11 Chapter 3

Lakshmi KVNS, Ph.
D Thesis, 2012
Chapter 3
Results
Lakshmi KVNS, Ph.D Thesis, 2012
80
Results
3.1 Enrichment, isolation, rapid typing and tentative

identification of purple bacteria from rhizosphere soils
of paddy fields and a few other habitats.
Anoxygenic phototrophic purple bacteria are usually isolated through
enrichments because of their existence in low numbers and their low
relative growth rates compared to total populations of bacteria inhabiting
rice fields.
3.1.1 Habitats: Seventy five (75) samples were collected from rhizosphere
soils of various paddy fields from different geographical locations of India.
Apart from these, three samples from brackish water habitat were also
analyzed for presence of purple bacteria.
3.1.2 Enrichment, isolation, rapid typing and tentative identification
of purple bacteria from paddy rhizosphere soils
3.1.2.1 Enrichments: Out of the 75 rhizosphere soil samples kept for
enrichment, all the samples except 5, gave enrichment for PNSB where
as only 32 samples (43%) gave PSB enrichments (Table 3.1.1). While the
color of all the PSB enrichments was reddish brown, the PNSB
enrichments were either reddish brown (22), yellowish brown (18), brown
(15), red (10), reddish brown to brown (4), pink (3) or greenish brown (3)
(Table 3.1.1).
3.1.2.2 Purification From all the positive enrichments obtained, 90
purple non sulfur and 33 purple sulfur bacterial isolates were obtained
in pure culture. Purification was done by repeated streaking on agar
slants. The isolates were given strain numbers (Table 3.1.1).
81
Results
3.1.2.3 Preservation All the purified strains were lyophilized and stored
at 40C.
3.1.2.4 Typing The purified strains were subjected to rapid typing (Fig.
3.1.1) based on color, colony morphology, microscopic observation,
carotenoid content and type of bacteriochlorophyll present (Table 3.1.2).
Representative strains (36) among these were selected and subjected to
16S rRNA gene sequence analysis and BLAST search to know their
sequence similarity with their closest type strains (Table 3.1.2).
3.1.2.4.1 Color of the strains Under anaerobic conditions, most of the
strains were either reddish brown (70) or yellowish brown (28), while
others were brown (19), red (3) or pink (3). Strains which were yellowish
brown changed to reddish brown when exposed to air and were pink
when grown under aerobic condition (Table 3.1.2).
3.1.2.4.2 Photosynthetic pigments Based on the absorption maxima
obtained in the whole cell absorption spectra, all strains showed
bacteriochlorophyll a (data not shown). Thirty seven strains which were
yellowish brown and brown in color had spheroidene series of
carotenoids with either spheroidene or spheroidenone as major pigments.
Thirty five strains which were either reddish brown or red or pink in
color had spirilloxanthin series of carotenoids. Forty one strains which
reddish brown in color had lycopene as major carotenoids and the
remaining brown colored strains (10) had rhodopin (Table 3.1.2).
82
Results
Fig.3.1.1 Identification of purple bacteria by a rapid typing procedure.
3.1.2.4.3
Colony
morphology
PNSB
strains
growing
photo-
organotrophically under anaerobic conditions showed eight different

colony morphologies on agar slants. Reddish brown colored strains
showed three different colony morphologies: small, round, convex,
smooth colonies; medium, round, convex, smooth colonies; large, round,
convex, smooth colonies. Reddish brown- pink colored colonies were
either medium, round, convex, smooth or large, round, convex, smooth.
Pink colored strains were small/medium/large, round, convex and
smooth.
Brown
colored
strains
showed
two
different
colony
83
Results
morphologies: medium, round, convex, smooth colonies; medium, round,

convex, smooth, sticky colonies. Yellowish brown colored strains showed
six different colony morphologies: small, round, convex, slimy colonies;
medium, round, convex, slimy colonies; small, round, convex, sticky
colonies; medium, round, convex, sticky colonies; medium, round,
convex, smooth colonies; medium, round, convex, rough colonies. The
slime producing colonies spread uniformly on the surface of the slants.
The yellowish brown colonies on exposure to air turned brown and when
grown aerobically formed pink colored colonies. All the thirty three
purple sulfur bacterial strains growing photolithoheterotrophically under
anaerobic conditions were reddish brown colored with their colony
morphology showing medium, round, convex, smooth colonies (Table
3.1.2).
3.1.2.4.4 Cell morphology, division and motility Of the 90 strains of
PNSB, 35 strains were rod shaped (few formed chains), motile and
multiplied by budding, few formed rosettes; 16 strains were spiral
shaped (few formed long chains), motile and divided by binary fission;
few of the spiral shaped strains formed spheroplasts; 18 strains were
oval/oval to rod shaped (few formed chains), non-motile and divided by
binary fission; 9 strains were oval/oval to rod shaped (few formed
chains), motile and divided by binary fission; 10 strains were curved to
rod shaped (few formed chains), motile and divided by binary fission; one
strain was vibrio-spiral shaped, motile and multiplied by binary fission.
84
Results
All 33 PSB strains were rod shaped, motile, multiplied by binary

fission and deposited sulfur granules inside the cells (Table 3.1.2).
3.1.2.4.5
Tentative
identification
Studies
on
phenotypic
characterization of the 123 strains, in terms of morphological and

cultural characteristics, led to the tentative identification of the strains
up to the genus level (Table 3.1.2). Rod shaped, motile cells which were
reddish brown/red/pink and multiplied by budding were tentatively
identified to be belonging to two genera Rhodopseudomonas (32 strains)
and Rhodoplanes (3 strains); 27 strains which were oval/oval to rod
shaped, multiplied by binary fission and yellowish brown in color were
identified to be belonging to two genera Rhodobacter and Rhodovulum.
Based on cell shape, color and microscopic observation, spiral shaped
strains were assigned to two different genera Rhododspirillum [(6 strains);
larger spirals, reddish brown] and Phaeospirillum [(10 strains); smaller
spirals, brown]. Ten strains which were slender-curved rods and
multiplied by binary fission and brown colored were identified as
belonging to the genus Rubrivivax. One strain which was pink colored,
vibrio-spiral shaped, motile and multiplied by binary fission was
assigned to the genus Rhodocista.
Thirty three strains which were rod shaped, motile, divided by binary
fission and bearing intracellular sulfur granules were tentatively
assigned to the genus Allochromatium.
85
Results
3.1.2.4.6 16S rRNA gene sequence analysis Among all the strains
(123) which were subjected to phenotypic characterization, the 16S rRNA
gene of 36 strains was sequenced and the similarities were examined
using the EzTaxon server. The data obtained revealed that these strains
showed highest pairwise sequence similarities (Table 3.1.2) with the type
strains of the species belonging to the genera Rhodobacter (8 strains),
Rhodovulum (1 strain), Rhodopseudomonas (8 strains), Rhodoplanes (1
strain), Phaeospirillum (4 strains), Rhododspirillum (4 strains), Rhodocista
(1
strain)
of
Alphaproteobacteria;
Betaproteobacteria
Gammaproteobacteria
Alphaproteobacteria,
and
and
Rubrivivax
(4
Allochromatium
were
distinct
Betaproteobacteria
from
and
(5
strains)
strains)
other
genera
of
of
of
Gammaproteobacteria,
respectively.
3.1.3 Enrichment, isolation, rapid typing of purple bacteria from
other habitats
Apart from the samples analyzed from the rice field habitats for purple
bacteria, two brown colored enrichment cultures obtained from sediment
of two different brackish water shrimp ponds and a sediment sample
collected from a brown pond were also analysed (Table 3.1.1). The
enrichment cultures of the 3 samples on purification yielded the strains
JA480T, JA481 and JA580T, respectively, which were further subjected to
16S rRNA gene sequence analysis (Table 3.1.2) and extensive polyphasic
characterization (Chapters 3.6; 3.7).
Place of
sample
collection
GPS
position
Sample
I.D
86
Date of
sample
collection
pH
Temp
(o C)
Results
Enrichments of purple bacteria

Purple non sulfur bacteria
Purple sulfur bacteria
Enrichment
Strains obtained
Enrichment
Strains obtained
+/Color
Name
Number
+/Color
Name
Number
Samples analyzed from rhizosphere soils of paddy fields

Nadergul
Kolluru
1727 N
7854 E
1618 N
8079 E
KI-1
6/8/07
7.5
30
rb
KI-2
25/10/07
7.5
30
KI-3
20/12/07
7.5
28
yb-rb
KII-1
13/8/08
7.5
30
KII-2
KII-3
14/10/08
22/12/08
7.5
7.5
30
30
+
+
rb-b
r
KIII-1
KIII-2
KIII-3
AI-1
18/8/09
13/10/09
27/12/09
3/7/07
7.0
7.5
7.5
7
30
30
28
30
+
+
+
+
di b
rb
yb
b-rb
AI-2
10/10/07
28
AI-3
AII-1
15/12/07
8/08/08
7
7
28
30
AII-2
5/10/08
AII-3
AIII-1
AIII-2
AIII-3
12/12/08
8/08/09
10/10/09
21/12/09
7
7
7
7
KL1
*JA477
T
rb
KIPs1
JA691
rb
KIPs2
JA692
rb
KIIPs1
JA693
+
+
rb
rb
KIIPs2
KIIPs3
JA694
JA711
K3P
K5R
K4Ru
KON
Ro
K4P
K6R
KL2
Yn
*JA317
*JA318T
*JA354
JA652
JA653
JA654
JA655
*JA478
JA666
JA656
JA657
JA658
*JA355
*JA452
*JA474
*JA570
+
+
+
+
rb
rb
rb
rb
KIIIPs1
KIIIPs2
KIIIPs3
AIPs1
JA695
JA713
JA714
JA686
yb-gb
K5Ru
Pry2
Rm
A4
A3
B5
KF
rb
AIPs2
JA687
+
+
yb-rb
yb
Pry1
Rl
JA659
JA660
rb
AIIPs1
JA688
30
rb
A/V2
JA661
rb
AIIPs2
JA689
28
30
30
28
+
+
+
+
rb
b
yb
r
A/V3
A5P
Ra
A/V
JA662
JA663
JA664
JA665
+
-
rb
-
AIIIPs
-
JA690
-
87
Results
Kakinada
1694 N
8223 E
Yn
20/10/07
7.5
30
Y6Ru
JA667
Ernakulam
1008 N
7621 E
PA1
17/11/07
30
rb-b
rb
L1/V12
*JA383
17/11/07
30
rb
PAL2
JA685
ChR
10/11/07
6.5
30
*JA353
*JA351
*JA415T
*JA352
*JA476
JA668
PA2
P3/L3
ILP
PA10
P2/L2
P1/L1
gi
rb
ChPs
JA684
CI-1
5/10/07
30
gb-yb
Cg
JA669
rb
CIPs
JA683
CI-2
CI-3
CII-1
CII-2
CII-3
CIII-1
CIII-2
DSr
25/12/07
14/01/08
1/10/08
10/11/08
21/12/08
1/8/09
22/11/09
17/5/07
7
7
7
7
7
7
7
8
30
30
30
30
28
30
30
30
+
+
+
+
+
+
+
+
rb
gb
rb
dib
rb
gb
rb
r
P4
P5
94R
C7Ru
T9
Cg2
CamV
1E
JA670
JA739
JA671
JA672
JA673
JA674
JA675
JA676
+
+
+
-
rb
rb
rb
-
CIIPs
CIIPs1
CIIIPs
-
JA681
JA740
JA682
-
EKr
25/5/07
7.5
28
rb
EKRb
JA677
FKhr
20/5/07
6 6.5
28
WA/H
JA678
GIr
18/5/07
6.5
28
rb
H1
*JA356
H1Pr
19/5/07
6 6.5
28
EQT
JA679
ChiR1
ChiR2
BOr
14/5/07
14/5/07
22/8/07
5.5 6
7.5
7
25
27
30
+
+
p
r
8BK
8Br1
JA680
JA696
Chennai
Kurnool
Suryapet
1306 N
8024 E
1582 N
7803 E
1713N
7962E
Kodad
1701 N
7996 E
Khola
1704 N
8009 N
Itchyapuram
1704 N
8009 E
Podugupadu
1449 N
7998 E
Rice field near 2023 N
Chilka lake
8427 E
Bhongiri
1704 N
8009 E
Basara
1888 N
7795 E
Ahmadnagar 1 2297 N
7865 E
Shinganapur
2297 N
7865 E
Buldhana
1998 N
7651E
Ahmadnagar 2 2297 N
7865 E
Chidambaram 1139 N
7969 E
Trivendrum
1051 N
7664 E
Vijayawada
1651 N
8062 E
88
Results
Bar
30/12/07
7.5
30
JA697
JA698
-
UnRb
B5P
-
Ahr
27/12/07
30
SSr
27/12/07
7.5
30
gb
SRb
JA699
Bd1r
Bd2r
Ahd
28/12/07
29/12/07
29/12/07
7
7
7.5
28
28
30
+
+
+
db
yb
r
6Ru
BRb
Rp1
JA700
JA701
JA702
Cb
15/11/07
26
dib
8Ru
JA703
rb
8Ps
JA708
Kr
18/11/07
25
rb
8Br2
JA704
Vjr1
30/09/08
30
yb-r
30
JA705
JA706
JA707
Rp2
VRb
V6P
Vjr2
30/09/08
32PS
32B
32PR
46B
32bD
*JA559
*JA524
*JA558
*JA569
*JA521
rb
32P
JA710
rb
32bP
JA709
Tada
1358 N
8003 E
32
26/12/08
28
yb-db
Vardayapada
1704 N
8009 E
1577 N
7802 E
1772 N
7916 E
1466 N
7817 E
1112 N
7865 E
32b
27/12/08
28
p-r
35
26/12/08
27
rb
32bS
35R
*JA522
*JA520
rb
35PS
35SR
*JA523
*JA560
W2
22/02/09
30
N6
8/02/09
30
rb
NRb1
JA712
TN58
22/05/09
28
yb
58Rb
*JA556
Panampadu
Janagaon
Nandyala
Pullai Mudali
Charod
89
Results
Thirumallaiva
sai
Thavalakuppa
m
1112 N
7865 E
1186 N
7979 E
TN59
22/05/09
27
rb
TN30
20/05/09
28
Munnargudi
1066 N
7945 E
TN93
24/05/09
27
TN94
25/05/09
0947 N
7889 E
Moolamattam, 1704 N
Nandyala
8009 E
TN131
26/05/09
1N
Kothapally,
Nandyala
1704 N
8009 E
Atmakodur,
Nandyala
1704 N
8009 E
1548 N
7848 E
Devipatnam
Parvapalli,
Nandyala
Bandiatmakur
Nandyala
Rice field nea

Alipi
Naupada,
Srikakulam
1704 N
8009 E
1551 N
7851 E
1051 N
7664 E
1857 N
8428 E
JA715
JA716
JA717
rb
59P
59
Trb1
rb
TPs2
JA736
27
131R
JA718
30
yb
131B
*JA557
24/09/09
30
yb
Trb2
JA719
2N
24/09/09
28
yb-gb
9Nf
*JA629
rb
NPs2
JA720
3N
24/09/09
28
NRb2
JA722
rb
NPs3
JA721
4N
24/09/09
30
5N
24/09/09
7.5
30
rb
5PB
*JA635
rb
NPs4
JA723
6N
24/09/09
30
P6P
JA727
rb
NPs5
JA724
7N
24/09/09
30
p-r
7Nf
*JA628
rb
7PS
*JA634
8N
24/09/09
30
dib
2NRu
*JA626
rb
NPs6
JA725
9N
10N
AlBw
24/09/09
24/09/09
25/09/09
7
7
7
30
30
26
+
+
+
yb
rb
di b
9Rb
10PNG
5N
JA728
JA729
*JA627
+
-
rb
-
NPs7
-
JA726
-
Sr1
20/11/09
7.5
28
rb
SrRp1
SrRp2
JA730
JA731
90
Results
BK29
27/12/09
26
rb
BK29
JA732
RA1
21/12/10
28
rb
RA2
JA733
RA2
28/01/10
27
rb
RA1
JA734
RA3
10/02/10
30
b-rb
30
rb
*JA630
*JA631
*JA632
JA735
27/04/10
1365 N Kal123
7941 E
Samples analyzed from other habitats
Nagapattinam, 10o44 N S1
09/03/03
Tamil Nadu 79o50 E
(brackish
shrimp pond)
Pappakovil,
12/09/06
10o44N S2
Tamil Nadu 79o49E
(brackish
shrimp pond)
Vethalai,
0916N Bp
27/05/09
Tamil Nadu 7906 E
(Sediment of a
brown pond)
PA3
Ru3
RA3
Kal1
8.2
30
S1
*JA480T
8.2
30
S2
*JA481
7.4
30
yb
Rv
*JA580 T
Bhitarkanika
2023 N
8427 E
Hyderabad
1738 N
7848 E
Rice field near 2023 N
Orissa
8427 E
Durg,
2118 N
Jharkand
8128 E
Tirupati
Table 3.1.1 Isolation of purple bacterial strains from rice rhizosphere soils and a few other habitats.
Abbreviations: ND, not determined; NA, not applicable; N, north; E, east.
R, red; p, pink; rb, reddish brown; b, brown; db, dark brown; dib, dirty brown; yb, yellowish brown; gb, greenish
brown.
Symbols: +, positive; -, negative.
*Strains whose 16S rRNA genes were sequenced.
The enrichment samples was obtained from Mr. G.V. Ashok Kumar.
Strain
name
Strain
No.
Colony
morphology &
color of broth
cultures
Pigments
91
Microscopic observations
(Shape, size, motility, mode of
division)
Results
16S rRNA gene sequence

similarity (%) with the nearest
type strain (EzTaxon result)/
Tentative taxa identified
EMBL
Accession
numbers
Purple bacterial strains obtained from rhizosphere soils of paddy fields

Alphaproteobacteria
3N
JA317T
5N
JA318 T R, Cv, Sm, M, RB
BChl a, Ly
JA477
R, Cv, Sm, M, RB
BChl a, Ly
JA478
R, Cv, Sm, M, RB
BChl a, Ly
KON
JA652
R, Cv, Sm, L, R
Bchl a, Sp
R, Cv, Sm, M, B
BChl a, Rp
vibrioid-spiral, 0.8-1.2 x 2.0-6.0 m,

chains, sp, motile, binary fission
vibrioid-spiral,0.1-0.3 x 4-16 m,
chains, motile, binary fission
vibrioid-spiral,0.1-0.4 x 4.0-8.0 m,
vibrioid-spiral, 0.2-0.5 x 3-10m,
rods, rosettes, motile, budding
98.2- Phaeospirillum
chandramohanii JA145T
99.8- Rhodospirillum
sulfurexigens JA143T
100- Rhodospirillum
Rhodopseudomonas sp.
AM901294
Rhodobacter sp.
Phaeospirillum sp.
AM901295
FN296204
FN296208
-
Ro
JA653
R, Cv, Sl, M, YB
BChl a, Se/Sn
K4P
JA654
R, Cv, Sm, M, B
BChl a, Rp
oval-rod, chains, non motile, binary

fission
spiral, chains, sp, motile, binary fission
K6R
JA655
R, Cv, Sm, M, RB
BChl a, Ly
spiral, chains, motile, binary fission
Rhodospirillum sp.
Pry2
JA657
R, Cv, Sm, S, RB
Bchl a, Sp
rods, motile, budding
Rm
JA658
R, Cv, Rg, M, YB
BChl a, Se/Sn
oval-rod, non motile, binary fission
Rhodobacter sp.
Pry1
JA659
R, Cv, Sm, S, P
Bchl a, Sp,
Rl
JA660
R, Cv, Sl, M, YB
BChl a, Se/Sn
Rhodobacter sp.
A/V2
JA661
R, Cv, Sm, M, RB
Bchl a, Sp

fission
A/V3
JA662
R, Cv, Sm, S, R
Bchl a, Sp
A5P
JA663
R, Cv, Sm, M, B
BChl a, Rp
spiral, chains, sp, motile, binary fission
Phaeospirillum sp.
Ra
JA664
R, Cv, Rg, M, YB
BChl a, Se/Sn
oval-rod, motile, binary fission
Rhodobacter sp.
92
Results
A/V
JA665
R, Cv, Sm, S, RB
Bchl a, Sp
A4
JA355
R, Cv, Sm, M, YB
BChl a, Se/Sn
100- Rhodobacter johrii JA192T
FN296201
A3
JA452
R, Cv, Sm, S, RB
Bchl a, Sp
JA474
R, Cv, Sm, L, RB
Bchl a, Sp
KF
JA570
R, Cv, Sl, M, YB
BChl a, Se/Sn
Cg
JA669
R, Cv, Sl, M, YB
BChl a, Se/Sn
99.7- Rhodopseudomonas
faecalis JCM 11668T
100- Rhodopseudomonas faecalis
JCM 11668T
99.7- Rhodobacter capsulatus
ATCC 11166T
Rhodobacter sp.
FN296206
B5
oval, 0.9-1.2 x 1.0-2.0m, motile,

binary fission
rods, 0.4-0.7 x 1.5-2.5m, motile,
budding
rods, 0.2-0.8 x 1.0-3.0m, motile,
budding
oval, chains, 0.91.2 x 1.02.0m, non
motile, binary fission
oval-rod, chains, motile, binary fission
P4
P5
JA670
JA739
R, Cv, Sm, L, RB
R, Cv, Sl, M, YB
Bchl a, Sp,
BChl a, Se/Sn
94R
T9
Cg2
JA671
JA673
JA674
R, Cv, Sm, M, RB
R, Cv, Sm, S, RB
R, Cv, Sl, M, YB
Bchl a, Sp
Bchl a, Sp
BChl a, Se/Sn
CamV
H1
JA675
JA356
Bchl a, Sp
Bchl a, Sp
Yn
L2/P2
JA666
JA352
R, Cv, Sm, S, RB
R, Cv, Sm, M, PRB
R, Cv, Sm, L, RB
R, Cv, Sl, M, YB
PA10
JA415 T R, Cv, Sm, M, RB
BChl a, Sp
L3/P3
JA353
R, Cv, Sm, M, P
BChl a, Sp
ILP
JA351
R, Cv, Sm, M, B
BChl a, Rp
L1/P1
JA476
R, Cv, Sl, M, YB
BChl a, Se/Sn
Bchl a, Sp
BChl a, Se/Sn

fission
fission
rods, rosettes, 0.4-0.6 x 1.5-2.0m,
motile, budding
oval-rod, chains,0.8-1.4 x 1.0-3.0m,
non motile, binary fission
FN296207
FN568343
-
Rhodobacter sp.
Rhodobacter sp.
faecalis JCM 11668T
98.9- Rhodobacter maris JA276 T
FN296203
rods, rosettes, associate to form T, V & Y

shapes, 1.01.2 x 3.05.0m, chains,
motile, budding
95.8- Rhodoplanes elegans

ATCC 51906T
FM202448
vibrio-spiral, 1-2 x 3.0-5.0m, motile,

binary fission
spiral, 0.8-1.4 x 2.0-6.0m, sp, motile,
binary fission
oval, chains, 0.6-1.2 x 1.0-3.0m, non
99.7- Rhodocista pekingenesis

JCM11669T
98.4- Phaeospirillum fulvum
NCIMB 11762T
99.7- Rhodobacter maris JA276 T
AM999778
AM999777
AM999776
FN296202
32bD
JA521
R, Cv, Sm, L, P-RB
Bchl a, Sp
32bS
JA522
R, Cv, Sm, M, P
Bchl a, Sp
35R
JA520
R, Cv, Sm, M, RB
BChl a, Ly
46B
JA569
R, Cv, St, S, YB
Bchl a, Se/Sn
32PR
JA558
Bchl a, Sp
32PS
JA559
R, Cv, Sm, M, PRB

R, Cv, Sm, M, B
gi
131B
JA668
JA557
R, Cv, Sm, S, RB
R, Cv, Sl, S, YB
Bchl a, Sp
BChl a, Se/Sn
58Rb
JA556
R, Cv, St, M, YB
BChl a, Se/Sn
59P
59
Trb1
JA715
JA716
JA717
R, Cv, Sm, M, B
R, Cv, Sm, S, RB
R, Cv, Sm, M, YB
BChl a, Rp
Bchl a, Sp
BChl a, Se/Sn
131R
8Br2
JA718
JA704
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
BChl a, Ly
BChl a, Ly
SrRp1
SrRp2
RA2
Kal1
5PB
JA730
JA731
JA733
JA735
JA635
R, Cv, Sm, S, RB
R, Cv, Sm, L, P
R, Cv, Rg, M, YB
R, Cv, Sm, L, RB
R, Cv, Sm, S, RB
Bchl a, Sp
Bchl a, Sp
BChl a, Se/Sn
Bchl a, Sp
Bchl a, Sp
P6P
PA3
JA727
JA630
R, Cv, Sm, M, B
R, Cv, Sm, M, B
BChl a, Rp
BChl a, Rp
BChl a, Rp
93
rods, 0.6-0.9 x 2.0-2.5m, motile,

budding
rods, 0.4-0.6 x 1.0-2.5m, motile,
budding
spiral, 0.1-0.3 x 5-16m, motile, binary
fission
oval, 0.8-1 x 1.2-1.5m, non motile,
binary fission
rods,0.4-0.6 x 1.5-2.0m, rosettes,
motile, budding
spiral, 0.6-1.0 x 2.0-6.0m, chains, sp,
oval- rods, 0.41.5 x 1.04.0m, non
oval, 0.9-1.4 x 1.0-2.0m, non motile,
binary fission
fission
rods, chains, rosettes, associate to form
T,V & Y shape, motile, binary fission
oval-rod, non motile, binary fission
rods, rosettes, 0.5-0.9 x 1.0-3.0m,
motile, budding
spiral, chains, motile, sp, binary fission,
spiral, chains, motile, sp, binary fission,
Results

JCM 11668T
JCM 11668T
photometricum DSM 122T
99.4- Rhodovulum
visakhapatnamense JA181T
JCM 11668T
98.6- Phaeospirillum
chandramohanii JA145T
98.6- Rhodobacter megalophilus
JA194T
100- Rhodobacter megalophilus
JA194T
Phaeospirillum sp.
Rhodobacter sp.
Rhodospirillum sp.
Rhodoplanes sp.
Rhodobacter sp.
faecalis JCM 11668T
Phaeospirillum sp.
98.3-Phaeospirillum
molischianum DSM120T
FN546953
FN546954
FN547821
FN568342
FN813495
FN813496
FN813493
FN813494
FN813499
FN813501
94
Results
9Rb
JA728
R, Cv, Sl, M, YB
BChl a, Se/Sn
oval, nonmotile, binary fission
Rhodobacter sp.
10PNG
JA729
R, Cv, Sm, S, RB
Bchl a, Sp
9Nf
JA629
R, Cv, Sm, S, RB
Bchl a, Sp
NRb2
JA722
R, Cv, Sl, M, YB
BChl a, Se/Sn
rods,0.4-0.6 x 2.5-3.5m, motile,

budding
oval, chains, motile, binary fission

JCM 11668T
Rhodobacter sp.
Trb2
JA719
R, Cv, Sl, M, YB
BChl a, Se/Sn
oval, chains, motile, binary fission
Rhodobacter sp.
NRb1
JA712
R, Cv, Sm, M, YB
BChl a, Se/Sn
Rhodobacter sp.
1E
JA676
R, Cv, Sm, M, R
Bchl a, Sp
EKRb
JA677
R, Cv, Sl, M, YB
BChl a, Se/Sn
Rhodobacter sp.
WA/H
JA678
R, Cv, Sm, S, RB
Bchl a, Sp
EQT
JA679
R, Cv, Sm, M, RB
Bchl a, Sp
RA1
JA734
R, Cv, Sm, M, YB
BChl a, Se/Sn
Rhodobacter sp.
BK29
JA732
R, Cv, Sm, M, RB
Bchl a, Sp

fission
8BK
JA680
R, Cv, Sm, M, RB
Bchl a, Sp
8Br1
JA696
R, Cv, Sm, M, RB
BChl a, Sp
Rhodoplanes sp.
UnRb
JA697
R, Cv, Sm, M, YB
BChl a, Se/Sn
rods, chains, rosettes,associate to form

T, V, Y shapes, motile, binary fission
oval-rod, motile, binary fission
Rhodobacter sp.
B5P
JA698
R, Cv, Sm, M, B
BChl a, Rp
Phaeospirillum sp.
SRb
JA699
R, Cv, Sl, M, YB
BChl a, Se/Sn
Rhodobacter sp.
6Ru
JA700
R, Cv, Sm, St, S, B
BChl a, Se

fission
curved rods, motile, binary fission
Rubrivivax sp.
BRb
JA701
R, Cv, Sl, M, YB
BChl a, Se/Sn

fission
Rhodobacter sp.
FN813500
-
95
Results
Rp1
JA702
R, Cv, Sm, S, RB
Bchl a, Sp
Rp2
JA705
R, Cv, Sm, S, RB
Bchl a, Sp,
VRb
JA706
R, Cv, Sl, M, YB
BChl a, Se/Sn
Rhodobacter sp.
V6P
JA707
R, Cv, Sm, M, B
BChl a, Rp

fission
spiral, chains, sp, motile, binary fission,
Phaeospirillum sp.
Ru3
JA631
R, Cv, Sl, S, YB
BChl a, Se/Sn
RA3
JA632
R, Cv, Sl, M, YB
BChl a, Se/Sn
oval- rods,0.6-0.8 x 1.0-1.3m, motile,

binary fission
oval- rods, chains, 0.9-1.2 x 1.0-3.0
m, motile, binary fission

ATCC 11166T
ATCC 11166T
FN813502
FN813503
Betaproteobacteria
7Nf
JA628
R, Cv, Sm, L, RB
Bchl a, Sp
R, Cv, Sm, St, M, B BChl a, Se
curved rods, chains, 0.2-0.3 x 1.04.0m, motile, binary fission

curved rods, 0.2-0.4 x 2.0-4.0m,
99.5- Rubrivivax benzoatilyticus

HE800189
T
JA2
99.8- Rubrivivax gelatinosus JCM HE582621
21318T
Rubrivivax sp.
-
K4Ru
JA354
K5Ru
JA656
Y6Ru
JA667
Rubrivivax sp.
C7Ru
JA672
R, Cv, Sm, St, S, B
BChl a, Se
Rubrivivax sp.
2NRu
JA626
5N
JA627
8Ru
JA703

100- Rubrivivax gelatinosus JCM FN813497

21318T
99.5-Rubrivivax benzoatilyticus
FN813498
T
JA2
Rubrivivax sp.
-
32B
JA524
99- Rubrivivax gelatinosus JCM

21318T
oval- rods, 2-2.5 x 2.5-6.0m, motile,

binary fission, So
99.4- Allochromatium vinosum

ATCC 17899T; Allochromatium
minutissimum DSM 1376T
FN546955
Gammaproteobacteria
35PS
JA523
R, Cv, Sm, M, RB
BChl a, Ly
FN547820
35SR
JA560
R, Cv, Sm, M, RB
BChl a, Ly
NPs6
JA728
R, Cv, Sm, M, RB
NPs7
JA726
KIPs1
96
Results
BChl a, Ly
oval- rods, 1.0 x 1.2-3.0m, motile,

binary fission, So
oval- rods, motile, binary fission, So

ATCC 17899T
Allochromatium sp.
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
JA691
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
KIPs2
JA692
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
KIIPs1
JA693
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
KIIPs2
JA694
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
KIIIPs
JA695
R, Cv, Sm, M, RB
BChl a, Ly
rods, motile, binary fission, So
Allochromatium sp.
PAL2
JA685
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
AIPs1
JA686
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
AIPs2
JA687
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
AIIPs1
JA688
R, Cv, Sm, M, RB
BChl a, Ly
oval- rods,motile, binary fission, So
Allochromatium sp.
7PS
JA634
R, Cv, Sm, M RB
BChl a, Ly

binary fission, So
AIIPs2
JA689
R, Cv, Sm, M, RB
BChl a, Ly
99.4-Allochromatium vinosum
Allochromatium sp.
AIIIPs
JA690
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
ChPs
JA684
R, Cv, Sm, M RB
BChl a, Ly
Allochromatium sp.
CIPs
JA683
R, Cv, Sm, M, RB
BChl a, Ly
Allochromatium sp.
CIIPs
CIIIPs
8Ps
JA681
JA682
JA708
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
BChl a, Ly
BChl a, Ly
BChl a, Ly

Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
FN813504
-
FN813505
97
Results
JPs
32bP
32P
NPs1
TPs1
NPs2
NPs3
NPs4
NPs5
JA711
JA709
JA710
JA713
JA714
JA720
JA721
JA723
JA724
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
R, Cv, Sm, M, RB
BChl a, Ly
BChl a, Ly
BChl a, Ly
BChl a, Ly
BChl a, Ly
BChl a, Ly
BChl a, Ly
BChl a, Ly
BChl a, Ly

oval-rods; motile, binary fission, So
oval - rods, motile, binary fission, So
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
Allochromatium sp.
TPs2
JA727
R, Cv, Sm, M, RB
BChl a, Ly
L1
JA383
R, Cv, Sm, M, RB
BChl a, Ly

binary fission, So

Allochromatium sp.
FN296205
Purple bacterial strains obtained from other habitats
S1
JA480T
R, Cv, Sm, S, LB
BChl a, Rpl
FN391894
92.9- Rhodospirillum rubrum
ATCC11170T
S2
JA481
R, Cv, Sm, S, LB BChl a, Rpl
92.9- Rhodospirillum rubrum
FN543109
ATCC11170T
Rv
JA580T
R, Cv, Sm, M,
BChl a, Se/Sn
96.3- Rhodovulum adriaticum
FN669139
T
YB
DSM 2781 and Rhodovulum
iodosum N1T
Table 3.1.2 Identification of purple bacterial strains based on 16S rRNA gene sequence analysis and/ or their
phenotypic characteristics.
Bchl a, Bacteriochlorophyll a; Se, spheroidene; Sn, spheroidenone; Sp, spirilloxanthin; Ly, lycopene, Rp, Rhodopin;
Rpl, Rhodopinal; YB, yellowish; brown; RB, reddish brown; P, pink; B, brown.
R, round; Cv, convex; Sm, smooth; St, sticky; Sl, slimy; Rg, rough; L, large; M, medium; S, small; YB, yellowish brown;
RB, reddish brown; LB, light brown; P, pink; B, brown.
0
S , sulfur granules inside the cell; sp, spheroplasts.
Taxa identified tentatively based on phenotypic characteristics.
vibriod; 0.3-0.5 x 1.2-2.5m; motile,
binary fission
vibriod; 0.3-0.5 x 1.2-2.5m; motile,
binary fission
oval- rods, 0.5-0.9 x 1-1.8m; non
98
Results
3.2 Distribution and enumeration of purple bacteria

from paddy soils of Andhra Pradesh
Three different rice fields belonging to different agro-climatic
conditions of Andhra Pradesh, located at Kolluru (coastal, alluvial soil,
surface water irrigation), Nadergul (semi-arid, black soil, ground water
irrigation), Kurnool (semi-arid, black soil, surface water irrigation)
were selected for quantitative analysis of purple bacteria, at different
stages of growth of rice plant (Table 3.2.1). Counts were repeatedly
performed by collecting the samples over three rice growing seasons
(that is for 3 consecutive years), at 3 different stages of plant growth
namely seedling stage, booting stage and harvest stage of the plant.
The rice rhizosphere soil in the initial two stages is water logged with
high moisture content where as the soil at the harvest stage of the
plant is relatively dry.
As there is no absolute definition for rhizosphere soil, roots and the
adhering soil was considered for collection of samples. During each
collection, samples were taken at random from 5 different sites of the
same field and pooled as one composite sample. Counts were
expressed as colony-forming units per gram dry soil (Table 3.2.1).
3.2.1 Enumeration of PNSB The rhizosphere soil samples were
enumerated for PNSB by serial dilution and pour plating which was
followed by wax overlay to create anaerobic conditions for their
growth. This method was found to be successful in enumerating PNSB
as all the samples which gave positive enrichments could also be
99
Results
enumerated. The colonies of purple non sulfur bacteria were round,

convex, small/medium/large and were yellowish brown/red/brown in
color. Estimates of populations of cultured purple non sulfur bacteria
ranged from 3 x 102 to 6 x 105 CFU/g dry soil (Table 3.2.1). From each
of the sets of samples analyzed, the purple non sulfur bacteria were
recorded in highest numbers in the booting stage, while the lowest
numbers were recorded after the harvest of the rice plant.
3.2.2 Enumeration of PSB During the first rice growing season,
though some samples yielded PSB through enrichments, the same
could not always be enumerated by either serial dilution-plating or
agar shake methods. Hence for the next two seasons, membrane
filtration method followed by the wax overlay method was followed.
Estimates of populations of cultured purple sulfur bacteria ranged
from 103 to 720 x 102 CFU/g dry soil (Table 3.2.1). The colonies of PSB
were round, large and reddish brown in color and their presence was
further confirmed by microscopic observations. Similar to the PNSB
counts, high numbers of PSB were also recorded in the booting stage,
while least numbers were recorded at the harvest stage of the rice
plant.
Sample
100
Stage of
paddy
cultivation
Sample
I.D
Date of
sample
collection
pH
Seedling
AI-1
3/7/07
Booting
Harvest
Seedling
Booting
Harvest
Seedling
Booting
Harvest
AI-2
AI-3
KI-1
KI-2
KI-3
CI-1
CI-2
CI-3
10/10/07
15/12/07
6/8/07
25/10/07
20/12/07
5/10/07
25/12/07
14/01/08
7
7
7.5
7.5
7.5
7
7
7
Seedling
AII-1
8/08/08
K
(Nadergul)
Booting
Harvest
Seedling
Booting
AII-2
AII-3
KII-1
KII-2
C
(Kurnool)
Harvest
Seedling
Booting
Harvest
Season 1
A
(Kolluru)
K
(Nadergul)
C
(Kurnool)
Season 2
A
(Kolluru)
Enrich
ment
Results
PNSB
Enumeration
Method (CFU/g dry
soil)
Enrich
ment
PSB
Enumeration
Method
(CFU/g dry
soil)
25 x 102
+
+
+
+
+
+
+
+
Serial
dilution
-plating
-do-do-do-do-do-do-do-do-
Serial
dilutionplating
-do-do-do-do-do-do-do-do-
*nil
4200x 102
16 x 102
50 x 102
1100x 102
10 x 102
40 x 102
45 x 102
3 x 102
+
+
+
+
-
-do-
16 x 102
5/10/08
12/12/08
13/8/08
14/10/08
7
7
7.5
7.5
+
+
+
+
-do-do-do-do-
100 x 102
10 x 102
100 x 102
220 x 102
+
+
+
Membrane
filtration
-do-do-do-do-
KII-3
CII-1
CII-2
22/12/08
1/10/08
10/11/08
7.5
7
7
+
+
+
-do-do-do-
60 x 102
220 x 102
210 x 102
+
+
-do-do-do-
50 x 102
400 x 102
CII-3
21/12/08
-do-
10 x 102
-do-
54 x 102
52x 102
*TLTC
*TLTC
*TLTC
10 x 102
55 x 102
40 x 102
210 x 102
Season 3
A
(Kolluru)
K
(Nadergul)
101
Results
Seedling
Booting
Harvest
Seedling
AIII-1
AIII-2
AIII-3
KIII-1
8/08/09
10/10/09
21/12/09
18/8/09
7
7
7
7.0
+
+
+
+
-do-do-do-do-
50 x 102
6000 x 102
10 x 102
15 x 102
+
+
-do-do-do-do-
100 x 102
20 x 102
Booting
KIII-2
13/10/09
7.5
-do-
2500 x 102
-do-
720 x 102
Harvest
KIII-3
27/12/09 7.5 +
-do15 x 102
+
-do10 x 102
C
Seedling
CIII-1
1/8/09
7
+
-do40 x 102
-do2
(Kurnool)
Booting
CIII-2
22/11/09 7
+
-do45 x 10
+
-do10 x 102
Harvest
CIII-3
NT NT
NT
NT
NT
NT
NT
Table 3.2.1 Enumeration of purple non sulfur and purple sulfur bacteria from selected rice rhizosphere
soils of Andhra Pradesh.
Symbols: +, positive; -, negative; NT, not tested.
*Numbers were too low to be counted.
102
Results
3.3 Polyphasic characterization of Phaeospirillum sp.

JA317T
3.3.1 Habitat: Sample that yielded the strain JA317T was collected from
the rhizosphere soil of paddy at Nadergul, Andhra Pradesh, India on 6th
August 2007 (GPS position: 17o 16'N, 78o 32'E). The sample had a pH of
7.4 and a temperature of 30C.
3.3.2 Cultural characteristics:
3.3.2.1 Broth cultures: The color of the photosynthetically [(anaerobic,
light (2,400 lux)] grown cultures was brown (Fig. 3.3.1a).
3.3.2.2 Colonial characteristics: Colonies of strain JA317T were round,
convex, smooth, medium sized and brown pigmented. Colony diameter
could reach up to 1.5 mm after 6 days of incubation at 30C (Fig. 3.3.1b).
3.3.3 Morphology and fine structure: Cells of strain JA317T were spiral
shaped (Fig. 3.3.2a), 2-6m long and 0.8-1.2m wide. The cells stained
Gram negative and were motile by means of a polar flagellum (Fig.
3.3.3a) and multiplied by binary fission. Spherical bodies called
spheroplasts were formed by the strain in the late log phase (Fig. 3.3.2a,
b). Spheroplasts were also visualised under Leica TCS SP2 laser
scanning confocal microscope by acridine orange staining (Fig. 3.3.2b).
Transmission electron microphotographs of ultrathin sections of the
strain revealed stacked type of internal membrane structures (Fig.
3.3.3b).
103
Results
(a)
(b)
Fig. 3.3.1 Broth culture (a) and colony morphology (b) of Phaeospirillum
sp. JA317T.
Fig.
(a)
(b)
3.3.2 Phase-contrast microphotograph (a) and confocal
microphotograph (b) of Phaeospirillum sp. JA317T showing
spheroplasts (indicated by arrows).
(a)
(b)
Fig. 3.3.3 Electron micrographs of Phaeospirillum sp. JA317T showing
a negatively stained cell with a polar flagellum (a) and an
ultra thin section of the strain showing the stacked nature
of the photosynthetic membranes (b).
104
Results
(a)
(b)
Fig. 3.3.4 Whole-cell absorption spectrum (a) and acetone spectrum
of extracted pigments (b) of Phaeospirillum sp. JA317T.
Fig. 3.3.5 Effect of salinity (%, w/v) on the growth of Phaeospirillum sp.
JA317T.
(a)
(b)
Fig. 3.3.6 Effect of pH (a) and temperature (b) on the growth of
Phaeospirillum sp. JA317T.
105
Results
3.3.4. Pigment composition: The in vivo absorption spectrum of intact

cells in sucrose exhibited maxima at 374, 491, 527 and 851 nm (Fig.
3.3.4a) confirming the presence of bacteriochlorophyll a and the
absorption spectrum for pigments extracted with acetone gave maxima at
360, 447, 474, 504 and 579 nm (Fig. 3.3.4b) indicating the presence of
lycopene and its derivatives. Carotenoid composition of strain as
assessed by HPLC analysis showed rhodopin (81%), lycopene (12%) and
rhodopin glucoside (7%).
3.3.5 FAME analysis: Whole-cell fatty acid analysis of the strain JA317T
revealed that C18:17c, C16:0 and C16:16c/C16:17c predominated. Minor
proportions of C12:0, C13:1 at 12-13, C14:03OH, C18:0, C18:15c were also
detected (Table 3.3.5).
3.3.6 G+C content: The DNA G + C content of strain JA317T was 63.3 +
0.8 mol% (by HPLC).
3.3.7 Physiological characteristics: Strain JA317T was able to grow
photoorganoheterotrophically [anaerobic, light (2,400 lux) with pyruvate
(0.03%
w/v)].
Photolithoautotrophy
[anaerobic,
light
(2400
lux),
Na2S.9H2O or Na2S2O3.5H2O (1mM each) and NaHCO3 (0.1%, w/v)],

chemolithoautotrophy [aerobic, dark, Na2S.9H2O or Na2S2O3.5H2O (1mM
each) and NaHCO3 (0.1%, w/v)], chemo-organoheterotrophy [aerobic,
dark, and pyruvate (0.3%, w/v)] and fermentative growth [anaerobic,
dark with glucose/ fructose/ pyruvate (0.3 % w/v)] could not be
demonstrated in the strain. Very few substrates supported the growth of

Carbon source/
e- donor
(concentration)
106
Results
Growth
O.D660 nm
+/ -
0.02
NA
Control [NaHCO3 (0.1%, w/v)]
0.02
NA
Acetate (0.3%, w/v)
0.34
Ascorbate(0.3%, w/v)
0.05
Aspartate(0.3%, w/v)
0.11
(+)
*Benzoate (1mM)
0.03
*Butyrate (0.1%, w/v)
*Caproate (0.1%, w/v)
0.13
Casamino acids (0.3%, w/v)
0.08
(+)
Citrate (0.3%, w/v)
0.01
Crotonate (0.1%, w/v)
0.03
Cysteine(0.3%, w/v)
0.05
Ethanol (0.1%, w/v)
0.07
Formate (0.3%, w/v)
0.06
Fructose (0.3%, w/v)
0.09
Fumarate (0.3%, w/v)
0.32
Glutamate (0.3%, w/v)
0.09
Glucose (0.3%, w/v)
0.08
*Glycerol (0.3%, w/v)
0.03
2-oxo glutarate (0.3%, w/v)
0.04
Lactate (0.3%, w/v)
Malate (0.3%, w/v)
0.05
Mannitol (0.3%, w/v)
0.08
Methanol (0.1%, w/v)
0.04
Peptone (0.05%, w/v)
0.05
Propanol (0.1%, w/v)
0.04
Control
[without
organic substrate]
inorganic/
Methionine (0.3%, w/v)
107
*Propionate (0.1%, w/v)
0.01
Pyruvate (0.3%, w/v)
0.48
Sorbitol (0.3%, w/v)
0.03
Succinate (0.3%, w/v)
0.34
Sucrose (0.3%, w/v)
0.06
Tartarate (0.3%, w/v)
0.02
Thioglycolate (0.3%, w/v)
0.02
*Valerate (0.1%, w/v)
0.02
* H2 (20%, v/v gas phase)
0.06
* Na2S.9H2O (0.5 mM)
0.02
* Sulfur (0.05%)
0.03
* Thiosulfate (5 mM)
0.02
* Sulfite (5 mM)
0.01
Starch (0.3%, w/v)
Results
Table 3.3.1 Carbon source/e-donor utilization by Phaeospirillum

sp. JA317T.
Substrates were provided by replacing pyruvate, succinate, acetate in the
triple carbon medium (Table 2.2). Experiment was performed in screw
capped tubes and the results expressed are average values of an
experiment done in triplicates after 96 h of light (2,400 lux) anaerobic
incubation at 30+2C. Symbols: + = Growth present; (+) = Weak growth; = No growth; NA = Not applicable.
* = 0.1% (w/v) Sodium bicarbonate was supplemented.

Sulfur sources
(concentration)
108
Results
Growth
O.D660 nm
+/ -
Control [without sulfur source]
0.04
NA
Cysteine (2 mM)
0.22
Magnesium sulfate (5 mM)
0.29
Methionine (2mM)
0.19
Sodium sulfate (5 mM)
0.32
Sodium sulfite (5 mM)
0.20
Sodium sulfide (1 mM)
0.08
Sulfur (2 mM)
0.29
Sodium thiosulfate (5 mM)
0.06
Table 3.3.2 Growth of Phaeospirillum sp. JA317T on various sulfur

sources.
Experimental details as given in Table 3.3.1, except that pyruvate
(0.3%, w/v) was used as carbon source and sulfur source varied as
indicated.
Nitrogen sources
Growth
(0.068%, w/v)
O.D660 nm
+/ Control [without nitrogen
0.08
NA
source]
Ammonium chloride
0.64
Aspartate
0.10
Glutamate
0.45
Glutamine
0.06
Sodium nitrate
0.04
Sodium nitrite
0.02
Urea
0.35
N2 [100% (v/v) gas phase]
0.17
Table 3.3.3 Growth of Phaeospirillum sp. JA317T on various

nitrogen sources.
Experimental details as given in Table 3.3.1, except that pyruvate (0.3%,
w/v) was used as carbon source and nitrogen source varied as indicated.

0
109
Vitamins
Results
Growth
O.D660 nm
0.05
+/ NA
0.23
*Without Thiamine
0.48
*Without Riboflavin
0.42
*Without Niacin
0.20
*Without Pantothenate
0.45
*Without
0.18
*Without Vitamin B12
0.22
*Without Biotin
0.42
* Without p-Amino benzoic

acid
Yeast extract
0.24
0.40
Control
[without
vitamin
source]
All
vitamins
Pyridoxal
phosphate
Table 3.3.4 Vitamin requirement by Phaeospirillum sp. JA317T.

w/v) was used as carbon source and yeast extract was replaced with
vitamin solution as indicated. Concentration of the vitamins used as
given in section 2.19.5.
*= other 7 vitamins were added; = all 8 vitamins were added.
Fatty acid
Composition (%)
Major fatty acids
32.5
C18:17c
C16:0
25.9
25.1
C16:16c/C16:17c
Minor fatty acids
C12:0
3.3
C13:1 at12-13
1.9
C18:0
1.4
C14:03OH
1.3
1.3
C18:15c
Unidentified
2.2
Table 3.3.5 Cellular fatty acid profile of Phaeospirillum sp.
JA317T.
110
Results
the strain (Table 3.3.1) as carbon source/e- donors under photoorganoheterotrophic
condition
which
included
acetate,
pyruvate,
succinate and fumarate. Aspartate and casa-amino acids could also

support growth but to a lesser extent. Those which could not be utilized
included
aspartate,
fructose,
malate,
2-oxoglutaric
caproate,
sucrose,
peptone,
gluconate,
acid,
glycerol,
casamino
acids,
methionine,
sorbitol,
glutamate,
glucose,
aspartate,
benzoate,
ascorbate,
thiogylcolate, butyrate, lactate, formate, propionate, valerate, crotonate,

glycolate, tartarate, caprylate, mannitol, methanol and ethanol.
Sulfate, sulfite, cysteine, methionine and elemental sulfur were used
as sulfur sources by the strain under photoheterotrophic conditions,
while sulfide and thiosulfate were not utilized (Table 3.3.2). Strain
JA317T could utilize dinitrogen, ammonium chloride, glutamate and urea
as nitrogen sources, but nitrate, nitrite, aspartate and glutamine did not
support growth (Table 3.3.3). The strain had requirement for complex
growth factors and even a combination of all the eight vitamins (Table
3.3.4) could not replace yeast extract. There is no salt requirement for
growth of the strain (Fig. 3.3.5). The pH range for growth of the strain
was 6.0-8.0 (optimum 7.0- 7.5) (Fig. 3.3.6a) and the temperature range
was 25-350C (optimum 300C) (Fig. 3.3.6b).
3.3.8 16S rRNA gene sequence similarity: The pairwise 16S rRNA gene
sequence similarities of strain JA317T with the nearest type strains were
found using EzTaxon server. The data obtained revealed that strain

JA317T
has
sequence
111
similarities
of
98.5,
Results
98.2,
97.7%
with
Phaeospirillum chandramohanii JA145T, Phaeospirillum molischianum

DSM120T and Phaeospirillum fulvum DSM113T respectively.
3.3.9 DNA-DNA hybridization: Genomic DNA-DNA hybridization of
strain JA317T was performed with the type strains of species of the
genus. The results indicated a reassociation of 55 +5 %, 38 + 7.4 % and
42 + 5.7 % (mean values from three experiments, including reciprocal
analyses)
occurred
with
the
type
strains
of
Phaeospirillum
chandramohanii; Phaeospirillum fulvum and Phaeospirillum molischianum

respectively.
3.3.10 16S rRNA gene sequence deposition: The GenBank/ EMBL/
DDBJ accession number for the 16S rRNA gene sequence of strain
JA317T is AM901294.
3.3.11 Culture deposition: Strain JA317T =NBRC 104938T =KCTC
5704T =ABRC-APB 24 T.
112
Results
3.4 Polyphasic characterization of Rhodoplanes sp.

JA415T
3.4.1 Habitat: Strain JA415T was isolated from a soil sample collected
on 17 November 2007, from a pokkali rice field, Vypeen Island,
Ernakulam, Kerala, on the west coast of India (GPS position: 10o 08 N
76o 21 E). The soil sample had a pH of 7.0 and a temperature of 30C.
3.4.2.1 Broth cultures: Cultures of strain JA415T were reddish brown
when grown phototrophically (Fig. 3.4.1a) and colorless when grown
chemotrophically.
3.4.2.2 Colonial characteristics: Colonies of phototrophic cultures of
strain JA415T were round, convex, smooth, medium sized and reddish
brown pigmented (Fig. 3.4.1b). Colonies of chemotrophically grown
cultures were pink in color. Colony diameter could reach up to 2 mm
after 6 days of incubation at 30C.
3.4.3 Morphology and fine structure: Cells of strain JA415T were rodshaped, 1.01.2 m wide and 3.05.0 m long (Fig. 3.4.1c). Strain
JA415T multiplied by budding and asymmetrical cell division without
forming
prosthecae.
Cells
of
strain
JA415T
associate
to
form
characteristic T, Y and V shapes, along with the formation of short

chains and rosettes (Fig. 3.4.1c). Strain JA415T was motile by means of a
single polar flagellum (Fig. 3.4.2a). Transmission electron micrographs of
113
Results
(a)
(b)
(c)
Fig. 3.4.1 Broth culture (a), colony morphology (b) and phase-contrast
microphotograph (c) [Bar, 5m] of Rhodoplanes sp. JA415T.
(a)
(b)
Fig. 3.4.2 Electron micrographs of Rhodoplanes sp. JA415T showing
negatively stained cell with a polar flagellum (a), Bar, 833nm
and ultrathin section of the strain showing lamellar type of the
photosynthetic membranes.
114
Results
(a)
(b)
Fig.3.4.3 Whole-cell absorption spectrum (a) and acetone spectrum
of extracted pigments (b) of Rhodoplanes sp. JA415T.
Fig.3.4.4 Effect of salinity (%, w/v) on the growth of Rhodoplanes

sp. JA415T.
(a)
(b)
Fig.3.4.5 Effect of pH (a) and temperature (b) on growth of
Rhodoplanes sp. JA415T.
115
Results
ultrathin sections of the strain revealed lamellar internal membrane

structures parallel to the cytoplasmic membrane (Fig. 3.4.2b).
3.4.4 Pigment composition: The whole-cell absorption spectrum of
strain JA415T showed absorption maxima at 380, 464, 491, 527, 596,
800 and 866 nm (Fig. 3.4.3a) and cell extracts of strain JA415T in
methanol showed an absorption maximum at 770 nm, confirming the
presence of bacteriochlorophyll a. No differences were observed in the
whole-cell absorption spectra of cells of strain JA415T when grown at
high [5000 lx (incandescent)] or low [500 lx (incandescent)] light
intensities (data not shown). Acetone extracts showed absorption
maxima at 359, 473, 503 and 570 nm, thus confirming the presence of
carotenoids of the spirilloxanthin series (Fig. 3.4.3b). The major
carotenoid of strain JA415T as detected by HPLC analysis was rhodopin
(77%);
minor
components
were
methyl
rhodopin,
lycopene
and
unidentified spirilloxanthins.
3.4.5 FAME analysis: Whole-cell fatty acid analysis revealed C18:17c as
the predominant fatty acid (61 %) in strain JA415T (Table 3.4.5).
Significant proportions of C16:0 (9 %), C18:0 (3.8 %), C18:0 2-OH (5.2 %) and
C19:0 cyclo8c (5.5 %) were also detected. Strain JA415T contained
ubiquinone-10 and rhodoquinone-10 as primary quinone components
Carbon source/
e- donor
(concentration)
116
Results
Growth
O.D660 nm
+/ -
Control
[without
inorganic/
organic substrate]
0.03
NA
0.03
NA
Acetate (0.3%, w/v)
0.37
0.05
0.09
*Benzoate (1mM)
0.03
0.16
0.03
0.18
Citrate (0.3%, w/v)
0.20
0.03
Cysteine(0.3%, w/v)
0.05
Ethanol (0.1%, w/v)
0.07
Formate (0.3%, w/v)
0.06
0.05
0.32
0.09
Glucose (0.3%, w/v)
0.08
0.03
0.04
Lactate (0.3%, w/v)
0.26
Malate (0.3%, w/v)
0.25
0.08
0.07
0.04
0.05
117
Results
0.04
0.15
0.45
0.03
0.44
Sucrose (0.3%, w/v)
0.06
0.03
0.03
0.04
0.05
* Na2S.9H2O (0.5 mM)
0.03
* Sulfur (0.05%)
0.03
0.02
* Sulfite (5 mM)
0.02
Table 3.4.1 Carbon source/e-donor utilization by Rhodoplanes

sp. JA415T.
Substrates were provided by replacing pyruvate in the single carbon
medium (Table 2.1). Experiment was performed in screw capped tubes
and the results expressed are average values of an experiment done in
triplicates after 72 h of light (2,400 lux) anaerobic incubation at 30+2C.
Symbols: + = Growth present; (+) = Weak growth; - = No growth; NA = Not
applicable.
* = 0.1% (w/v) Sodium bicarbonate was supplemented
Sulfur sources
(concentration)
118
Results
Growth
O.D660 nm
0.18
+/ NA
Cysteine (2 mM)
0.15
0.30
0.24
0.31
0.20
Sulfur (2 mM)
0.20
0.35
Sodium thioglycolate (5 mM)
0.17
Table 3.4.2 Growth of Rhodoplanes sp. JA415T on various sulfur

sources.
(0.3%, w/v) was used as carbon source and sulfur sources varied as
indicated.
Nitrogen sources (0.068%,

w/v)
Growth
O.D660 nm
0.12
+/ NA
Ammonium chloride
0.40
Glutamate
0.10
Glutamine
0.28
Sodium nitrate
0.09
Sodium nitrite
Urea
0.04
0.16
Control
[without
nitrogen
source]
Table 3.4.3 Growth of Rhodoplanes sp. JA415T on various nitrogen

sources.
(0.3%, w/v) was used as carbon source and nitrogen sources varied as
indicated.
119
Results
Vitamins
Growth
O.D660 nm
0.05
+/ NA
0.30
*Without Thiamine
0.28
*Without Riboflavin
0.37
*Without Niacin
*Without
Pyridoxal
phosphate
0.35
0.37
*Without Biotin
0.31
0.65
Control
[without
source]
All vitamins
vitamin

acid
Yeast extract
Table 3.4.4 Vitamin requirement by Rhodoplanes sp. JA415T.

given in section 2.19.5. *= other 7 vitamins were added; = all 8 vitamins
were added.
Fatty acid
Major fatty acids
C18:17c
C16:0
Minor fatty acids
C19:0 cyclo 8c
C18:0
C18:0 2OH
C12:0
C17:0 iso
C18:1 iso H
C20:1 7c
C16:17c
Summed features*
1
2
3
4
Composition (%)
61.0
9.0
5.5
3.8
5.2
2.0
1.4
1.2
3.5
1.9
1.9
1.41
1.56
2.63
Table 3.4.5 Cellular fatty acid profile of Rhodoplanes sp. JA415T.

*Summed features represent groups of two fatty acids that could not be separated by
GLC with the MIDI system. Summed feature 1, C16:17c / 16:16c; 2,
C17:1isoI/anteisoB; 3, C19:111c/ C19:19c; 4, C19:17c/ C19:16c.
120
Results
(molar ratio approximately 0.5). Menaquinones were absent. The

combination of the presence of ubiquinone-10 and rhodoquinone-10 and
the absence of menaquinones is specific for members of the genus
Rhodoplanes.
3.4.6 G+C content: The genomic DNA G+C content of strain JA415T was
67.2 mol% (by HPLC).
photoorganoheterotrophically [anaerobic, light (2400 lx), with pyruvate
(0.03% w/v)] and chemo-organoheterotrophically [aerobic, dark, with
pyruvate (0.3% w/v) as carbon source]. Photolithoautotrophy [anaerobic,
light (2400 lx), Na2S. 9H2O or Na2S2O3. 5H2O (1mM each) and NaHCO3
(0.1 %, w/v)], chemolithoautotrophy [aerobic, dark, Na2S. 9H2O or
Na2S2O3. 5H2O (1mM each) and NaHCO3 (0.1 %, w/v)] and fermentative
growth [anaerobic, dark, with glucose, fructose or pyruvate (0.3 %, w/v)]
could not be demonstrated. For photoheterotrophic growth, strain
JA415T could utilize acetate, lactate, citrate, fumerate, casamino acids,
butyrate,
malate,
propionate,
pyruvate
and
succinate
as
carbon
source/e- donors (Table 3.4.1). Some of those which could not be utilized
included aspartate, benzoate, formate, glycolate, mannitol, methanol, 2oxoglutaric acid, glycerol, glutamate, caproate, sucrose, gluconate,
methionine,
ascorbate,
caprylate and ethanol.
thiogylcolate,
valerate,
crotonate,
tartarate,
121
Results
Sulfate, sulfite, thiosulfate and methionine were used as sulfur

sources but cysteine, elemental sulfur, sulfide and thioglycolate did not
support growth (Table 3.4.2). Strain JA415T could utilize ammonium
salts and glutamate as nitrogen sources but others did not support
growth (Table 3.4.3). Growth with nitrate as sole nitrogen source was
observed without visible dinitrogen gas production. Diazotrophic growth
and acetylene reduction could be demonstrated. Niacin, pantothenate
and para-aminobenzoate were required as growth factors (Table 3.4.4).
Strain JA415T did not require salt (NaCl) for growth, but the growth yield
increased (~30%) in the presence of NaCl (range 02.5 %, w/v; optimum
1.5 %, w/v; Fig. 3.4.4). The pH and temperature range for the strain was
6.07.5 (optimum 7) and 25300C (optimum 300C), respectively (Fig.
3.4.5a, b).
sequence similarity of strain JA415T, found using EzTaxon server,
revealed that the strain has sequence similarities of 96.4, 96.2, 95.7 and
94.9% with the type strains of Rhodoplanes elegans, Rhodoplanes roseus,
Rhodoplanes cryptolactis and Rhodoplanes serenus, respectively.
3.4.9 16S rRNA gene sequence deposition: The GenBank/EMBL/DDBJ
accession number for the 16S rRNA gene sequence of strain JA415T is
FM202448.
3.4.10 Culture deposition: Strain JA415T =KCTC 5711T=NBRC 104972T
=ABRCAPB 61T.
122
Results
3.5 Polyphasic characterization of Rhodospirillum sp.

JA318T
3.5.1 Habitat: The strain JA318T was isolated from the rhizosphere soil
of paddy, Nadergul, Andhra Pradesh, India which was collected on 6th
August 2007 (GPS position: 17o 16'N, 78o 32'E). The sample had a pH of
7.4 and a temperature of 30C.
3.5.2 Cultural characteristics
3.5.2.1 Broth cultures: Photosynthetically [anaerobic, light (2,400 lux)]
grown cell cultures were reddish brown in color (Fig. 3.5.1a).
5.2.2. Colonial characteristics: Colonies of strain JA318T were round,
convex, smooth, medium sized and reddish brown pigmented (Fig.
3.5.1b). Colony diameter could reach up to 1.5 mm after 6 days of
incubation at 30C.
3.5.3 Morphology and fine structure: Cells of strain JA318T were spiral
shaped, 4-16m long and 0.1-0.3m wide (Fig. 3.5.1c). They stained
gram negative and were motile by means of peritrichous flagella (Fig.
3.5.2a). The cells multiplied by binary fission. Transmission electron
microphotographs of ultrathin sections of the strain revealed stacked
type of internal membrane structures which were present parallel and
perpendicular to the cytoplasmic membrane (Fig. 3.5.2b).
3.5.4 Pigment composition: The in vivo absorption spectrum of intact
cells in sucrose exhibited maxima at 374, 491, 527, 791 and 851 nm
(a)
(a)
123
(b)
Results
(c)
(b)
(c)
Fig. 3.5.1 Broth culture (a), colony morphology (b) and phase
contrast
microphotograph
(c)
(Bar,
5m)
of
Rhodospirillum sp. JA318T.
(a)
(b)
Fig. 3.5.2 Electron micrographs of Rhodospirillum sp. JA318T
showing negatively stained flagellated cell (a) and
ultrathin section of the strain showing the stacked
nature of the photosynthetic membranes.
124
Results
(a)
(b)
of extracted pigments (b) of Rhodospirillum sp. JA318T.
Fig.3.5.4 Effect of salinity (%, w/v) on the growth of Rhodospirillum

sp. JA318T.
(a)
(b)
Fig.3.5.5 Effect of pH (a) and temperature (b) on growth of
Rhodospirillum sp. JA318T.
125
Results
(Fig. 3.5.3a) confirming the presence of bacteriochlorophyll a and the

357, 480, 507 and 579 nm (Fig. 3.5.3b) confirming the presence of
rhodopinal series of carotenoids. Carotenoid composition of strain
JA318T was
analysed
by
HPLC
analysis
showing
1,
2,
1,
2-
tetrahydrolycopene (37%), rhodopin (35%), anhydro rhodovibrin (18%),

spirilloxanthin (6%) and rhodovibrin (4%).
3.5.5 FAME analysis: Whole-cell fatty acid analysis revealed that
C18:17c, C16:0, C16:16c/C16:17c predominated in strain JA318T. Minor
proportions of C12:0, C13:1 at 12-13, C14:03OH, C18:0, C18:15c were also
detected (Table 3.5.5).
3.5.6 G+C content: The DNA G + C content of strain JA318T was 59.460.2 mol% (by HPLC).
(0.03%
w/v)].
[anaerobic,
light
(2400
lux),
Na2S.9H2O or Na2S2O3.5H2O (1mM each) and NaHCO3 (0.1% w/v)],

chemolithoautotrophy [aerobic, dark, Na2S2O3.5H2O (1mM) and NaHCO3
(0.1% w/v)], chemoorganoheterotrophy [aerobic, dark, and pyruvate
(0.3%, w/v)] and fermentative growth [anaerobic, dark with glucose/
fructose/ pyruvate (0.3 % w/v)] could not be demonstrated in the strain.
The substrates that were utilized as carbon source/e- donors under
photorganoheterotrophic
condition
(Table
3.5.1)
included
acetate,
Carbon source/
e- donor
(concentration)
126
Results
Growth
O.D660 nm
+/ -
Control
[without
inorganic/
organic substrate]
0.02
NA
0.02
NA
Acetate (0.3%, w/v)
0.34
0.05
0.09
*Benzoate (1mM)
0.03
0.25
0.03
0.08
Citrate (0.3%, w/v)
0.01
0.23
Cysteine(0.3%, w/v)
0.05
Ethanol (0.1%, w/v)
0.27
Formate (0.3%, w/v)
0.22
0.09
0.32
0.14
(+)
Glucose (0.3%, w/v)
0.12
(+)
0.03
0.24
Lactate (0.3%, w/v)
0.26
Malate (0.3%, w/v)
0.25
0.28
0.14
(+)
0.06
127
Results
0.05
0.07
0.43
0.04
0.34
Sucrose (0.3%, w/v)
0.07
Starch (0.3%, w/v)
0.01
0.29
0.05
0.22
0.03
* Na2S.9H2O (0.5 mM)
0.02
* Sulfur (0.05%)
0.02
0.02
* Sulfite (5 mM)
0.02
Table 3.5.1 Carbon source/e-donor utilization by Rhodospirillum

sp. JA318T.
incubation at 30+2C. Symbols: + = Growth present; - = No growth; (+) =
Weak growth; NA = Not applicable.
* = 0.1% (w/v) Sodium bicarbonate was supplemented
Sulfur sources
(concentration)
128
Results
Growth
O.D660 nm
+/ -
0.04
NA
Cysteine (2 mM)
0.25
0.29
Methionine (2mM)
0.22
0.23
0. 02
0.69
Sulfur (2 mM)
0.04
0.59
Sodium thioglycolate (5 mM)
0.30
Table 3.5.2 Growth of Rhodospirillum sp. JA318T on various sulfur

sources.
indicated.
Nitrogen sources
(0.068%, w/v)
Growth
O.D660 nm
0.06
+/ NA
0.28
Aspartate
0.23
Glutamate
0.29
Glutamine
0.41
Sodium nitrate
0.40
Sodium nitrite
0.02
Urea
0.05
0.17
Control [without nitrogen

source]
Ammonium chloride
Table 3.5.3 Growth of Rhodospirillum sp. JA318T on various

nitrogen sources.
w/v) was used as carbon source and nitrogen source varied as indicated.
129
Vitamins
Results
Growth
O.D660 nm
0.02
+/ NA
0.25
*Without Thiamine
0.04
*Without Riboflavin
0.22
*Without Niacin
0.07
0.45
*Without
Pyridoxal
phosphate
0.19
0.22
*Without Biotin
0.24

acid
Yeast extract
0.09
0.30
Control [without
source]
All vitamins
vitamin
Table 3.5.4 Vitamin requirement by Rhodospirillum sp. JA318T.

Experimental details as given in Table 3.5.1, except that pyruvate (0.3%, w/v)
was used as carbon source and yeast extract was replaced with vitamin
solution as indicated. Concentration of the vitamins used as given in section
2.19.5.
Fatty acid
Major fatty acids
Composition (%)
C18:17c
38.4
C16:0
32.8
Minor fatty acids

C 5c
18:1
4.2
4.2
C10:0
C 2OH
2.8
2.7
C16:1 7c/ C16:1 6c

C
3-OH
2.4
1.9
18:0
18:1
16:0
12:0
C14:0
C19:0 iso
C15:02OH
C18:0 3OH
1.8
1.8
1.7
1.5
1.1
Table 3.5.5 Cellular fatty acid profile of Rhodospirillum sp. JA318T.
130
Results
butyrate, pyruvate, fumarate, malate, valerate, succinate, crotonate,

glutarate, tartarate, lactate, formate, ethanol and mannitol. Those which
could not be utilized included arginine, aspartate, caproate, caprylate,
fructose, glycerol, glycolate, propionate, sorbitol, glutamate, peptone,
casamino acids, glucose, benzoate, sucrose, gluconate, methionine,
ascorbate and methanol.
Sulfate, cysteine, methionine, thioglycolate, sulfide and thiosulfate
were used as sulfur sources by the strain under photoheterotrophic
conditions (Table 3.5.2), whereas elemental sulfur and sulfite did not
support the growth. Strain JA318T could utilize ammonium chloride,
nitrate, glutamate, aspartate, dinitrogen and glutamine as nitrogen
sources while nitrite and urea did not support the growth (Table 3.5.3).
Thiamine, niacin and PABA were used as source of vitamins (Table
3.5.4). There is no salt requirement for the strain JA318T (range 0-1.5%;
Fig. 3.5.4). The pH range is 6.0-8.0 (optimum 7.0; Fig. 3.5.5a). The
temperature optimum is at 300C (range 25-350C; Fig. 3.5.5b).
JA318T has sequence similarities of 99.9, 96.5, 96.1% with respect to
Rhodospirillum
sulfurexigens
JA143T,
Rhodospirillum
photometricum
DSM122T and Rhodospirillum rubrum ATCC11170T respectively.
131
Results
3.5.9 DNA-DNA hybridization: Genomic DNA-DNA hybridization of

strain JA318T was performed with its nearest phylogenetic neighbor of
the genus, Rhodospirillum sulfurexigens JA143T. The results indicated a
homology of 52% (mean value from three experiments, including
reciprocal analyses). With this technique, a maximum difference of 16
units was obtained between the reciprocal values and the mean standard
deviation was 8 units and deviations up to 2025 units are still
acceptable (Goris et al., 1998).
3.5.10
16S
rRNA
gene
sequence
deposition:
The
GenBank/EMBL/DDBJ accession number for the 16S rRNA gene

sequence of strain JA318T is AM901295.
3.5.11 Culture deposition: Strain JA318T =KCTC 5960T= NBRC
107573T =ABRC APB-23 T.
132
Results
3.6 Polyphasic characterization of strain JA480T

3.6.1 Habitat: Strain JA480T was purified from an enrichment culture
obtained from a sediment sample collected from a brackish shrimp pond
at Vadkkupoygainallur (GPS 10o4406N and 79o5012E), a village near
Nagapattinam, Tamilnadu, India on 9th March 2003. The sample had a
pH of 8.2, a salinity of 0.5% NaCl (w/v) and a temperature of 30C.
3.6.2.1 Broth cultures: Phototrophically [anaerobic, light (2,400 lux)]
grown cultures of strain JA480T were light brown (Fig. 3.6.1a).
3.6.2.2 Colonial characteristics: On agar slant, colonies of strain
JA480T were round, convex, smooth and light brown colored (Fig.
3.6.1b). Size of the colonies reached up to 1mm diameter after 7 days of
incubation under fluorescent light (2,400 lux) at 30oC.
3.6.3 Morphology and fine structure: Individual cells of strain JA480T
were vibriod, 0.3-0.5 m wide and 1.2-2.5 m long and multiplied by
binary fission (Fig. 3.6.1c). Flagellar motility could not be observed in the
strain JA480T at any stage of phototrophic growth under phase contrast
microscope. Initially the transmission electron microscopic analysis also
showed cells without flagella. A more careful search confirmed the
presence of monopolar biflagellate cells (Fig. 3.6.3) of strain JA480T.
However, their number was very low (1 out of 100). Soft agar (0.5% w/v)
stabs were also used to test the motility of the cells, where the strain
JA480T diffused throughout the medium (Fig. 3.6.2a). Phototaxis was
133
Results
(a)
(b)
(c)
Fig. 3.6.1 Broth culture (a), colony morphology (b) and phasecontrast microphotograph (c) [Bar, 5m] of strain
JA480T.
(a)
(b)
Fig.3.6.2 Motility assay tubes (a), stabbed with the cultures
of Phaeospirillum chandramohanii JA145T (control) and
the strain JA480T; Demonstration of phototaxis (b) in
the strain JA480T [tube incubated in dark (i) and that
illuminated from bottom (ii)].
1.3 m
500 nm
Fig. 3.6.3 Electron micrographs of negatively stained cells of

strain JA480T showing both flagella and absence of
flagella.
(a)
134
Results
(b)
Fig. 3.6.4 (a), (b). Electron micrographs of ultra thin sections

of strain JA480T, showing chimeric ICM structures.
V = vesicles; L = lamellae.
(a)
(b)
Fig. 3.6.5 Whole-cell absorption spectrum (a) and acetone
spectrum of extracted pigments (b) of strain JA480T.
Fig.3.6.6 Effect of salinity (%, w/v) on the growth of strain

JA480T.
(a)
(b)
Fig.3.6.7 Effect of pH (a) and temperature (b) on growth of strain
JA480T.
135
Results
demonstrated in the strain (Fig. 3.6.2b), where the tube incubated in

dark (completely covered with an aluminum foil) had no growth while in
the tube where illumination was provided only from the bottom, the cells
(after 5 days of incubation) moved in the direction of light. Transmission
electron micrographs of ultrathin sections of strain JA480T revealed
chimeric type of intracellular cytoplasmic membrane (ICM) structures,
where both lamellar stacks and vesicles are present in a single cell (Fig.
3.6.4).
3.6.4 Pigment composition: Whole cell absorption spectrum of strain
JA480T showed absorption maxima at 380, 488, 524, 593, 794 and 863
nm (Fig. 3.6.5a) confirming the presence of bacteriochlorophyll a.
Spectrum of acetone extracted cells of strain JA480T showed absorption
maxima at 360, 474, 504, 579 nm (Fig. 3.6.5b), indicating rhodopinal
series of carotenoids (Britton et al, 2004). Carotenoid composition of the
strain as determined by C18-HPLC analysis, showed the presence of
rhodopin (80%), 4, 4-diapolycopene (17%) and tetrahydrolycopin (3%).
Carotenoid glycosides could not be detected from the acetone extracts of
the strain, as tested on thin-layer chromatography (Dichloromethyl:
hexane: methanol (3:1.5:0.5); sprayed with anthrone reagent [0.2%
anthrone in 90% H2SO4]) or using HPLC.
revealed that C18:17c and C16:0 were major fatty acids (Table 3.6.5) and
minor quantities of C14:0, C16:03OH, C16:17c/C16:16c were also detected.
Carbon source/
e- donor
(concentration)
136
Results
Growth
O.D660 nm
+/ -
0.01
NA
Control (HCO3- 0.1%, w/v)
0.01
NA
Acetate (0.3%, w/v)
0.20
0.03
0.02
*Benzoate (1mM)
0.04
0.01
Citrate (0.3%, w/v)
0.01
0.03
Cysteine(0.3%, w/v)
0.01
Ethanol (0.1%, w/v)
Formate (0.3%, w/v)
0.03
0.02
0.01
0.02
Glucose (0.3%, w/v)
0.01
0.03
Lactate (0.3%, w/v)
Malate (0.3%, w/v)
0.10
(+)
0.02
0.01
0.02
Control [without
organic substrate]
inorganic/
137
Results
0.01
0.23
0.03
0.25
Sucrose (0.3%, w/v)
0.04
0.01
0.14
0.01
* Na2S.9H2O (0.5 mM)
0.02
* Sulfur (0.05%)
0.01
0.02
* Sulfite (5 mM)
0.01
Starch (0.3%, w/v)

Table 3.6.1 Carbon source/e-donor utilization by strain JA480T.

incubation at 30+2C. Symbols: + = Growth present; - = No growth; (+) =
Weak growth; NA = Not applicable.
* = 0.1% (w/v) Sodium bicarbonate was supplemented.
Sulfur sources
(concentration)
138
Results
Growth
O.D660 nm
+/ -
0.02
NA
Cysteine (2 mM)
0.02
0.02
Methionine (2mM)
0.01
0.02
0.03
0.30
0.06
Sulfur (2 mM)
Table 3.6.2 Growth of strain JA480T on various sulfur sources.

indicated.
Nitrogen sources
(0.068%, w/v)
Growth
O.D660 nm
0.03
+/ NA
Ammonium chloride
0.28
Aspartate
0.02
Glutamate
0.05
Glutamine
0.34
Sodium nitrate
0.04
Sodium nitrite
0.01
Urea
0.03
0.02
Control
(without
nitrogen
source)
Table 3.6.3 Growth of strain JA480T on various nitrogen sources.

(0.3%, w/v) was used as carbon source and nitrogen source varied as
indicated.
139
Vitamins
Results
Growth
O.D660 nm
0.10
+/ NA
0.23
*Without Thiamine
0.25
*Without Riboflavin
0.21
*Without Niacin
0.16
0.26
*Without
Pyridoxal
phosphate
0.15
0.20
*Without Biotin
0.10
0.11
0.20
Control
[without
vitamin source]
All vitamins
*Without
benzoic acid
Yeast extract
p-Amino
Table 3.6.4 Vitamin requirement by the strain JA480T.

given in section 2.19.5.
Fatty acid
Major fatty acids
C16:0
C18:17c
Composition (%)
22.0
55.0
Minor fatty acids

C12:0
C13:1 at 12-13
C14:0
C16:03OH
C16:1 5c
C16:17c/ C16:16c
C16:1 7c alcohol
C17:0 anteiso
C17:1 6c
C18:15c
0.7
1.4
3.5
2.9
0.7
3.0
0.8
1
0.8
1.3
Table 3.7.5 Cellular fatty acid profile of the strain JA480T.

C9:0 3OH, C10:0 2OH, C11:0, C11:0 iso 3OH, C14:0 3OH/ C16:1 iso I, C15:0 iso, C15:0
anteiso, C15:0 2OH, C17:0, C17:1 8c, C19:0 anteiso occurred in trace amounts
(<0.5 %).
140
Results
3.6.6 G+C content: The DNA G + C content of strain JA480T was 67.8
mol% (by HPLC).
(0.03%,
w/v)].
[anaerobic,
light
(2400
lux),
Na2S.9H2O or Na2S2O3.5H2O (1mM each) and NaHCO3 (0.1%, w/v)],

(0.1%, w/v)], chemo-organoheterotrophy [aerobic, dark, and pyruvate
(0.3%, w/v)] and fermentative growth [anaerobic, dark with glucose/
fructose/ pyruvate (0.3 %, w/v)] could not be demonstrated. Limited
number of substrates supported the growth of the strain (Table 3.6.1) as
carbon source/e- donors under photoorganoheterotrophic condition
which included acetate, pyruvate, valerate and succinate. Those which
could not be utilized included 2-oxoglutaric acid, glycerol, sorbitol,
glutamate, aspartate, caproate, peptone, casamino acids, glucose,
benzoate, fructose, sucrose, gluconate, methionine, aspartate, ascorbate,
thiogylcolate, butyrate, lactate, formate, propionate, fumerate, crotonate,
glycolate, tartarate, caprylate, mannitol, methanol and ethanol.
Sulfide is obligatory and used as sulfur source, while sulfate, sulfite,
thiosulfate, methionine, elemental sulfur and cysteine did not support
phototrophic growth of the strain (Table 3.6.2). Strain JA480T could be
grown with ammonium chloride and glutamine as nitrogen sources
while, molecular nitrogen, urea, glutamate, aspartate, nitrite and nitrate
141
Results
did not support phototrophic growth (Table 3.6.3). Biotin and p-amino
benzoic acid are required as growth factors (Table 3.6.4). There was no
requirement of NaCl for growth but the strain tolerates up to 0.5% NaCl
(Fig. 3.6.6). The pH range for the strain was 7.0-8.0 with an optimum at
7.5 (Fig. 3.6.7a). Optimum growth of the strain occurs at 25-30C while
the temperature range was 20-40 C (Fig. 3.6.7b).
JA480T has sequence similarities of 92.6, 92.1 and 91.6% with
Rhodospirillum
photometricum
DSM122T,
Rhodospirillum
rubrum
ATCC11170T and Rhodospirillum sulfurexigens JA143T respectively.

3.6.10
16S
rRNA
gene
sequence
deposition:
The
GenBank/EMBL/DDBJ accession number for the 16S rRNA gene

sequence of strain JA480T is FN 391894.
3.6.11 Culture deposition: Strain JA480T =KCTC 5960T= NBRC
107573T=ABRC-APB 59T.
142
Results
3.7 Polyphasic characterization of Rhodovulum sp. JA580T

3.7.1 Habitat: Sediment sample that yielded the strain JA580T was
collected from a brown pond, near the village Vethalai, Tamil Nadu, India
(GPS position: 0916 N 7906 E), on 27
th
May 2009. The sample had a
pH of 7.4, a salinity of 2 % (w/v, NaCl) and a temperature of 30C.

3.7.2 Cultural characteristics
3.7.2.1 Broth cultures: The color of the photosynthetically [anaerobic,
light 2,400 lux)] grown cultures was yellowish brown (Fig. 3.7.1a).
3.7.2.2 Colonial characteristics: Colonies of strain JA580T were round,
convex, smooth, medium sized and yellowish brown pigmented (Fig.
3.7.1b). Colony diameter reached up to 1mm after 4 days of incubation
at 30C.
T
3.7.3 Morphology and fine structure: Cells of strain JA580 are oval to
rod shaped (Fig. 3.7.1c), 1-1.8m long and 0.5-0.9 m wide. The cells
were non-motile and multiplied by binary fission. Transmission electron
microphotographs of ultrathin sections of the strain revealed vesicular
internal membrane structures (Fig. 3.7.2).
3.8.4 Pigment composition: The in vivo absorption spectrum of intact
cells in sucrose exhibited maxima at 380, 479, 590, 803 and 863 nm
(Fig. 3.7.3a) confirming the presence of bacteriochlorophyll a. The
303, 309, 348, 456 and 486nm (Fig. 3.7.3b) indicating the presence of
(a)
(b)
143
Results
(c)
Fig. 3.7.1 Broth culture (a), colony morphology (b) and phase
contrast microphotograph (c) of Rhodovulum sp. JA580T.
(a)
(b)
Fig. 3.7.2 Electron micrographs of ultrathin sections of
Rhodovulum sp. JA580T (a, b) showing the vesicular type
of the photosynthetic membranes.
144
Results
(a)
(b)
of extracted pigments (b) of Rhodovulum sp. JA580T.
OD660
0.3
0.2
0.1
0
0
0.5 1 1.5
2 2.5 3
3.5 4
Salinity (%)
0.4
0.3
0.2
0.1
0
OD660
OD660
Fig.3.7.4 Effect of salinity on the growth of Rhodovulum sp. JA580T.
15
20
25
30
35
40
O
Temperature ( C)
0.4
0.3
0.2
0.1
0
5 5.5 6 6.5 7 7.5 8 8.5 9
pH
(a)
(b)
Fig.3.7.5 Effect of pH (a) and temperature (b) on growth of Rhodovulum
sp. JA580T.
145
Results
carotenoids of the spheroidene series. Carotenoid composition of the

strain as assessed by HPLC showed the presence of spheroidene (56%),
demethylspheroidene (12%), hydroxyspheroidene (8%), spheroidenone
(17%), hydroxyspheroidenone (5%) and neurosporene (2%).
revealed that C
18:1
7c, C
18:0
,C
18:1
9c along with C
18:1
7c 11-methyl and
C16:0 are the major fatty acids. Minor proportions of C10:03OH, C12:0,
C
12:0
3OH, C
16:1
7c/C
16:1
6c, C
16:0
10-methyl, C
18:1
5c, C
19:0
cyclo 8c,
C20:17c were also detected (Table 3.7.5).

T
3.7.6 G+C content: The DNA G + C content of strain JA580 was 62.3
mol% (by HPLC).


(0.03% w/v)]. Chemo-organoheterotrophy [aerobic, dark, and pyruvate
(0.3%,
w/v)],
photolithoautotrophy
[anaerobic,
light
(2400
lux),
Na2S.9H2O or Na2S2O3.5H2O (1mM each) and NaHCO3 (0.1% w/v)],

(0.1% w/v)] and fermentative growth [anaerobic, dark with glucose/
fructose/pyruvate (0.3 % w/v)] could not be demonstrated. The
substrates which supported the growth of the strain (Table 3.7.1) as
carbon source/e- donors under photo-organoheterotrophic condition
included acetate, casamino acids, fumerate, glutamate, malate, peptone,
Carbon source/
e- donor
(concentration)
146
Results
Growth
O.D660 nm
+/ -
Control [without inorganic/

organic substrate]
0.03
NA
0.03
NA
Acetate (0.3%, w/v)
0.30
0.04
0.09
(+)
*Benzoate (1mM)
0.01
0.01
0.03
0.18
Citrate (0.3%, w/v)
0.04
0.03
Cysteine(0.3%, w/v)
0.045
Ethanol (0.1%, w/v)
0.14
(+)
Formate (0.3%, w/v)
0.03
0.06
0.34
0.29
Glucose (0.3%, w/v)
0.07
0.13
(+)
0.04
Lactate (0.3%, w/v)
0.02
Malate (0.3%, w/v)
0.25
0.08
0.10
(+)
0.25
0.04
147
Results
0.01
0.45
0.13
(+)
0. 43
Sucrose (0.3%, w/v)
0.26
0.12
(+)
0.04
*H2 (20%, v/v gas phase)
0.01
*Na2S.9H2O (0.5 mM)
0.04
*Sulfur (0.05%)
0.03
*Thiosulfate (5 mM)
0.03
*Sulfite (5 mM)
0.01
Table 3.7.1 Carbon source/e-donor utilization by Rhodovulum

sp. JA580T.
Substrates were provided by replacing pyruvate in the single carbon
medium (Table 2.3). Experiment was performed in screw capped tubes
and the results expressed are average values of an experiment done in
triplicates after 72 h of light (2,400 lux) anaerobic incubation at 30+2C.
Symbols: + = Growth present; - = No growth; (+) = Weak growth; NA = Not
applicable.
*= 0.1% (w/v) Sodium bicarbonate was supplemented.
Sulfur sources
(concentration)
148
Results
Growth
O.D660 nm
+/ -
0.01
NA
0.02
0.04
Methionine (2mM)
0.00
0.04
0.02
0.18
Sulfur (2 mM)
0.03
0.23
Control
[without
source]
Cysteine (2 mM)
sulfur
Table 3.7.2 Growth of Rhodovulum sp. JA580T on various sulfur

sources.
indicated.
Nitrogen sources
(0.068%, w/v)
Growth
O.D660 nm
0.18
+/ NA
0.30
Aspartate
0. 01
Glutamate
0.30
Glutamine
0.31
Sodium nitrate
0.25
Sodium nitrite
0.02
Urea
0.05
0.17
Control [without nitrogen

source]
Ammonium chloride
Table 3.7.3 Growth of Rhodovulum sp. JA580T on various

nitrogen sources.
(0.3%, w/v) was used as carbon source and nitrogen source varied
as indicated.
149
Vitamins
Results
Growth
O.D660 nm
0.05
+/ NA
0.03
*Without Thiamine
0.08
*Without Riboflavin
0.02
*Without Niacin
0 07
0.05
*Without
0.01
0.02
*Without Biotin
0.02
*Without p-Amino benzoic

acid
Yeast extract
0.04
0.34
Control [without
source]
All vitamins
vitamin
Pyridoxal
phosphate
Table 3.7.4 Vitamin requirement by Rhodovulum sp. JA580T.

(0.3%, w/v) was used as carbon source and yeast extract was replaced
with vitamin solution as indicated. Concentration of the vitamins used
as given in section 2.19.5.
Fatty acid
Major fatty acids
C16:0
C18:0
C18:17c 11-methyl
C18:19c
C18:17c
Minor fatty acids
C10:0 3OH
C12:0
C12:0 3OH
C16:0 10-methyl
C16:1 7c/ C16:1 6c
C18:15c
C19:0 cyclo 8c
Composition (%)
9.0
15.7
10.0
14.2
21.0
2.7
1.8
2.0
1.3
3.6
4.8
4.4
Table 3.7.5 Cellular fatty acid profile of Rhodovulum sp. JA580T.
150
Results
pyruvate, succinate and sucrose. Those which could not be utilized

included ascorbate, 2-oxoglutaric acid, caproate, crotonate, cysteine,
glucose, fructose, formate, benzoate, butyrate, citrate, aspartate, lactate,
valerate, mannitol, methionine, proponal and propionate.
Sulfide or thiosulfate is obligatory for growth and were used as sulfur
sources, while sulfite, thiogylcolate, methionine and cysteine did not
support phototrophic growth of the strain (Table 3.7.2). Strain JA580
could utilize ammonium chloride, nitrate, glutamate and glutamine as

nitrogen sources while nitrite, aspartate, aspargine and dinitrogen did
not support the growth (Table 3.7.3). The strain required complex growth
factors for growth (Table 3.7.4). There was no requirement of salt (NaCl)
T
for the growth of strain JA580 [salinity range and optimum were from 0T
3 % and 1-1.5% (w/v) NaCl respectively] (Fig. 3.7.4). Strain JA580 grew
at a pH range of 6.58.0 (optimum 7.07.5) and temperature of 2535C
(optimum 30C) (Fig. 3.7.5a, b).
sequence similarity of strain JA580T with the nearest type strains was
JA580T has a sequence similarity of 96.3, 96.3, 96.2, 96.2, 95.9, 95.9%
with Rhodovulum adriaticum DSM 2781T, Rhodovulum iodosum N1T,
Rhodovulum
robiginosum
N2T,
Rhodovulum
steppense
A-20sT,
151
Results
Rhodovulum sulfidophilum DSM 1374T, Rhodovulum visakhapatnamense

JA181T, respectively.
3.7.9 16S rRNA gene sequence deposition: The GenBank/EMBL/DDBJ
accession number for the 16S rRNA gene sequence of strain JA580T is
FN669139.
3.7.10 Culture deposition: Strain JA580T =NBRC 107612T =KCTC
T
5963 = ABRC APB 62T.
152
Results
3.8 Plant growth promoting attributes of purple

phototrophic bacteria of paddy fields.
In order to select purple anoxygenic phototrophic bacteria that could
improve plant growth and thereby agricultural yield, several strains of
PNSB isolated from rhizosphere soils of various paddy fields were
screened for their plant growth promoting potentials, viz. diazotrophic
growth and acetylene reduction activity (nitrogenase); capability to
release soluble phosphates from their insoluble forms (phosphate
solubilization); excrete phytohormones such as indole-3-acetic acid and
limit the growth and proliferation of other microbes (antimicrobial
activity).
3.8.1 Diazotrophic growth and nitrogenase activity of PNSB strains:
In the present study, 68 purple non sulfur bacterial strains isolated from
various paddy fields were tested for their ability to grow diazotrophically
using nitrogen gas as a sole nitrogen source. Among them, 11 strains
(JA354, JA420, JA477, JA478, JA667, JA703, JA520, JA559, JA670,
JA671 and JA731) did not show any diazotrophic growth. The activity of
the nitrogenase enzyme in the remaining diazotrophically grown strains
(57), which belonged to 7 genera (Rhodopseudomonas, Rhodoplanes,
Rhodobacter, Rubrivivax, Rhodospirillum, Phaeospirillum and Rhodocista),
was tested by acetylene reduction activity (ARA). The activity of these
strains varied from 9 to 837 moles C2H4.mg BChl-1.h-1. High ARAs of
837,
630,
588,
826
and
616
moles
C2H4.mg
BChl-1.h-1
153
S.
No
Strain
No.
Diazotrophy
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
JA652
JA678
JA356
JA665
JA669
JA666
JA477
JA653
JA556
JA557
JA569
JA658
JA660
JA664
JA558
JA657
JA635
JA659
JA676
JA718
JA705
JA662
JA668
JA670
JA673
JA697
JA731
JA733
JA730
JA700
JA735
JA675
JA661
JA631
JA632
JA734
JA522
JA420
JA667
JA478
JA520
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Results
Nitrogenase
activity
(moles C2H4. mg BChl-1.
h-1)
588
509
630
837
826
277
529
27
17
19
165
616
330
90
173
102
506
31
9
51
165
16
126
50
256
357
355
505
505
125
108
65
146
146
-
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
JA559
JA354
JA703
JA728
JA318T
JA626
JA729
JA524
JA680
JA352
JA671
JA696
JA415T
JA630
JA521
JA521
JA701
JA353
JA702
JA570
JA679
JA716
JA452
JA145
JA317T
JA629
JA559
154
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Results
70
28
46
68
11
33
28
41
27
40
55
42
29
29
60
68
81
59
106
56
29
65
153
Table 3.8.1 Acetylene reduction activity (ARA) of

purple non sulfur bacteria.
+, presence; -, absence
155
Results
were exhibited by the strains of genera Rhodopseudomonas (JA665,

JA356, and JA652) and Rhodobacter (JA669, JA660), respectively (Table
3.8.1).
3.8.2 Phosphate solubilization activity of PNSB strains: The potential
of PNSB strains to solubilize precipitated phosphates such as triclacium

phosphate (TCP) was assessed both qualitatively and quantitatively.
3.8.2.1 Qualitative tests: Eight strains JA356, JA652, JA661, JA662,
JA678, JA702, JA735 (of genus Rhodopseudomonas) and JA352 (of

genus Rhodobacter) were screened for their phosphate solubilization
capabilities by plate and broth assays. In the plate assay performed
using the bromophenol blue indicator dye, only 2 strains (JA652, JA661)
gave positive result with a visible yellow zone surrounding the colonies.
However, in liquid medium 7 strains (except for JA352) could grow using
tri calcium phosphate (TCP) as a sole source of phosphorous (Table
3.8.2), for several subcultures.
3.8.2.2 Quantitative test: The amount of phosphate solubilized by the
7 phosphate solubilizing PNSB was studied by constructing a standard

graph.
While
the
strain
JA652
showed
the
highest
phosphate
solubilization activity (16.2 ppm), the amount of soluble phosphates

discharged by the other strains JA356, JA661,JA662, JA678, JA702 and
JA735 was 8.4, 10.4, 10.8, 8.4, 8.8 and 10.8 ppm respectively (Table
3.8.2). However, the solubilization of phosphates by the strains was not
S.
No
Strain
No.
156
Qualitative tests
Plate
assay
Broth assay
Test (TCP
Positive
Negative
as sole P
control
control
source)
(+KH2PO4) (-KH2PO4)
+
+
-
JA356
JA735
JA702
JA661
JA678
Results
*Quantitative test
Amount of soluble
phosphates in the
uninoculated test = 0.8
ppm
Final
Phosphate
pH
solubilized (ppm)
[Final-initial ]
(Initial
pH 7.0)
7.0
8.4
7.4
10.8
7.4
8.8
5.0
10.4
7.0
8.4
JA662
8.0
10.8
JA652
5.5
16.2
JA352
NA
NA
Table 3.8.2 Phosphate solubilization activity of a few strains of purple

non sulfur bacteria.
-, negative; +, positive; NA, not applicable; TCP, triclacium phosphate.
* Solubilized phosphates were estimated by the method of Olsen & Sommers, 1982.
Decline in the pH of the medium, associated with organic acid production.
(a)
(b)
Fig. 3.8.1 Bioassay plates (a), (b) showing clearing zones
indicated by arrows) around the discs.
157
Results
accompanied with decline in pH of the medium except for the strains

JA652 and JA661, where the pH dropped to 5.0-5.5.
3.8.3 Antimicrobial activity of PNSB strains: Culture supernatants of
29 PNSB strains of the genera Rhodopseudomonas, Rhodobacter,

Rhodoplanes and Rubrivivax were screened for their antimicrobial
activities against 6 indicator strains, by agar disc diffusion method (3
Gram negative and 3 Gram positive bacteria were used as indicators).
While 69% (20 strains) of the strains tested showed inhibitory activity
against at least one indicator organism, the remaining strains did not
produce any zones of inhibition. The pH of the culture supernatants was
between 5.0-8.0. The antibacterial spectrum of all the strains showing
inhibitory
activity
is
presented
in
the
Table
3.8.3.
Among
the
antibacterial metabolite producing strains, 10% strains showed activity

against both Gram positive and Gram negative indicators, 52% strains
could limit the growth of only Gram negative bacteria (E. coli JC113,
Pseudomonas sp. JC115, Shewanella sp. JC5T) and 4% strains inhibited
the growth of Gram positive bacteria (Staphylococcus aureus JC114).
Majority of the strains were shown to inhibit the growth of Pseudomonas
sp. JC115 and Shewanella sp. JC5T. Zones of inhibition (Fig. 3.8.1)
produced by the strains measured between 5 to 13mm.

S.
Produ
No. cer
strain
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
pH of
culture
supern
atants
JA556
JA671
JA705
JA679
JA415
JA356
JA635
JA558
JA652
JA678
JA662
JA522
JA632
JA731
JA661
JA701
JA700
JA631
JA626
JA730
JA731
JA675
JA735
JA696
JA733
JA680
JA697
JA666
JA702
Table 3.8.3
158
Results
Diameter (mm) of inhibition zone formed against indicator

organisms
Gram negative indicators
Gram positive indicators
E. coli
JC113
Shewanel
la sp.
JC5T
Pseudomona
s sp. JC115
6.5
7.0
7.0
5.5
7.4
7.0
7.4
8.0
5.5
11
7.0
5
8.0
5.5
5
6.0
8
7.4
7.0
7.0
7.0
7.4
5
7.0
10
6.5
6.5
8
7.4
13
7.4
13
6.0
12
7.4
11
6.5
11
7.0
7.0
11
7.4
12
Antimicrobial activitiy of
bacteria.
Staphylococcu
s aureus
JC114
Lactobacillus
sp. JC116
Bacillus
subtilis
JC3
11
8
12
12
10
12
9
8
12
9
8
a few strains of purple non sulfur
8
-
159
Results
3.8.4 Indole-3-Acetic Acid (IAA) production by PNSB strains:
29 strains belonging to the genera Rhodopseudomonas, Rhodobacter,

Rhodoplanes and Rubrivivax were screened for their ability to produce
the plant growth regulator, IAA. Screening was done by growing each of
the strains with varying concentrations of tryptophan, i.e. 0, 2, 5mM.
While only 3 strains showed IAA production in the absence of
tryptophan, a large number of strains produced IAA in the presence of 2
(24 strains) and 5mM (26 strains) of tryptophan (Table 3.8.4). While the
strains JA735, JA731, JA626 showed remarkably high IAA production
(OD530nm> 0.45), 3 strains did not produce any detectable IAA.
In summary, 5 PNSB strains (JA735, JA652, JA702, JA662 and
JA678) exhibited all the four traits tested for, while the strains JA356,
JA661 and JA731 exhibited three of the traits tested. At least 11 strains
exhibited more than one type of plant growth promoting activities tested.
S.No. Strain
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
JA556
JA671
JA705
JA679
JA415
JA356
JA635
JA558
JA652
JA678
JA662
JA522
JA632
JA731
JA661
JA701
JA700
JA631
JA626
JA730
JA731
JA675
JA735
JA696
JA733
JA680
JA697
JA666
JA702
160
Results
*IAA production at different concentration of

tryptophan (mM)
0
2
5
+
+
+
++
(+)
+
++
+
+
++
++
++
+
++
+++
+
++
++
(+)
(+)
++
++
++
++
++
+
+++
++
+++
(+)
++
+++
++++
++++
+
++
++++
+
+
++++
++++
(+)
(+)
++++
+++
(+)
(+)
++
++
+
++
++
+++
Table 3.8.4 IAA production by purple non sulfur bacteria.

-, negative; (+), O.D 0.01- 0.10; +, O.D 0.11- 0.20; ++, O.D>0.21- 0.30; +++, O.D
0.31-0.40; ++++, O.D> 0.41.
* IAA production was detected by the method of Loper & Schroth, 1986.
The optical density of the pink colored complex formed was measured at 530nm
colorimetrically.

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Lakshmi KVNS, Ph.

Lakshmi KVNS, Ph.D Thesis, 2012

3.1 Enrichment, isolation, rapid typing and tentative

Lakshmi KVNS, Ph.D Thesis, 2012

Lakshmi KVNS, Ph.D Thesis, 2012

Fig.3.1.1 Identification of purple bacteria by a rapid typing procedure.

organotrophically under anaerobic conditions showed eight different

Lakshmi KVNS, Ph.D Thesis, 2012

morphologies: medium, round, convex, smooth colonies; medium, round,

Lakshmi KVNS, Ph.D Thesis, 2012

All 33 PSB strains were rod shaped, motile, multiplied by binary

characterization of the 123 strains, in terms of morphological and

Lakshmi KVNS, Ph.D Thesis, 2012

Lakshmi KVNS, Ph.D Thesis, 2012

Enrichments of purple bacteria

Samples analyzed from rhizosphere soils of paddy fields

Lakshmi KVNS, Ph.D Thesis, 2012

Lakshmi KVNS, Ph.D Thesis, 2012

Lakshmi KVNS, Ph.D Thesis, 2012

Rice field nea

Lakshmi KVNS, Ph.D Thesis, 2012

Lakshmi KVNS, Ph.D Thesis, 2012

16S rRNA gene sequence

Purple bacterial strains obtained from rhizosphere soils of paddy fields

JA318 T R, Cv, Sm, M, RB

vibrioid-spiral, 0.8-1.2 x 2.0-6.0 m,

oval-rod, chains, non motile, binary

spiral, chains, motile, binary fission

rods, motile, budding

oval-rod, non motile, binary fission

rods, rosettes, motile, budding

oval-rod, chains, non motile, binary

rods, motile, budding

spiral, chains, sp, motile, binary fission

oval-rod, motile, binary fission

Lakshmi KVNS, Ph.D Thesis, 2012

rods, motile, budding

100- Rhodobacter johrii JA192T

oval, 0.9-1.2 x 1.0-2.0m, motile,

JA415 T R, Cv, Sm, M, RB

rods, rosettes, motile, budding

rods, rosettes, associate to form T, V & Y

95.8- Rhodoplanes elegans

vibrio-spiral, 1-2 x 3.0-5.0m, motile,

99.7- Rhodocista pekingenesis

Lakshmi KVNS, Ph.D Thesis, 2012

R, Cv, Sm, L, P-RB

R, Cv, Sm, M, PRB

rods, 0.6-0.9 x 2.0-2.5m, motile,

100- Rhodopseudomonas faecalis

Lakshmi KVNS, Ph.D Thesis, 2012

oval, nonmotile, binary fission

rods, rosettes, motile, budding

rods,0.4-0.6 x 2.5-3.5m, motile,

99- Rhodopseudomonas faecalis

oval, chains, motile, binary fission

oval-rod, chains, motile, binary fission

rods, rosettes, motile, budding

oval-rod, chains, motile, binary fission

rods, rosettes, motile, budding

rods, rosettes, motile, budding

oval-rod, chains, non motile, binary

rods, motile, budding

rods, chains, rosettes,associate to form