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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).

Lakshmi KVNS, Ph.D Thesis, 2012

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).

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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).

Lakshmi KVNS, Ph.D Thesis, 2012

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
-

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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,
chains, motile, binary fission
vibrioid-spiral, 0.2-0.5 x 3-10m,
chains, motile, binary fission
rods, rosettes, motile, budding

98.2- Phaeospirillum
chandramohanii JA145T
99.8- Rhodospirillum
sulfurexigens JA143T
99.8- Rhodospirillum
sulfurexigens JA143T
100- Rhodospirillum
sulfurexigens JA143T

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

Rhodopseudomonas sp.

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,

rods, rosettes, motile, budding

Rhodopseudomonas 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

oval-rod, chains, non motile, binary


fission
rods, motile, budding

Rhodopseudomonas sp.

Rhodopseudomonas sp.

A/V3

JA662

R, Cv, Sm, S, R

Bchl a, Sp

rods, motile, budding

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.

Lakshmi KVNS, Ph.D Thesis, 2012

92

Results

A/V

JA665

R, Cv, Sm, S, RB

Bchl a, Sp

rods, motile, budding

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

rods, rosettes, motile, budding


oval-rod, chains, non motile, binary
fission
rods, rosettes, motile, budding
rods, motile, budding
oval-rod, chains, non motile, binary
fission
rods, rosettes, motile, budding
rods, rosettes, 0.4-0.6 x 1.5-2.0m,
motile, budding
rods, rosettes, motile, budding
oval-rod, chains,0.8-1.4 x 1.0-3.0m,
non motile, binary fission

Rhodopseudomonas sp.

FN296207
FN568343
-

Rhodopseudomonas sp.
Rhodobacter sp.

Rhodopseudomonas sp.
Rhodopseudomonas sp.

Rhodobacter sp.

Rhodopseudomonas sp.
99.5- Rhodopseudomonas
faecalis JCM 11668T

Rhodopseudomonas sp.
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
motile, binary fission

99.7- Rhodocista pekingenesis


JCM11669T
98.4- Phaeospirillum fulvum
NCIMB 11762T
99.7- Rhodobacter maris JA276 T

AM999778

AM999777

AM999776
FN296202

Lakshmi KVNS, Ph.D Thesis, 2012

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,
motile, binary fission
rods, rosettes, motile, budding
oval- rods, 0.41.5 x 1.04.0m, non
motile, binary fission
oval, 0.9-1.4 x 1.0-2.0m, non motile,
binary fission
spiral, chains, motile, binary fission
rods, rosettes, motile, budding
oval-rod, chains, non motile, binary
fission
spiral, chains, motile, binary fission
rods, chains, rosettes, associate to form
T,V & Y shape, motile, binary fission
rods, motile, budding
rods, rosettes, motile, budding
oval-rod, non motile, binary fission
rods, rosettes, motile, budding
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

100- Rhodopseudomonas faecalis


JCM 11668T
100- Rhodopseudomonas faecalis
JCM 11668T
99.6- Rhodospirillum
photometricum DSM 122T
99.4- Rhodovulum
visakhapatnamense JA181T
99- Rhodopseudomonas faecalis
JCM 11668T
98.6- Phaeospirillum
chandramohanii JA145T

Rhodopseudomonas sp.
98.6- Rhodobacter megalophilus
JA194T
100- Rhodobacter megalophilus
JA194T

Phaeospirillum sp.

Rhodopseudomonas sp.

Rhodobacter sp.

Rhodospirillum sp.
Rhodoplanes sp.

Rhodopseudomonas sp.
Rhodopseudomonas sp.

Rhodobacter sp.

Rhodopseudomonas sp.
99.1- Rhodopseudomonas
faecalis JCM 11668T

Phaeospirillum sp.
98.3-Phaeospirillum
molischianum DSM120T

FN546953
FN546954
FN547821
FN568342
FN813495
FN813496
FN813493
FN813494
FN813499
FN813501

Lakshmi KVNS, Ph.D Thesis, 2012

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

rods, rosettes, motile, budding

Rhodopseudomonas 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

99- Rhodopseudomonas faecalis


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

oval-rod, chains, motile, binary fission

Rhodobacter sp.

1E

JA676

R, Cv, Sm, M, R

Bchl a, Sp

rods, rosettes, motile, budding

Rhodopseudomonas sp.

EKRb

JA677

R, Cv, Sl, M, YB

BChl a, Se/Sn

oval-rod, chains, motile, binary fission

Rhodobacter sp.

WA/H

JA678

R, Cv, Sm, S, RB

Bchl a, Sp

rods, rosettes, motile, budding

Rhodopseudomonas sp.

EQT

JA679

R, Cv, Sm, M, RB

Bchl a, Sp

rods, rosettes, motile, budding

Rhodopseudomonas sp.

RA1

JA734

R, Cv, Sm, M, YB

BChl a, Se/Sn

Rhodobacter sp.

BK29

JA732

R, Cv, Sm, M, RB

Bchl a, Sp

oval-rod, chains, non motile, binary


fission
rods, motile, budding

Rhodopseudomonas sp.

8BK

JA680

R, Cv, Sm, M, RB

Bchl a, Sp

rods, motile, budding

Rhodopseudomonas 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

spiral, chains, motile, binary fission

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

oval-rod, chains, non motile, binary


fission
curved rods, motile, binary fission

Rubrivivax sp.

BRb

JA701

R, Cv, Sl, M, YB

BChl a, Se/Sn

oval-rod, chains, non motile, binary


fission

Rhodobacter sp.

FN813500
-

Lakshmi KVNS, Ph.D Thesis, 2012

95

Results

Rp1

JA702

R, Cv, Sm, S, RB

Bchl a, Sp

rods, rosettes, motile, budding

Rhodopseudomonas sp.

Rp2

JA705

R, Cv, Sm, S, RB

Bchl a, Sp,

rods, motile, budding

Rhodopseudomonas sp.

VRb

JA706

R, Cv, Sl, M, YB

BChl a, Se/Sn

Rhodobacter sp.

V6P

JA707

R, Cv, Sm, M, B

BChl a, Rp

oval-rod, chains, non motile, binary


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

99.7- Rhodobacter capsulatus


ATCC 11166T
99.8- Rhodobacter capsulatus
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,
motile, binary fission
curved rods, motile, binary fission

99.5- Rubrivivax benzoatilyticus


HE800189
T
JA2
99.8- Rubrivivax gelatinosus JCM HE582621
21318T

Rubrivivax sp.
-

K4Ru

JA354

R, Cv, Sm, St, M, B BChl a, Se

K5Ru

JA656

Y6Ru

JA667

R, Cv, Sm, St, M, B BChl a, Se

curved rods, motile, binary fission

Rubrivivax sp.

C7Ru

JA672

R, Cv, Sm, St, S, B

BChl a, Se

curved rods, motile, binary fission

Rubrivivax sp.

2NRu

JA626

R, Cv, Sm, St, M, B BChl a, Se

5N

JA627

R, Cv, Sm, St, M, B BChl a, Se

8Ru

JA703

R, Cv, Sm, St, M, B BChl a, Se

curved rods, chains, 0.2-0.5 x 2.06.0m, motile, binary fission


curved rods, chains, 0.1-0.3 x 2.06.0m, motile, binary fission
curved rods, motile, binary fission

100- Rubrivivax gelatinosus JCM FN813497


21318T
99.5-Rubrivivax benzoatilyticus
FN813498
T
JA2

Rubrivivax sp.
-

32B

JA524

R, Cv, Sm, St, M, B BChl a, Se

curved rods, chains, 0.1-0.2 x 3.07.0m, motile, binary fission

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

Lakshmi KVNS, Ph.D Thesis, 2012

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

99.3- Allochromatium vinosum


ATCC 17899T

Allochromatium sp.

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

JA691

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

KIPs2

JA692

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

KIIPs1

JA693

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

KIIPs2

JA694

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

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

oval- rods, motile, binary fission, So

Allochromatium sp.

AIPs1

JA686

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

AIPs2

JA687

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

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

oval- rods, 2.0 x 2.0-5.0m, motile,


binary fission, So

AIIPs2

JA689

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

99.4-Allochromatium vinosum
ATCC 17899T; Allochromatium
minutissimum DSM 1376T

Allochromatium sp.

AIIIPs

JA690

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

ChPs

JA684

R, Cv, Sm, M RB

BChl a, Ly

oval- rods, motile, binary fission, So

Allochromatium sp.

CIPs

JA683

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, motile, binary fission, So

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

oval- rods, motile, binary fission, So


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

Allochromatium sp.
Allochromatium sp.

Allochromatium sp.

FN813504
-

FN813505

Lakshmi KVNS, Ph.D Thesis, 2012

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

oval- rods, motile, binary fission, So

L1

JA383

R, Cv, Sm, M, RB

BChl a, Ly

oval- rods, 2.5 x 2.5-4.0m, motile,


binary fission, So

99.4- Allochromatium vinosum


ATCC 17899T; Allochromatium
minutissimum DSM 1376T

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
motile, binary fission

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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).

Lakshmi KVNS, Ph.D Thesis, 2012

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).

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012


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)

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012


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.

Lakshmi KVNS, Ph.D Thesis, 2012


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.


Experimental details as given in Table 3.3.1, 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.
*= 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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012


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.

Lakshmi KVNS, Ph.D Thesis, 2012

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 Cultural characteristics:
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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

Carbon source/
e- donor
(concentration)

116

Results

Growth
O.D660 nm

+/ -

Control
[without
inorganic/
organic substrate]
Control [NaHCO3 (0.1%, w/v)]

0.03

NA

0.03

NA

Acetate (0.3%, w/v)

0.37

Ascorbate(0.3%, w/v)

0.05

Aspartate(0.3%, w/v)

0.09

*Benzoate (1mM)

0.03

*Butyrate (0.1%, w/v)

0.16

*Caproate (0.1%, w/v)

0.03

Casamino acids (0.05%, w/v)

0.18

Citrate (0.3%, w/v)

0.20

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)

0.05

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)

0.26

Malate (0.3%, w/v)

0.25

Mannitol (0.3%, w/v)

0.08

Methionine (0.3%, w/v)

0.07

Methanol (0.1%, w/v)

0.04

Peptone (0.05%, w/v)

0.05

Lakshmi KVNS, Ph.D Thesis, 2012

117

Results

Propanol (0.1%, w/v)

0.04

*Propionate (0.1%, w/v)

0.15

Pyruvate (0.3%, w/v)

0.45

Sorbitol (0.3%, w/v)

0.03

Succinate (0.3%, w/v)

0.44

Sucrose (0.3%, w/v)

0.06

Tartarate (0.3%, w/v)

0.03

Thioglycolate (0.3%, w/v)

0.03

*Valerate (0.1%, w/v)

0.04

* H2 (20%, v/v gas phase)

0.05

* Na2S.9H2O (0.5 mM)

0.03

* Sulfur (0.05%)

0.03

* Thiosulfate (5 mM)

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

Lakshmi KVNS, Ph.D Thesis, 2012

Sulfur sources
(concentration)

118

Results

Growth
O.D660 nm
0.18

+/ NA

Cysteine (2 mM)

0.15

Magnesium sulfate (5 mM)

0.30

Sodium sulfate (5 mM)

0.24

Sodium sulfite (5 mM)

0.31

Sodium sulfide (1 mM)

0.20

Sulfur (2 mM)

0.20

Sodium thiosulfate (5 mM)

0.35

Sodium thioglycolate (5 mM)

0.17

Control [without sulfur source]

Table 3.4.2 Growth of Rhodoplanes sp. JA415T on various sulfur


sources.
Experimental details as given in Table 3.4.1, except that pyruvate
(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

N2 [100% (v/v) gas phase]

0.16

Control

[without

nitrogen

source]

Table 3.4.3 Growth of Rhodoplanes sp. JA415T on various nitrogen


sources.
Experimental details as given in Table 3.4.1, except that pyruvate
(0.3%, w/v) was used as carbon source and nitrogen sources varied as
indicated.

Lakshmi KVNS, Ph.D Thesis, 2012

119

Results

Vitamins

Growth
O.D660 nm
0.05

+/ NA

0.30

*Without Thiamine

0.28

*Without Riboflavin

0.37

*Without Niacin

*Without Pantothenate

*Without
Pyridoxal
phosphate
*Without Vitamin B12

0.35

0.37

*Without Biotin

0.31

0.65

Control
[without
source]
All vitamins

vitamin

* Without p-Amino benzoic


acid
Yeast extract

Table 3.4.4 Vitamin requirement by Rhodoplanes sp. JA415T.


Experimental details as given in Table 3.4.1, 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. *= 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.

Lakshmi KVNS, Ph.D Thesis, 2012

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).
3.4.7 Physiological characteristics: Strain JA415T was able to grow
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,

Lakshmi KVNS, Ph.D Thesis, 2012

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).
3.4.8 16S rRNA gene sequence similarity: The pairwise 16S rRNA gene
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

(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.

Lakshmi KVNS, Ph.D Thesis, 2012

124

Results

(a)
(b)
Fig. 3.5.3 Whole-cell absorption spectrum (a) and acetone spectrum
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.

Lakshmi KVNS, Ph.D Thesis, 2012

125

Results

(Fig. 3.5.3a) confirming the presence of bacteriochlorophyll a and the


absorption spectrum for pigments extracted with acetone gave maxima at
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).
3.5.7 Physiological characteristics: Strain JA318T 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, 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,

Lakshmi KVNS, Ph.D Thesis, 2012

Carbon source/
e- donor
(concentration)

126

Results

Growth
O.D660 nm

+/ -

Control
[without
inorganic/
organic substrate]
Control [NaHCO3 (0.1%, w/v)]

0.02

NA

0.02

NA

Acetate (0.3%, w/v)

0.34

Ascorbate(0.3%, w/v)

0.05

Aspartate(0.3%, w/v)

0.09

*Benzoate (1mM)

0.03

*Butyrate (0.1%, w/v)

0.25

*Caproate (0.1%, w/v)

0.03

Casamino acids (0.3%, w/v)

0.08

Citrate (0.3%, w/v)

0.01

Crotonate (0.1%, w/v)

0.23

Cysteine(0.3%, w/v)

0.05

Ethanol (0.1%, w/v)

0.27

Formate (0.3%, w/v)

0.22

Fructose (0.3%, w/v)

0.09

Fumarate (0.3%, w/v)

0.32

Glutamate (0.3%, w/v)

0.14

(+)

Glucose (0.3%, w/v)

0.12

(+)

*Glycerol (0.3%, w/v)

0.03

2-oxo glutarate (0.3%, w/v)

0.24

Lactate (0.3%, w/v)

0.26

Malate (0.3%, w/v)

0.25

Mannitol (0.3%, w/v)

0.28

Methanol (0.1%, w/v)

0.14

(+)

Peptone (0.05%, w/v)

0.06

Methionine (0.3%, w/v)

Lakshmi KVNS, Ph.D Thesis, 2012

127

Results

Propanol (0.1%, w/v)

0.05

*Propionate (0.1%, w/v)

0.07

Pyruvate (0.3%, w/v)

0.43

Sorbitol (0.3%, w/v)

0.04

Succinate (0.3%, w/v)

0.34

Sucrose (0.3%, w/v)

0.07

Starch (0.3%, w/v)

0.01

Tartarate (0.3%, w/v)

0.29

Thioglycolate (0.3%, w/v)

0.05

*Valerate (0.1%, w/v)

0.22

* H2 (20%, v/v gas phase)

0.03

* Na2S.9H2O (0.5 mM)

0.02

* Sulfur (0.05%)

0.02

* Thiosulfate (5 mM)

0.02

* Sulfite (5 mM)

0.02

Table 3.5.1 Carbon source/e-donor utilization by Rhodospirillum


sp. JA318T.
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; - = No growth; (+) =
Weak growth; NA = Not applicable.
* = 0.1% (w/v) Sodium bicarbonate was supplemented

Lakshmi KVNS, Ph.D Thesis, 2012

Sulfur sources
(concentration)

128

Results

Growth
O.D660 nm

+/ -

Control [without sulfur source]

0.04

NA

Cysteine (2 mM)

0.25

Magnesium sulfate (5 mM)

0.29

Methionine (2mM)

0.22

Sodium sulfate (5 mM)

0.23

Sodium sulfite (5 mM)

0. 02

Sodium sulfide (1 mM)

0.69

Sulfur (2 mM)

0.04

Sodium thiosulfate (5 mM)

0.59

Sodium thioglycolate (5 mM)

0.30

Table 3.5.2 Growth of Rhodospirillum sp. JA318T on various sulfur


sources.
Experimental details as given in Table 3.5.1, except that pyruvate
(0.3%, w/v) was used as carbon source and sulfur source varied as
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

N2 [100% (v/v) gas phase]

0.17

Control [without nitrogen


source]
Ammonium chloride

Table 3.5.3 Growth of Rhodospirillum sp. JA318T on various


nitrogen sources.
Experimental details as given in Table 3.5.1, except that pyruvate (0.3%,
w/v) was used as carbon source and nitrogen source varied as indicated.

Lakshmi KVNS, Ph.D Thesis, 2012

129

Vitamins

Results

Growth
O.D660 nm
0.02

+/ NA

0.25

*Without Thiamine

0.04

*Without Riboflavin

0.22

*Without Niacin

0.07

*Without Pantothenate

0.45

*Without
Pyridoxal
phosphate
*Without Vitamin B12

0.19

0.22

*Without Biotin

0.24

* Without p-Amino benzoic


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.
*= other 7 vitamins were added; = all 8 vitamins were added.
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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).
3.5.8 16S rRNA gene sequence similarity: The pairwise 16S rRNA gene
sequence similarities of strain JA318T with the nearest type strains were
found using EzTaxon server. The data obtained revealed that strain
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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 Cultural characteristics:
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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

(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.

Lakshmi KVNS, Ph.D Thesis, 2012

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.
3.6.5 FAME analysis: Whole-cell fatty acid analysis of the strain JA480T
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Ascorbate(0.3%, w/v)

0.03

Aspartate(0.3%, w/v)

0.02

*Benzoate (1mM)

*Butyrate (0.1%, w/v)

*Caproate (0.1%, w/v)

0.04

Casamino acids (0.3%, w/v)

0.01

Citrate (0.3%, w/v)

0.01

Crotonate (0.1%, w/v)

0.03

Cysteine(0.3%, w/v)

0.01

Ethanol (0.1%, w/v)

Formate (0.3%, w/v)

0.03

Fructose (0.3%, w/v)

0.02

Fumarate (0.3%, w/v)

0.01

Glutamate (0.3%, w/v)

0.02

Glucose (0.3%, w/v)

0.01

*Glycerol (0.3%, w/v)

0.03

2-oxo glutarate (0.3%, w/v)

Lactate (0.3%, w/v)

Malate (0.3%, w/v)

0.10

(+)

Mannitol (0.3%, w/v)

0.02

Methanol (0.1%, w/v)

0.01

Peptone (0.05%, w/v)

0.02

Control [without
organic substrate]

inorganic/

Methionine (0.3%, w/v)

Lakshmi KVNS, Ph.D Thesis, 2012

Propanol (0.1%, w/v)

137

Results

0.01

Pyruvate (0.3%, w/v)

0.23

Sorbitol (0.3%, w/v)

0.03

Succinate (0.3%, w/v)

0.25

Sucrose (0.3%, w/v)

0.04

0.01

*Valerate (0.1%, w/v)

0.14

* H2 (20%, v/v gas phase)

0.01

* Na2S.9H2O (0.5 mM)

0.02

* Sulfur (0.05%)

0.01

* Thiosulfate (5 mM)

0.02

* Sulfite (5 mM)

0.01

*Propionate (0.1%, w/v)

Starch (0.3%, w/v)


Tartarate (0.3%, w/v)
Thioglycolate (0.3%, w/v)

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


Substrates were provided by replacing pyruvate, succinate, acetate in the
triple carbon medium (Table 2.4). 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; - = No growth; (+) =
Weak growth; NA = Not applicable.
* = 0.1% (w/v) Sodium bicarbonate was supplemented.

Lakshmi KVNS, Ph.D Thesis, 2012

Sulfur sources
(concentration)

138

Results

Growth
O.D660 nm

+/ -

Control [without sulfur source]

0.02

NA

Cysteine (2 mM)

0.02

Magnesium sulfate (5 mM)

0.02

Methionine (2mM)

0.01

Sodium sulfate (5 mM)

0.02

Sodium sulfite (5 mM)

0.03

Sodium sulfide (1 mM)

0.30

0.06

Sulfur (2 mM)
Sodium thiosulfate (5 mM)

Table 3.6.2 Growth of strain JA480T on various sulfur sources.


Experimental details as given in Table 3.6.1, except that pyruvate
(0.3%, w/v) was used as carbon source and sulfur source varied as
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

N2 [100% (v/v) gas phase]

0.02

Control

(without

nitrogen

source)

Table 3.6.3 Growth of strain JA480T on various nitrogen sources.


Experimental details as given in Table 3.6.1, except that pyruvate
(0.3%, w/v) was used as carbon source and nitrogen source varied as
indicated.

Lakshmi KVNS, Ph.D Thesis, 2012

139

Vitamins

Results

Growth
O.D660 nm
0.10

+/ NA

0.23

*Without Thiamine

0.25

*Without Riboflavin

0.21

*Without Niacin

0.16

*Without Pantothenate

0.26

*Without
Pyridoxal
phosphate
*Without Vitamin B12

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.


Experimental details as given in Table 3.6.1, 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.
*= other 7 vitamins were added; = all 8 vitamins were added.
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 %).

Lakshmi KVNS, Ph.D Thesis, 2012

140

Results

3.6.6 G+C content: The DNA G + C content of strain JA480T was 67.8
mol% (by HPLC).
3.6.7 Physiological characteristics: Strain JA480T 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, Na2S2O3.5H2O (1mM) 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. 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

Lakshmi KVNS, Ph.D Thesis, 2012

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).
3.6.9 16S rRNA gene sequence similarity: The pairwise 16S rRNA gene
sequence similarities of strain JA480T with the nearest type strains were
found using EzTaxon server. The data obtained revealed that strain
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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
absorption spectrum for pigments extracted with acetone gave maxima at
303, 309, 348, 456 and 486nm (Fig. 3.7.3b) indicating the presence of

Lakshmi KVNS, Ph.D Thesis, 2012

(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.

Lakshmi KVNS, Ph.D Thesis, 2012

144

Results

(a)
(b)
Fig. 3.7.3 Whole-cell absorption spectrum (a) and acetone spectrum
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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%).
3.7.5 FAME analysis: Whole-cell fatty acid analysis of the strain JA580T

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).


3.7.7 Physiological characteristics: Strain JA580T was able to grow

photoorganoheterotrophically [anaerobic, light (2,400 lux) with pyruvate


(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)],


chemolithoautotrophy [aerobic, dark, Na2S2O3.5H2O (1mM) and NaHCO3
(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,

Lakshmi KVNS, Ph.D Thesis, 2012

Carbon source/
e- donor
(concentration)

146

Results

Growth
O.D660 nm

+/ -

Control [without inorganic/


organic substrate]
Control [NaHCO3 (0.1%, w/v)]

0.03

NA

0.03

NA

Acetate (0.3%, w/v)

0.30

Ascorbate(0.3%, w/v)

0.04

Aspartate(0.3%, w/v)

0.09

(+)

*Benzoate (1mM)

0.01

*Butyrate (0.1%, w/v)

0.01

*Caproate (0.1%, w/v)

0.03

Casamino acids (0.3%, w/v)

0.18

Citrate (0.3%, w/v)

0.04

Crotonate (0.1%, w/v)

0.03

Cysteine(0.3%, w/v)

0.045

Ethanol (0.1%, w/v)

0.14

(+)

Formate (0.3%, w/v)

0.03

Fructose (0.3%, w/v)

0.06

Fumarate (0.3%, w/v)

0.34

Glutamate (0.3%, w/v)

0.29

Glucose (0.3%, w/v)

0.07

*Glycerol (0.3%, w/v)

0.13

(+)

2-oxo glutarate (0.3%, w/v)

0.04

Lactate (0.3%, w/v)

0.02

Malate (0.3%, w/v)

0.25

Mannitol (0.3%, w/v)

0.08

Methanol (0.1%, w/v)

0.10

(+)

Peptone (0.05%, w/v)

0.25

Propanol (0.1%, w/v)

0.04

Methionine (0.3%, w/v)

Lakshmi KVNS, Ph.D Thesis, 2012

147

Results

*Propionate (0.1%, w/v)

0.01

Pyruvate (0.3%, w/v)

0.45

Sorbitol (0.3%, w/v)

0.13

(+)

Succinate (0.3%, w/v)

0. 43

Sucrose (0.3%, w/v)

0.26

Tartarate (0.3%, w/v)

0.12

(+)

*Valerate (0.1%, w/v)

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.

Lakshmi KVNS, Ph.D Thesis, 2012

Sulfur sources
(concentration)

148

Results

Growth
O.D660 nm

+/ -

0.01

NA

0.02

Magnesium sulfate (5 mM)

0.04

Methionine (2mM)

0.00

Sodium sulfate (5 mM)

0.04

Sodium sulfite (5 mM)

0.02

Sodium sulfide (1 mM)

0.18

Sulfur (2 mM)

0.03

Sodium thiosulfate (5 mM)

0.23

Control
[without
source]
Cysteine (2 mM)

sulfur

Table 3.7.2 Growth of Rhodovulum sp. JA580T on various sulfur


sources.
Experimental details as given in Table 3.7.1, except that pyruvate
(0.3%, w/v) was used as carbon source and sulfur source varied as
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

N2 [100% (v/v) gas phase]

0.17

Control [without nitrogen


source]
Ammonium chloride

Table 3.7.3 Growth of Rhodovulum sp. JA580T on various


nitrogen sources.
Experimental details as given in Table 3.7.1, except that pyruvate
(0.3%, w/v) was used as carbon source and nitrogen source varied
as indicated.

Lakshmi KVNS, Ph.D Thesis, 2012

149

Vitamins

Results

Growth
O.D660 nm
0.05

+/ NA

0.03

*Without Thiamine

0.08

*Without Riboflavin

0.02

*Without Niacin

0 07

*Without Pantothenate

0.05

*Without

0.01

*Without Vitamin B12

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.


Experimental details as given in Table 3.7.1, 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.
*= other 7 vitamins were added; = all 8 vitamins were added.
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.

Lakshmi KVNS, Ph.D Thesis, 2012

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).
3.7.8 16S rRNA gene sequence similarity: The pairwise 16S rRNA gene

sequence similarity of strain JA580T with the nearest type strains was
found using EzTaxon server. The data obtained revealed that strain
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,

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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
-

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012


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
-

Lakshmi KVNS, Ph.D Thesis, 2012

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.

Lakshmi KVNS, Ph.D Thesis, 2012

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.