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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

tO

4.6 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/g

wet 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 o

and 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.

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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.

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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.

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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 n

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N2-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

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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.

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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 )

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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

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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 .

References

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3. Baumann P, Furniss AL, Lee JV (1984) Genus I Vibrio Pacini 1854, 411 ^L. In: Krieg NR (ed) Bergey's manual of systematic bacteriology, vol. 1. Williams and Wilkins Co, Baltimore, pp 518-538

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8. Dicker HJ, Smith DW (1980) Enumeration and relative importance of acetylene-reducing (nitrogen-fixing) bacteria in a Delaware salt marsh. Appl Environ Microbiol 39:1019-1025 9. Egidius E, Wilk R, Andersen K, Hoff KA, Hjeltnes B (1986) Vibrio salmonicida sp. nov., a

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12. Guerinot ML, West PA, Lee JV, Colwell RR (1982) Vibrio diazotrophicus sp. nov., a marine nitrogen-fixing bacterium. Int J Syst Bacteriol 32:350-357

13. Hada HS, West PA, Lee JV, Stemmler J, Colwell RR (1984) Vibrio tubiashii sp. nov., a pathogen of bivalve mollusks. Int J Syst Bacteriol 34:1-4

14. Haines JR, Atlas RM, Gritfiths RP, Morita RY (1981) Denitrification and nitrogen fixation in Alaskan continental shelf sediments. Appl Environ Microbiol 41:412-421

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17. Jones KL, Rhodes-Roberts ME (1980) Physiological properties of nitrogen-scavenging bacteria from the marine environment. J Appl Bacteriol 49:421--433

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27. Patriquin D, Knowles R (1972) Nitrogen fixation in the rhizosphere of marine angiosperms. Mar Biol 16:49-58

28. Patriquin DG, McClung CR (1978) Nitrogen accretion, and the nature and possible significance

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47:227-242

29. Reichelt JL, Nealson KH, Hastings JW (1977) The specificity of symbiosis: Pony fish and luminescent bacteria. Arch Microbiol 112:157-161

30. Ruby EG, Morin JG (1978) Specificity ofsymbiosis between deep-sea fishes and psychrotrophic luminous bacteria. Deep Sea Res 25:161-167

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in fish. Curt Microbiol 6:343-348

32. Shieh WY, Simidu U, Maruyama Y (1987) Isolation of a nitrogen-fixing Vibrio species from

the roots of eelgrass (Zostera marina). J Gen Appl Microbiol 33:321-330

33. Shieh WY, Simidu U, Maruyama Y (1988) Nitrogen fixation by marine agar-degrading bac- teria. J Gen Microbiol 134:1821-1825

34. Shieh WY, Simidu U, Maruyama Y (1988) New marine nitrogen-fixing bacteria isolated from

an eelgrass (Zostera marina) bed. Can J Microbiol 34:886-890

35. Shier WY, Simidu U, Maruyama Y (1989) Nitrogenase activity of heterotrophie bacteria

associated with the roots of eelgrass (Zostera marina). Nippon Suisan Gakkaishi (formerly

Bull Japan Soc Sci Fish) 55:853-857

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N2-Fixing Bacteria in an Eelgrass Bed 259 39. Waterbury JB, Calloway CB, Turner RD (1983) A cellulolytic nitrogen-fixing bacterium cul- tured from the gland of Deshayes in shipworms (Bivalvia: Teredinidae). Science 221:1401-

1403

40. West PA, Brayton PR, Twilley RR, Bryant TN, Colwell RR (1985) Numerical taxonomy of nitrogen-fixing "decarboxylase-negative" Vibrio species isolated from aquatic environments. Int J Syst Bacteriol 35:198-205

41. Wiebe WJ, Johannes RE, Webb KL (1975) Nitrogen fixation in a coral reefcommunity. Science 188:257-259

42. W3(nn-Williams DD, Rhodes ME (1974) Nitrogen fixation in seawater. J Appl Bacteriol 37: 203-216

43. Zuberer DA, Silver WS (1974) Mangrove-associated nitrogen fixation. In: Walsh GE, Snedaker SC, Teas HJ (eds) The biology and management of mangroves. University of Florida, Gaines- ville

44. Zuberer DA, Silver WS (1978) Biological dinitrogen fixation (acetylene reduction) associated with Florida mangroves. Appl Environ Microbiol 35:567-575

數據

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)
Table 2.  MPN and plate counts of various bacterial groups in Aburatsubo Inlet  seawater (cells/ml)  Bacterial  group and  counting  Month  method  (in  1987)

參考文獻

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