 Anova Table 5b.
143
Figure 4.6(i): Effect of nitrogen source on Sm1
Figure 4.6(ii): Effect of nitrogen source on Sm2
Figure 4.6(iii): Effect of nitrogen source on Sm3
144
Figure 4.6(iv): Effect of nitrogen source on Sm4 (***highest production)
Figure 4.6(v): Effect of nitrogen source on Sm5
Figure 4.6(vi): Effect of nitrogen source on Sm6
145
Figure 4.6(vii): Effect of nitrogen source on Sm7
Figure 4.6(viii): Effect of nitrogen source on Sm8
Figure 4.6(ix): Effect of nitrogen source on Sm9
146
Figure 4.6(x): Effect of nitrogen source on Sm10
Figure 4.6(xi): Effect of nitrogen source on Sm11
Figure 4.6(xii): Effect of nitrogen source on Sm12
147
Figure 4.6(xiii): Effect of nitrogen source on Sm13
Figure 4.6(xiv): Effect of nitrogen source on Sm14
Figure 4.6(xv): Effect of nitrogen source on Sm15
148
Figure 4.6(xvi): Effect of nitrogen source on prodigiosin production from Serratia isolates (Fcal2123.57>Ftab2.085 at 5% with 44df using peptone as nitrogen source)
4.6 Screening for the production of enzymes by Serratia
marcescens isolates
Commercial enzyme production has grown in recent times in volume and
number of products in response to expanding markets and increasing demand for
novel biocatalysts. Microbial enzymes produced by the same organism are of great
importance as they have several industrial applications such as in detergent
manufacturing, dairy food processes, in water treatment and therapeutics. They are
essentially extracellular products and their formation is generally affected by nutrients
and other physical parameters.
4.6.1 Screening for protease production by Serratia isolates
All the fifteen Serratia isolate obtained from a hundred soil and water samples
were screened for the production of protease on skim milk agar medium at 30˚C. On
the basis of the formation of large clear zones around the colonies, 11 isolates were
identified as potential protease producing organisms. The test was carried out in
triplicates aseptically.
149
4.6.1.1 Type of protease produced
After the identification of the 11 protease producing isolates, they were tested
on skim milk agar of four varying pH values of 6, 7, 8 and 9 to ascertain the type or
nature of protease produced by each organism.
Sm2, Sm3, Sm5, Sm6, Sm7, Sm11, Sm12 and Sm13 produced distinct clear
zones at the neutral pH 7 (Figure 4.7(i)) and were considered to be neutral protease
producers. However, Sm1, Sm9, Sm10, Sm11 and Sm13 showed clear zone formation
at pH 8 as well (Figure 4.7(ii)). This indicated that the protease produced by them
was slightly alkaline in nature. Hence, Sm2, Sm3, Sm5, Sm6, Sm7 and Sm12
produced neutral proteases only. The ability to produce neutral protease by Serratia
has been suggested to cause the possibility of tissue damage during Serratia-induced
diseases (Lyerly and Kreger, 1979). In addition to this, it was also seen that Sm11
and Sm13 produced clear zones on skim milk agar at pH 9 (Figure 4.7(iii)). This
confirmed that Sm11 and Sm13 were producers of more alkaline proteases when
compared to Sm1, Sm9 and Sm10. These alkaline proteases have been found to be
extremely useful in the manufacture of biocleaners for the detergent industries
worldwide (Joseph and Palaniyandi, 2011).
Figure 4.7(i): Protease production by Serratia isolates at pH 7
150
Figure 4.7(ii): Protease production by Serratia isolates at pH 8
Figure 4.7(iii): Protease production by Serratia isolates at pH 9
4.6.2 Screening for lipase production by Serratia isolates
The 15 isolates of Serratia were grown on Tween80 lipolytic media (Shukla
and Gupta, 2007). Only two of the isolates, Sm1 and Sm3 formed a white precipitate
around the colony thereby showing a positive result for the production of extracellular
lipase.
All the isolates showed the presence of a white precipitate. This indicated the
production of lipase by all Serratia isolates. However, two isolates Sm1 and Sm3
showed more white precipitate when compared to the others due to the increased
diameter of the precipitate indicative of increased lipase production (Table 4.12). The
test was conducted in triplicates to confirm the result and an average of all three
values was taken. These were selected for lipolytic assay. The lipase assay test for the
two isolates was conducted and the lipase activity was found to be moderate in case of
both Sm1 and Sm3 as shown in Table 4.12.
151
Table 4.12 Screening of Serratia isolates for lipolytic activity at 30˚C
Isolate No. Lipase Activity based on zone of clearance
Lipase Assay (ìg/ml/min)
Sm1 +++ 0.8 Sm2 - - Sm3 +++ 0.89 Sm4 - - Sm5 + - Sm6 + - Sm7 - - Sm8 - - Sm9 - - Sm10 ++ - Sm11 - - Sm12 ++ - Sm13 ++ - Sm14 + - Sm15 + -
*the test was carried out in triplicates the average value was determined, +++ Highest lipase activity, ++Moderate lipase activity, +Low activity
4.6.3 Screening for chitinase production by Serratia isolates
Screening for the extracellular enzyme, chitinase was carried out by
inoculating all the isolates on colloidal chitin agar. Two of the isolates showed a clear
zone formation around the line of streak, indicating hydrolysis of chitin by the action
of chitinase producing Serratia isolates. They were Sm1 and Sm5 respectively
illustrated in Figure 4.8.
Figure 4.8: Chitinase production by Serratia isolates
152
4.7. Screening for biosurfactant production by Serratia isolates
Biosurfactants are amphiphilic compounds of microbial origin with potential
applications in the petroleum, cosmetic and food industries. Many microorganisms
have been reported to produce biosurfactants which are key components for
biodegradation of slightly soluble organic compounds. The bioavailability could be
increased directly by adding the surfactant during the degradation stage or indirectly
by adding a biosurfactant producing microorganism to the contaminated area.
4.7.1. Hemolytic activity assay
Hemolytic activity on blood agar was seen by many of the Serratia isolates. A
distinct clear zone around the line of streak was evident in the case of each of the
following isolates of Serratia, Sm1, Sm2, Sm3, Sm6, Sm7, Sm8, Sm11, Sm13 and
Sm15 respectively (Figure 4.9). Most of the isolates that show hemolysis of red blood
cells have been reported to show biosurfactant activity simultaneously because they
are essentially made of lipoproteins and lipopeptides. These components are
responsible for the property of hemolysis of mammalian blood (Sakalle and
Rajkumar, 2009).
Figure 4.9: Hemolysis demonstrated by Serratia isolates
4.7.2. Oil Displacement Method
All the isolates that showed visible hemolysis in the above experiment were
selected for the oil displacement technique. The cultures were grown in minimal
medium enriched with sucrose. A clear halo obtained was obtained due to the
displacement of the oil by the surfactants present in the isolates. This held true for all
153
the isolates tested. The diameter of the drop before and after displacement was
calculated and the result has been shown in Table 4.13. The ODA range was
displayed at 0.95-2.84cm2. Greatest difference in displacement area was obtained for
Sm3 recorded at 2.54.
Table 4.13 Biosurfactant activity of Serratia marcescens isolates by drop
displacement test
Isolate No.
Diameter of oil drop (mm) Oil area displacement (ðr2
cm2)*
Difference
in area
(cm2) Before displacement
After displacement
Before displacement
After displacement
Sm1 10 15 0.79 1.77 0.98 Sm2 14 17 1.54 2.27 0.73 Sm3 8 19 0.5 2.84 2.34 Sm6 10 14 0.79 1.54 0.75 Sm7 6 15 0.28 1.77 1.49 Sm8 10 13 0.79 1.33 0.54
Sm11 8 11 0.5 0.95 0.45 Sm13 13 14 1.33 1.54 0.21 Sm15 12 13 1.13 1.33 0.2
*value corrected to two decimal places
4.7.3. Emulsification Activity
Emulsification is the process of mixing two or more liquids that are normally
immiscible liquids. The emulsification activity of the biosurfactant producing isolates
was tested against crude vegetable oil. The foam was produced by vigorously
vortexing the crude biosurfactant solution.
The emulsification activity was studied for all isolates showing a positive test
for hemolysis. Four of the isolates Sm1, Sm2, Sm3 and Sm7 displayed considerable
activity calculated to be 50, 62.5, 75 and 50 respectively (Table 4.14). The results
obtained displayed excellent emulsification properties by the Serratia isolates in
comparison to Pseudomonas fluorescens as depicted in the paper by Abouseoud et al.
(2008). Isolates Sm2 and Sm3, showed more than 50% biosurfactant activity against
crude oil.
154
Table 4.14 Emulsification activity of biosurfactant produced by Serratia
marcescens isolates against crude oil
Isolate No. Height of emulsion layer (in cm)
Total height (in cm) Emulsification index
Sm1 0.5 1.0 50.0 Sm2 0.5 0.8 62.5 Sm3 0.6 0.8 75.0 Sm6 0.3 0.8 37.5 Sm7 0.4 0.8 50.0 Sm8 0.3 0.9 33.33
Sm11 0.1 0.5 20.0 Sm13 0.3 0.8 37.5 Sm15 0.3 0.8 37.5
4.8 Applications
4.8.1 Prodigiosin:
The pigment has no defined role in physiology but has been reported to have
antimicrobial properties (Samrot et al., 2011). Out of all the isolates, four were
selected that produced the best amount of pigment and they were Sm1, Sm5, Sm6 and
Sm8 based on the values obtained by conducting the optimization experiments.
The agar well diffusion assay was carried out against Gram positive and Gram
negative bacteria namely Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli and Klebsiella pneumonia respectively using ethanolic extracts of the
pigment obtained from the aforementioned selected isolates. These bacteria were
selected because of their recent emergence as nosocomial pathogens and their
potential multi-drug resistance capability. The zones of inhibition of microbial growth
due to pigment extract were measured in millimeters as shown in Table 4.15. It was
evident that Staphylococcus aureus, the Gram positive bacteria showed maximum
sensitivity to the pigment extract followed by E.coli, Klebsiella pneumonia and
Pseudomonas aeruginosa. Sm5, Sm6 and Sm8 showed significant resistance towards
S.aureus and E.coli indicated by their p values. Figure 4.10 illustrates the technique
of agar-well diffusion and shows the zones of clearance distinctly.
The tube dilution method was carried out to quantitatively interpret
antibacterial activity of the pigment against the test organisms. The extract
155
concentrations were 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78µg/ml
respectively for each pigment produced by Sm1, Sm5, Sm6 and Sm8. The Minimum
inhibitory concentrations (MICs) are defined as the lowest concentration of an
antimicrobial that will inhibit the visible growth of a microorganism after overnight
incubation (Andrews, 2001). The MIC values after 24 and 48 hours of incubation of
the test organisms in the presence of the pigment extracted from the selected isolates
are given in Table 4.16. The Minimum bactericidal concentrations (MBCs) are
defined as the lowest concentration of antimicrobial that will prevent the growth of an
organism after subculture on to antibiotic-free media. This was carried out
immediately after MIC to determine the lowest concentration of pigment that
inhibited bacterial growth on solid media (Table 4.16).
Table 4.15 Antibacterial activity of prodigiosin produced by Serratia marcescens isolates
Isolate no. Diameter of zone of clearance (mm)
E.coli S.aureus K.pneumoniae P.aeruginosa
Sm1 15** 15** 10* 13*
Sm5 25*** 28*** 15* 12*
Sm6 27*** 27*** 20** 17**
Sm8 22*** 25*** 20** 17** *the test was carried out in triplicates and an average value for the diameter was taken, *p<0.5 **p<0.1 ***p<0.01
Figure 4.10: Agar-well diffusion technique for determination of antibacterial
activity of the pigment
156
In the case of S. aureus, Sm1 showed MIC and MBC to be 12.5µg/ml and the
remaining isolates showed lower MIC values of 3.125µg/ml for Sm5 and 6.25µg/ml
for Sm6 and Sm8 respectively. This indicated that Gram positive S. aureus could be
inhibited at concentrations as low as 3.125µg/ml of pigment concentration depicting
the pigment to be effective against Gram positive bacteria (Samrot et al., 2011). In
case of E. coli, inhibition by pigment was seen at 12.5µg/ml concentration thus
showing moderate levels of inhibition. The MBC in case of Sm6 and Sm8 were
12.5µg/ml and Sm1 and Sm5 showed an MBC value of 25µg/ml. For K. pneumoniae,
resistance was seen at 25µg/ml concentration for Sm1 and Sm6. The remaining two
showed inhibition at 12.5µg/ml. MBC in all cases was one dilution lower than the
MIC value. Sm1 and Sm6 showed an MBC value of 50µg/ml whereas Sm5 and Sm8
showed the MBC value to be 25µg/ml. P. aeruginosa showed maximum resistance to
pigment when compared to other test bacteria with concentration of pigment going up
to 100µg/ml in case of Sm1 and Sm5. This indicated that an increased concentration
of the pigment was required to bring about inhibition of highly pathogenic Gram
negative bacteria. The MBC value for Sm1 and Sm5 were shown to be 100µg/ml
whereas for Sm6 and Sm8 the value was 50µg/ml. The values which are in bold font
indicate a change in value after incubation time of 24 hrs. to 48 hrs. as well as a
change in MBC value if at all observed. Results showed that the most effective MIC
and MBC value for inhibiting S.aureus was 3.125µg/ml produced by SM5 which was
highly significant indicated by its p value (multivariant anova) followed by Sm8 at
6.25 µg/ml MIC value. For E.coli, prodigiosin produced by Sm1, Sm6 and Sm8 were
most effective at a moderate MIC and MBC value of 12.5µg/ml. For K.pneumoniae
and P.aeruginosa however, concentrations of prodigiosin required were much higher
for effective inhibition. All the MIC and MBC values of the selective Serratia
marcescens strains have been depicted in Table 4.16.
Table 4.16 MIC and MBC values of prodigiosin produced by Serratia strains against test organisms
Isolate no. Test organism
Concentration of prodigiosin (µg/ml) Minimum Inhibitory Concentration (MIC)
After 24 hrs. After 48 hrs. MBC after 24 hrs.
Sm1 S.aureus 12.5 12.5** 12.5**
E.coli 12.5 12.5** 25*
157
K.pneumoniae 25 25* 50 P.aeruginosa 50 100 100
Sm5 S.aureus 3.125 3.125*** 3.125***
E.coli 12.5 12.5** 25* K.pneumoniae 12.5 12.5** 25* P.aeruginosa 50 50 100
Sm6 S.aureus 3.125 6.25*** 6.25***
E.coli 12.5 12.5** 12.5** K.pneumoniae 12.5 25* 50 P.aeruginosa 12.5 25* 50
Sm8 S.aureus 6.25 6.25*** 6.25***
E.coli 12.5 12.5** 12.5** K.pneumoniae 12.5 12.5** 25* P.aeruginosa 12.5 25* 50
*p<0.5 **p<0.1 ***p<0.01
The pigment has therefore shown a lot of promise with respect to its potential
application in the therapeutic industry against pathogenic bacteria. By further
studying the mechanism of action of the pigment in inhibiting bacterial growth, the
pigment can be modified to attach to target specific sites for better action.
4.8.2 Biosurfactant
The bioemulsifiers are usually produced as cultures reach the stationary stage
of growth (Ron and Rosenberg, 2002). The biosurfactant activity of the isolates was
compared against three hydrocarbons, namely vegetable oil, petrol and diesel.
Biosurfactant activity against vegetable oil and diesel was observed to be more when
compared to diesel. Maximum activity against petrol and diesel was seen in the case
of Sm8 (71.4% against petrol) and Sm3 (61.53% against diesel) which were very
significant according to their p values as seen in Figure 4.11. Overall, greatest
biosurfactant activity was seen against vegetable oil as compared to the two sources
of fuel. This property of the isolates can be used for treatment of oil spills and
treatment of sludge where hydrocarbon concentration is significantly high.
Bioremediation of soil is another area where biosurfactants help in breaking down
toxic hydrocarbons like diesel and petrol. Values lower than 50% were considered as
organisms with poor surface activity properties.
158
The production of biosurfactant is very vital in reducing the concentration of
individual or mixed environmental pollutants by reducing the surface and interfacial
tensions (Mukherjee et al., 2006). The attributes that make them commercially
promising alternatives to chemically synthesized surfactants are their reduced toxicity,
higher biodegrading capacity, hence, greater environmental compatibility, better
foaming properties (useful in mineral processing), and stable activity at extremes of
pH, salinity and temperature (Desai and Banat, 1997).
*p<0.5 **p<0.1 ***p<0.01
Figure 4.11: Comparison of Emulsification Index (E24) of biosurfactant against
different hydrocarbons