Microb Ecol (1989) 18:249-259
MICROBIAL ECOLOGY
9 Springer-Veflag New York Inc. 1989
Enumeration and Characterization of Nitrogen-Fixing
Bacteria in an Eelgrass
(Zostera marina)
Bed
Wung Yang Shieh, ~,* Usio Simidu, ~ and Yoshiharu M a r u y a m a 2
~Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164; and 2Departrnent of
Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Bunkyo,
Tokyo 113, Japan
Abstract.
Marine nitrogen-fixing bacteria distributed in the eelgrass bed
and seawater o f Aburatsubo Inlet, Kanagawa, Japan were investigated using
anaerobic and microaerobic enrichment culture methods. The present en-
richment culture methods are simple and efficient for enumeration and
isolation o f nitrogen-fixing bacteria from marine environments. Most-
probable-number (MPN) values obtained for nitrogen-fixing bacteria ranged
from 1.1 x 102
tO4.6 x 102/ml for seawater, 4.0 x 104 to 4.3 x 105/g
wet wt for eelgrass-bed sediment, and 2.1 x 105 to 1.2
• 107/gwet wt for
eelgrass-root samples. More than 100 strains o f halophilic, nitrogen-fixing
bacteria belonging to the family Vibrionaceae were isolated from the M P N
tubes. These isolates were roughly classified into seven groups on the basis
o f their physiological and biochemical characteristics. The majority o f the
isolates were assigned to the genus
V i b r i oand one group to the genus
P h o t o b a c t e r i u m .
However, there was also a group that could not be iden-
tified to the generic level. All isolates expressed nitrogen fixation activities
under anaerobic conditions, and no organic growth factors were required
for their activities.
Introduction
For many years, biological nitrogen fixation occurring in marine environments
was believed to be mainly due to cyanobacteria [37]. However, the presence
o f bacterial nitrogen fixation has been demonstrated in many diverse marine
habitats since the 1970s. Most environments where bacterial nitrogen fixation
has been found are benthic, such as seagrass [5, 6, 27, 36], salt marsh [26, 28],
mangrove [15, 43, 44], and coral reef [41] communities, and a variety o f
estuarine and marine sediments [14, 16, 23]. There are also reports that bacterial
nitrogenase activity is associated with diatoms and other plankters in the pelagic
zone [ 19, 21 ]. It also has been demonstrated that heterotrophic nitrogen fixation
might occur in association with oxygen-poor microzones in nitrogen-depleted
* Present address:
Institute of Oceanography, National Taiwan University, P.O. Box 23-13, Taipei,
Taiwan, Republic of China.
aerobic marine waters [24, 25]. These studies indicate that nitrogen-fixing
bacteria may introduce significant amounts o f a m m o n i u m nitrogen into various
marine environments.
Several studies have been done concerning the enumeration of nitrogen-
fixing bacteria in seawater [10, 21, 42], estuarine and oceanic sediments [20,
21], and salt marsh soils [8, 28]. Nitrogen-fixing bacteria associated with the
roots of marsh grass
Spartina alterniflora
and the gastrointestinal tracts o f sea
urchins have also been enumerated [11, 28]. Early attempts were made using
plate counts [21, 42]. However, these methods proved to be ineffective because
many strains isolated from agar media without added combined nitrogen proved
to be nitrogen-scavenging bacteria that were unable to fix nitrogen when assayed
by the acetylene reduction method [17]. The most-probable-number (MPN)
counts of nitrogen-fixing bacteria have also been done by analyzing nitrogenase
(acetylene reduction) activity in the cultures that were inoculated with 0.2 um
filters used to concentrate the bacteria in the initial seawater samples [10].
However, only low numbers of nitrogen-fixing bacteria were counted in these
studies. For example, counts o f 10-t-102 cells/liter for seawater from Chesa-
peake Bay and the coast o f Puerto Rico were reported by Guerinot and Colwell
[ 10]. Nitrogen-fixing bacteria requiring NaC1 for growth have been isolated
from a variety of marine sources [ 10, 22, 39, 40]. However, nitrogenase activity
of these bacteria was low, and few of them showed significant levels of nitrog-
enase activity (> 10 nmol CzH4/mg dry wt cells/hour) within a few hours o f
incubation.
During a study of nitrogen fixation in an eelgrass bed in Aburatsubo Inlet,
Kanagawa, Japan, we isolated a species o f marine nitrogen-fixing
Vibrio
from
eelgrass
(Zostera marina)
roots using an anaerobic enrichment culture method
[32]. This bacterium showed rapid growth in a nitrogen-free liquid m e d i u m
and it expressed nitrogenase activity after only a few hours o f incubation under
anaerobic conditions. In the present study, anaerobic and mieroaerobic en-
richment culture methods, basically similar to that of our previous report [32],
were applied in the M P N technique for further enumeration and isolation o f
marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater o f
Aburatsubo Inlet. More than 100 strains o f marine nitrogen-fixing bacteria
were isolated from eelgrass roots, eelgrass-bed sediment, and seawater. Their
taxonomic characteristics and nitrogen fixation properties were also examined.
Materials and Methods
Sample Collection and Treatment
Samples of sediment and eelgrass roots were collected from an eelgrass bed in Aburatsubo Inlet, Kanagawa, Japan, in July and August of 1986, and seawater samples were collected from two stations located in this inlet in June and August of 1987. All samples were processed within a few hours. After root samples were washed free o f sediment in sterile seawater, 1 g wet wt samples o f the roots were washed once again by vigorous shaking in 50 ml o f sterile seawater containing 1 p p m Tween 80 (root rinse water). The roots were again washed three times in sterile seawater, and then homogenized with 9 ml o f sterile seawater (root homogenate solution). Samples of seawater, sediment, root rinse water, and root homogenate solution were then used for bacterial counts.
N2-Fixing Bacteria in an Eelgrass Bed 251
Media
To determine suitable carbon sources for obtaining the most active nitrogen-fixing bacteria, we
examined 10 sugars and organic acids for their effects on the nitrogenase activities associated with eelgrass roots and eelgrass-bed sediment. Glucose and sucrose were most effective in enhancing the activities (data not shown). They were therefore used as the carbon sources in making nitrogen- free (N-free) and nitrogen-deficient (N-deficient) media for enrichment o f nitrogen-fixing bacteria. The N-free liquid media contained the following components: (1) basal m e d i u m (NaC128 g, MgSO4. 7H20 5 g, CaCI2"2HzO 10 nag, Na2MoO~.2H:O 10 rag, Tris 50 retool, distilled water 800 ml) adjusted to pH 8,0; (2) K2HPO4 0.5 g dissolved in 100 ml o f distilled water; and (3) glucose or sucrose 5 g, FeCI3-6H20 15 mg dissolved in 100 ml o f distilled water. These were mixed after autoclaving separately. The N-deficient liquid media were prepared by supplementing N-free liquid media with 1 mg o f yeast extract (Difco) per liter. The polypepton-yeast-glucose agar (PYG-A)
m e d i u m contained the following two components: (1) Polypepton (Daigo, Tokyo, Japan) 2.0 g,
yeast extract (Difco, Detroit, Michigan, USA) 0.5 g, Tris 50 mmoi, and agar (Difco) 15 g dissolved in 900 ml o f seawater and adjusted to p H 7.6; (2) 5 g glucose dissolved in 100 ml of distilled water. The two components were mixed after autoclaving separately. The polypepton-yeast agar (PY-A) m e d i u m contained 2.0 g Polypepton (Daigo), 0.5 g yeast extract (Difco), and 15 g agar (Difco) in
1 liter of 90% seawater, adjusted to p H 7.6.
Bacterial Enumeration
One milliliter seawater, 1 g wet wt sediment, 1 ml root rinse water, and 1 ml root homogenate solution were serially diluted with 9 ml o f sterile seawater. Plate counts o f heterotrophic bacteria were done by spreading 0.1 ml aliquots of each dilution onto duplicate PY-A or PYG-A plates. Incubation was carried out at 25"C for 7 days under either aerobic (air) or anaerobic (GasPak System, BBL, Cockeysville, Maryland, USA) conditions. The PY-A a n d P Y G - A media were used for aerobic and anaerobic cultures, respectively. The most-probable-number (MPN) o f heterotro- phic bacteria in seawater and nitrogen-fixing bacteria in all samples was estimated from series with three tubes per dilution using the tables o f Cochran [7]. For heterotrophic bacteria, 1 ml aliquots o f each diluent were added to 5 ml o f P Y broth (PY-B) m e d i u m and incubated at 25"C for 7 days under aerobic conditions. For nitrogen-fixing bacteria, a I ml aliquot o f each diluent was transferred to a 20 ml test tube containing 5 ml of each o f the following liquid media: (1) N-free glucose (-NFG), (2) N-deficient glucose (NDG), (3) N-free sucrose t'NFS), (4) N-deficient sucrose (NDS). After the tubes were sealed with rubber stoppers, the air in each tube was replaced with N2 (100%)
or N2/O2 (97%:3~ All cultures were incubated in the dark at 250C for 3-7 days. The criterion
used to record an M P N tube as positive was the development of visible turbidity because non- nitrogen-fixing bacteria (usually oligotrophic bacteria) might develop invisible (but never visible) turbidity in the present N-free or N-deficient media.
Isolation o f Nitrogen-Fixing Bacteria
M P N tubes that developed visible turbidity were again serially diluted with solution containing 25 g NaC1 and 5 g MgSO4-7H20 in 1 liter of distilled water. A 0.1 ml aliquot of each diluent o f the cultures was transferred to fresh N-free or N-deficient m e d i u m and the anaerobic or microaer- obic enrichment culture procedures described above were repeated. For the final isolation o f nitrogen-fixing bacteria, the cultures o f the highest dilution with positive growth from the second enrichment were spread on PY-A plates and the plates were incubated at 25"C for 2-5 days under aerobic conditions. All colonies from the plating of the same enrichment culture were usually o f the same type, indicating that they might have been derived from the same cell. However, two types of colonies also occurred on some of the plates. Individual colonies were picked and purified by repeated streakings on PY-A plates. The nitrogenase activity of the isolates was measured by the acetylene-reduction assay method. A variety o f marine nitrogen-fixing bacteria were isolated using the present enrichment culture methods.
Measurement o f Nitrogenase Activity
The nitrogenase activity of the isolates was measured by the acetylene-reduction assay method using a Shimadzu G-4CM gas chromatograph [32]. Cells from PY-A plates were inoculated into 20 ml tubes containing 5 ml NFG medium. The air in the tube was replaced with N2 (100%) or
Nz/Oz
(97%:3%; v/v) after it was sealed with a rubber stopper. Incubation was carried out at 25~ in the dark. The tubes that developed turbidities (optical density at 600 nm) of 0.10--0.20 were injected with 2 ml of acetylene with a gas-tight syringe. Incubation was continued for a day, and then 0.2 ml of gas in each tube was removed with a gas-tight syringe for the analysis of ethylene production.Characterization of Nitrogen-Fixing Isolates
Characterization of the nitrogen-fixing isolates was carried out according to the methods described in our previous report [34] with some modifications. The presence of agarase was examined by incubating the isolates on PY agar plates for 5 days, and strains that caused liquefaction or depressions were recorded as agarase-positive. Some strains that showed ambiguous reactions in this test were clarified by the iodine test [1]. The ability to produce acid and gas from different carbohydrates was determined in stab media as described previously [34]. However, broth media free of agar were also used instead of stab media for some agar-degrading isolates which produced acid from agar [33]. Poly-~-hydroxybutyrate (PHB) accumulation was tested in a medium con- taining the following two components: (1) 0.5 g of yeast extract (Difco), and 50 mmol Tris, dissolved in 900 ml of seawater and adjusted to pH 8.0; (2) 2 g of glucose dissolved in 100 ml of distilled water. The two components were mixed after autoclaving separately. Growth at different concen- trations of NaC1 was determined in yeast extract (0.4%, w/v; Difco) broth containing 0, 3, 6, and
10% NaC1.
R e s u l t s and D i s c u s s i o n
Bacterial Enumeration
T a b l e s 1 a n d 2 s u m m a r i z e t h e M P N c o u n t s o f n i t r o g e n - f i x i n g b a c t e r i a as well as t h e p l a t e c o u n t s o f h e t e r o t r o p h i c b a c t e r i a i n t h e eelgrass b e d a n d s e a w a t e r s a m p l e s . N i t r o g e n - f i x i n g b a c t e r i a w e r e f o u n d i n all s a m p l e s u s i n g t h e p r e s e n t a n a e r o b i c a n d m i c r o a e r o b i c e n r i c h m e n t c u l t u r e m e t h o d s . T h e M P N v a l u e s o f n i t r o g e n - f i x i n g b a c t e r i a r a n g e d f r o m 1.1 • 102 to 4 . 6 • 102 f o r s e a w a t e r (cells/ m l ) a n d 4 . 0 • 104 t o 4.3 x 105 f o r e e l g r a s s - b e d s e d i m e n t (cells/g w e t wt). F o r e e l g r a s s - r o o t s a m p l e s , t h e s e v a l u e s (cells/g w e t wt) r a n g e d f r o m 2.2 x 105 t o 1.2 • 107 f o r r o o t r i n s e w a t e r a n d 2.1 x 105 t o 4.6 x 105 f o r r o o t h o m o g e n a t e s o l u t i o n . I n t h e p r e s e n t s t u d y , h a l o p h i l i c b a c t e r i a p o s i t i v e i n n i t r o g e n a s e ( a c e t y l e n e r e d u c t i o n ) a c t i v i t y w e r e i s o l a t e d f r o m all t h e e n r i c h m e n t c u l t u r e t u b e s t h a t d e v e l o p e d v i s i b l e t u r b i d i t y . S o m e c u l t u r e s (ca. 10%) u s e d i n t h e i s o l a t i o n o f n i t r o g e n - f i x i n g b a c t e r i a w e r e n o t p u r e a n d c o n t a i n e d n o n n i t r o g e n - f i x i n g b a c - teria. H o w e v e r , n i t r o g e n - f i x i n g b a c t e r i a a l w a y s a p p e a r e d as t h e p r e d o m i n a n t g r o u p s i n t h e s e m i x e d c u l t u r e s : c o l o n y - f o r m i n g u n i t s ( C F U ) o f n i t r o g e n - f i x i n g b a c t e r i a w e r e a l w a y s m u c h h i g h e r t h a n t h o s e o f n o n n i t r o g e n - f i x i n g b a c t e r i a . T h e r e s u l t s i n d i c a t e t h a t t h e p r e s e n t c o u n t i n g m e t h o d s a r e i n d e e d a p p l i c a b l e for t h e e v a l u a t i o n o f n i t r o g e n - f i x i n g b a c t e r i a i n m a r i n e e n v i r o n m e n t s . G u e r i n o t a n d C o l w e l l [10] r e p o r t e d a e r o b i c p l a t e c o u n t s o f h e t e r o t r o p h i c b a c t e r i a i nN2-Fixing Bacteria in an Eelgrass Bed 253 Table 1. MPN and plate counts of various bacterial groups in the eelgrass bed of Aburatsubo Inlet (cells/g wet vet sediment or root sample)
Bacterial counts
Bacterial group Root
and counting Month Root rinse homogenate
method (in 1986) Medium ~ Sediment water solution
Plate counts of heterotrophs
Aerobic July PY-A 2.2 • 107 3.4 • 107
Aerobic Aug. PY-A 2.6 • l0 T 2.4 x l0 T
Anaerobic July PYG-A 4.2 x 106 6.2 x 106
Anaerobic Aug. PYG-A 2.5 x 106 5.5 • 106
MPN counts of N:-fixing bacteria
2.6 x 10 7 2.8 x 10 7 3.8 x 10 6 2.8 x 10 6 Anaerobic July NFS 9.0 • 104 1.2 x 107 2.4 • l0 s Anaerobic July NDS 2.3 • l0 s 5.5 x 105 2.4 x 105 Anaerobic Aug. NFG 9.0 x 104 4.6 x 105 4.3 x 105 Anaerobic Aug. NDG 1.5 • l0 s 2.2 x 105 2.3 x 10 s Microaerobic July NFS 2.4 x 105 1.2 x 106 4.6 x 105 Microaerobic July NDS 4.0 x 104 2.3 • 106 2.4 x 10 n Microaerobic Aug. NFG 9.0 x 104 2.2 x 105 2.1 • 105 Microaerobic Aug. NDG 4.3 x 105 7.5 • 105 4.3 • 10 ~
a PY-A, PY agar medium; PYG-A, PYG agar medium; NFS, liquid N-free sucrose medium; NFG, liquid N-free glucose medium; NDS, liquid N-deficient sucrose medium; NDG, liquid N-deficient glucose medium s e a w a t e r f r o m C h e s a p e a k e B a y ( f r o m 1.9 x 104 t o 5.1 • 104 c e l l s / m l ) w h i c h w e r e s i m i l a r to t h o s e i n A b u r a t s u b o I n l e t s e a w a t e r ( f r o m 3.3 x 104 t o 1.6 x 105 c e l l s / m l ; T a b l e 2). H o w e v e r , i n t h e i r s t u d y , t h e M P N v a l u e s o f n i t r o g e n - f i x i n g b a c t e r i a f o r s e a w a t e r s a m p l e s c o l l e c t e d f r o m C h e s a p e a k e B a y ( f r o m 2 • 10 -3 t o 1.05 • 10 -1 c e l l s / m l ) w e r e f a r l o w e r t h a n t h o s e i n A b u r a t s u b o I n l e t s e a w a t e r ( f r o m 1.1 • 102 t o 4.6 x 102 c e l l s / m l ) o b t a i n e d h e r e ( T a b l e 2). T h e r e l a t i v e l y l o w M P N v a l u e s t h e y o b t a i n e d m a y b e e x p e c t e d b e c a u s e t h e y u s e d e n r i c h m e n t m e d i a c o n t a i n i n g 100 m g o f y e a s t e x t r a c t p e r l i t e r . A l t h o u g h t h e h i g h a m o u n t o f y e a s t e x t r a c t m i g h t s u p p o r t m o r e d i v e r s e s p e c i e s o f n i t r o g e n - f i x i n g b a c t e r i a , b a c t e r i a o t h e r t h a n n i t r o g e n - f i x i n g b a c t e r i a m i g h t g r o w i n t h e s e m e d i a m u c h m o r e r a p i d l y t o s u r p a s s t h e l a t t e r .
Nitrogen Fixation Activity
A t o t a l o f 127 s t r a i n s o f n i t r o g e n - f i x i n g b a c t e r i a w e r e i s o l a t e d f r o m t h e a b o v e s o u r c e s . A l l t h e s e i s o l a t e s r e d u c e d a c e t y l e n e i n t o e t h y l e n e u n d e r a n a e r o b i c c o n d i t i o n s ( > 2 0 0 n m o l C : H 4 / c u l t u r e / d a y ) . M o s t o f t h e i s o l a t e s s h o w e d sig- n i f i c a n t l e v e l s o f n i t r o g e n a s e a c t i v i t y a f t e r o n l y a few h o u r s o f i n c u b a t i o n w h e n u s i n g t h e p r e v i o u s m e a s u r e m e n t m e t h o d [34]. A l l i s o l a t e s g r e w i n t h e N F G m e d i u m u n d e r N2. T h i s i n d i c a t e s t h a t t h e y c o u l d fix N2 a s t h e s o l e n i t r o g e n s o u r c e u n d e r a n a e r o b i c c o n d i t i o n s , a n d n o o r g a n i c g r o w t h f a c t o r s w e r e r e q u i r e d b y t h e s e i s o l a t e s . S o m e o f t h e i s o l a t e s h a v e b e e n d e m o n s t r a t e d t o b e t o l e r a n t t o l o w l e v e l s o f o x y g e n [34], b u t n o n e o f t h e m s h o w e d s i g n i f i c a n t g r o w t h i n t h i s m e d i u m u n d e r a i r . O n t h e o t h e r h a n d , all i s o l a t e s g r e w i n P Y G m e d i u m u n d e r b o t h a e r o b i c (air) a n d a n a e r o b i c ( G a s P a k S y s t e m , B B L ) c o n d i t i o n s . T h e s e
Table 2. MPN and plate counts of various bacterial groups in Aburatsubo Inlet seawater (cells/ml) Bacterial group and counting Month method (in 1987)
Bacterial counting value in
Medium ~ Station I Station II
Plate counts of heterotrophs
Aerobic June PY-A 1.6 x 10 ~ 3.3 x 10'
Aerobic Aug. PY-A 1.2 x 105 6.0 • 104
Anaerobic June PYG-A 1.3 x 104 1.8 • 103
Anaerobic Aug. PYG-A 3.8 x 104 2.5 x 103
MPN counts of heterotrophs
Aerobic June PY-B 2.3 x 105 2.1 x 105
Aerobic Aug. PY-B 1.5 x 106 7.5 x l0 s
MPN counts of N2-fixing bacteria
Anaerobic June NFG 2.4 x 10 z 1.1 x 102
Anaerobic Aug. NFG 1.5 • 102 4.6 • 102
a PY-A, PY agar medium; PYG-A, PYG agar medium; PY-B, PY broth medium; NFG, liquid N-free glucose medium
results indicate that all isolates were typical facultatively anaerobic, nitrogen- fixing bacteria. Microaerophilic, nitrogen-fixing bacteria requiring NaC1 for growth h a v e been isolated f r o m the gland o f D e s h a y e s in s h i p w o r m s [39] a n d
the roots o f m a r s h grass
Spartina alterniflora
[22] in the past several years.H o w e v e r , n o n e o f these bacteria c o u l d be isolated using the present m i c r o a e r - obic culture m e t h o d s . S o m e nitrogen-fixing bacteria isolated f r o m the rhizo- sphere o f terrestrial plants [ 18, 38] h a v e been d e m o n s t r a t e d to fix nitrogen only in the presence o f c o m b i n e d nitrogen or certain v i t a m i n s b u t these bacteria were also unable to be isolated in this study. C o n s i d e r i n g the high M P N c o u n t s o f facultatively anaerobic, nitrogen-fixing bacteria w i t h o u t the r e q u i r e m e n t o f organic g r o w t h factors, these bacteria m i g h t be i m p o r t a n t in p r o v i d i n g inputs o f a m m o n i u m nitrogen into the present s t u d y sites. I n o u r p r e v i o u s r e p o r t [35], we suggested that the in situ nitrogenase activity o f e e l g r a s s r h i z o m e - r o o t c o m - plexes m i g h t be p r i m a r i l y attributed to r o o t - a s s o c i a t e d nitrogen-fixing bacteria. W e also suggested t h a t facultatively a n a e r o b i c nitrogen-fixing bacteria, includ- ing the present isolates, m i g h t be the m o s t i m p o r t a n t nitrogen fixers associated with eelgrass roots.
Characterization of Nitrogen-Fixing lsolates
All the nitrogen-fixing isolates were g r a m - n e g a t i v e rods w h i c h were m o t i l e b y m e a n s o f a single p o l a r flagellum in P Y - B m e d i u m . T h e y f e r m e n t e d glucose a n d required NaC1 for growth. These characteristics clearly indicate t h a t all isolates belong to the family Vibrionaceae. All isolates were r o u g h l y d i v i d e d into seven groups (Table 3), b a s e d m a i n l y o n the results o f the following tests: P H B a c c u m u l a t i o n , utilization o f / ~ - h y d r o x y b u t y r a t e , a n d p r o d u c t i o n o f agar- ase, oxidase, catalase, a n d gas f r o m glucose fermentation.
N 2 - F i x i n g B a c t e r i a i n a n E e l g r a s s B e d 2 5 5 T a b l e 3 . C h a r a c t e r i s t i c s o f m a r i n e n i t r o g e n - f i x i n g b a c t e r i a i s o l a t e d i n t h i s s t u d y B a c t e r i a l g r o u p a I I I I I I I V V V I V I I ( N o . o f s t r a i n s ) ( 5 ) ( 4 ) ( 1 0 ) ( 1 2 ) ( 7 3 ) ( 1 5 ) ( 8 ) C h a r a c t e r i s t i c P H B a c c u m u l a t i o n + b + . . . . . G a s f r o m g l u c o s e . . . + A g a r a s e - - + + - - - C a t a l a s e + + + + + + - O x i d a s e + + v + + - - G e l a t i n a s e + + + - v - + L e c i t h i n a s e + v . . . . . L i p a s e + + + + v - - A m y l a s e v v w + + - - D N A s e + v w v + - + C h i t i n a s e w . . . . A r g i n i n e d i h y d r o l a s e + v - - v - - S e n s i t i v i t y t o 0 / 1 2 9 ~ ( 1 5 0 # g ) v v v v v + - G r o w t h a t 4 " C v v v - v v - 2 0 ~ + + + + + + + 4 0 ~ - v v v v v + G r o w t h i n 0 % N a C 1 . . . . 3 % N a C 1 + + + + + + + 6 % N a C 1 + + + + + + + 1 0 % N a C 1 - v - - v - + A c i d p r o d u c t i o n f r o m G l u c o s e + + + + + + + S u c r o s e + + - - + + + M a n n i t o l + v + + + + + I n o s i t o l - v - - v + - I ) - A r a b i n o s e . . . + C e l l o b i o s e + + + + + + + M e l i b i o s e + v + + v + + L a c t o s e - v v w v w + U t i l i z a t i o n a s s o l e c a r b o n s o u r c e / % H y d r o x y b u t y r a t e - + . . . . . G l c u o s e + + + + + + + M a n n i t o l + v + + + + + G a l a c t o s e + + + + v + + M a n n o s e + v v - v + v L - R h a m n o s e -- v -- -- v -- v G l y c e r o l + v v - v v + I n o s i t o l + v - - v + - T r e h a l o s e + + - + v - - D u l c i t o l . . . + T a r t r a t e . . . + A c e t a t e + + v - v - - C i t r a t e v + + - v - + ~ A l l s t r a i n s w e r e p o s i t i v e f o r g l u c o s e f e r m e n t a t i o n a n d n i t r o g e n a s e a n d w e r e n e g a t i v e f o r G r a m s t a i n , s w a r m i n g , p i g m e n t a t i o n a n d l u m i n e s c e n c e + , p o s i t i v e ; - , n e g a t i v e ; w , w e a k l y p o s i t i v e ; v , v a r i a b l e b e t w e e n s t r a i n s c 2 , 4 - d i a m i n o - 6 , 7 - d i i s o p r o p y l p t e r i d i n e ( p h o s p h a t e )
The strains included in group I were plump, straight rods. T h e y accumulated
P H B as an intracellular product and produced lecithinase. They utilized glucose,
mannose, and glycerol but not ~-hydroxybutyrate as the sole carbon sources.
Gas was not produced during glucose fermentation. These strains could be
assigned to the genus
Photobacterium
[2]. They were not luminous; however,
this cannot exclude them from the genus because bioluminescence is no longer
treated as a generic-key characteristic o f
Photobacterium
[2]. Inclusion o f ni-
trogen-fixing strains into the genus
Photobacterium
requires a redefinition o f
the genus because the current definition
of Photobacterium
states that no species
fix N2 [2]. Strains belonging to this group have been isolated from sediment
and eelgrass roots but not from seawater.
G r o u p II contained strains that were able to accumulate PHB and utilize
B-hydroxybutyrate as the sole carbon source. All strains included in this group
were positive in oxidase and catalase reactions, and they did not produce gas
during glucose fermentation. This group could be placed into the genus
Vibrio
[3]. All strains included in this group were isolated from eelgrass roots; none
o f these bacteria was isolated from samples o f sediment or seawater.
Strains that could neither accumulate appreciable amounts o f P H B nor utilize
~-hydroxybutyrate were placed into the other five groups. Groups III, IV, V,
and VI contained strains that produced acid but no gas during the fermentation
o f glucose. All these groups could also be assigned to the genus
Vibrio.
Groups
III and IV contained strains that produced agarase but did not produce acid
from sucrose. They might represent new species o f the genus
Vibrio
because
all recognized
Vibrio
species lack agarase activity. The strains included in group
III liquefied agar. When the strains were streaked on PY agar plates and grown
at 25~
deep holes were formed by the colonies in a few days. T h e agar in the
plates was completely liquefied after incubation for 1-2 months. Some strains
belonging to this group have been demonstrated to fix nitrogen anaerobically
using agar as the sole carbon source [33]. Strains included in this group were
isolated from either sediment or seawater, and none o f them was isolated from
eelgrass roots. G r o u p IV contained strains that softened but did not liquefy
agar. Colonies on PY-A plates were circular and were surrounded by shallow
depressions; their agarase reaction was confirmed using the iodine test [ 1 ]. All
strains included in group IV utilized trehalose and they could neither hydrolyze
gelatin nor utilize citrate. These characteristics clearly distinguished t h e m from
group III. This group and groups V - V I I were isolated from all the present
samples, including seawater, sediment, and eelgrass roots.
G r o u p V included the majority o f the nitrogen-fixing isolates. The strains
produced oxidase, amylase, and DNAse, but not agarase. Most o f the strains
also hydrolyzed gelatin. This group could be further divided into several
subgroups because so many characteristics among the group were variable.
G r o u p VI included strains that were oxidase-negative. Acid production from
D-Arabinose distinguished this group from all the other groups. All strains
included in this group did not produce DNAse, amylase, lipase, and gelatinase.
They did not utilize acetate and citrate as the sole carbon sources. These strains
might represent a new species o f
Vibrio
because only two oxidase-negative
species are included in this genus [3], neither o f which has been reported to
produce nitrogenase.
Vibrio diazotrophicus
[ 12] and
Vibrio natriegens
[40] are
Nz-Fixing Bacteria in an Eelgrass Bed 257 t h e o n l y t w o r e c o g n i z e d s p e c i e s o f Vibrio c a p a b l e o f f i x i n g N2. O f t h e t w o s p e c i e s , t h e f o r m e r c a n b e i n c l u d e d i n g r o u p V a n d t h e l a t t e r is s i m i l a r to t h e s t r a i n s o f g r o u p II. T h e V i b r i o - l i k e , n i t r o g e n - f i x i n g b a c t e r i u m d e s c r i b e d in o u r p r e v i o u s r e p o r t [32] c a n a l s o b e i n c l u d e d i n g r o u p II. T h e s t r a i n s i n c l u d e d i n g r o u p V I I c o n t a i n e d c u r v e d r o d s t h a t p r o d u c e d n o t o n l y a c i d b u t a l s o g a s d u r i n g t h e f e r m e n t a t i o n o f g l u c o s e . C a t a l a s e a n d o x i d a s e w e r e n e g a t i v e . T h e y u t i l i z e d d u l c i t o l a n d t a r t r a t e a s s o l e c a r b o n s o u r c e s . A m - y l a s e a n d l i p a s e ( T w e e n 80 h y d r o l y s i s ) w e r e n o t p r o d u c e d . A t p r e s e n t , t h i s g r o u p c a n n o t b e i d e n t i f i e d t o t h e g e n e r i c l e v e l . T h e s t r a i n s i n c l u d e d i n t h i s g r o u p e i t h e r r e p r e s e n t a n e w g e n u s o f t h e f a m i l y V i b r i o n a c e a e o r t h e y a r e n e w m e m b e r s o f t h e g e n u s Vibrio [34]. M e m b e r s o f t h e h a l o p h i l i c , f a c u l t a t i v e l y a n a e r o b i c b a c t e r i a c o m p r i s e o n e o f t h e p r e d o m i n a n t b a c t e r i a l g r o u p s i n m a n y m a r i n e h a b i t a t s . S o m e o f t h e m a r e k n o w n a s p a t h o g e n s o f h u m a n s [4] o r m a r i n e a n i m a l s [9, 13, 31]. T h e i r s y m - b i o t i c r e l a t i o n s h i p w i t h l u m i n o u s m a r i n e f i s h e s h a s a l s o r e c e i v e d c o n s i d e r a b l e a t t e n t i o n [29, 30]. T h e p r e s e n t r e s u l t s , a s w e l l a s t h o s e o f o t h e r s [10, 40], i n d i c a t e t h a t h a l o p h i l i c , f a c u l t a t i v e l y a n a e r o b i c b a c t e r i a m a y a l s o b e i m p o r t a n t i n p r o - v i d i n g a m m o n i u m n i t r o g e n i n t o e s t u a r i n e a n d c o a s t a l e n v i r o n m e n t s .
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