Enhancement of Nitrogen-fixing Activity of

http://wwwsoc.nii.ac.jp/jsme2/

Microbes Environ. Vol. 22, No. 1, 59–70, 2007

Enhancement of Nitrogen-fixing Activity of Enterobacteriaceae Strains

Isolated from Sago Palm (Metroxylon sagu) by Microbial Interaction with

Non-nitrogen Fixers

ARCHANA SHRESTHA1*, KOKI TOYOTA1, MASANORI OKAZAKI

1, YUKO SUGA2, MARCELO A. QUEVEDO3,

ALAN B. LORETO3 and ALGERICO A. MARISCAL

3

1 Graduate school of Bio-Applications and System Engineerings, Tokyo University of Agriculture and Technology,

2–24–16, Nakamati, Koganei City, Tokyo 184–8588, Japan2 National Agricultural Research Center for Western Region, Kyoto, Japan3 Philippine Root Crop Research and Training Center (PhilRootcrops), Leyte State University, Baybay, Leyte,

Philippines

(Received September 25, 2006—Accepted January 9, 2007)

This study was done to determine microbial interactions between nitrogen-fixing bacteria (NFB) and different

indigenous bacteria, both of which were isolated from the same samples of sago palm. Co-culture of NFB and

indigenous bacteria, isolated using nutrient agar medium, into a nitrogen-free Rennie medium showed signifi-

cantly higher nitrogen-fixing activity (NFA) than single inoculations of NFB did in almost all combinations. A

reduced oxygen status also enhanced the NFA of NFB. All NFB preferred simple sugars as their substrates for

NFA and showed extremely low levels of NFA in starch, hemicellulose, and pectin-containing media. NFA was

markedly stimulated in the consortium of starch-degrading Bacillus sp. strain B1 and NFB, ranging from 0–1.5

to 150–270 nmol C2H4 culture−1 h−1. The consortium of hemicellulose-degrading Agrobacterium sp. strain HMC1

or Flexibacter sp. strain HMC2 and NFB also showed enhanced NFA, ranging from 0–0.1 to 16–38 nmol. In

contrast, no enhanced NFA was detected in the consortium of pectin-degrading Burkholderia sp. strain BT1 or

Paenibacillus sp. strain P1 and NFB. These results may indicate that beneficial microbial interactions occur in

sago palm to enhance nitrogen-fixing activity through collaborative utilizations of starch, hemicellulose and their

degradation products.

Key words: starch-degradation, pectin-degradation, hemicellulose-degradation, PCR-DGGE, nifH

In agriculture, crop productivity is mainly governed by

the use of inorganic N-fertilizers, which are expensive and

damaging to the environment32). Finding alternative ways of

supplying nitrogen to grasses and cereals has been a major

research challenge in tropical agriculture1,3). From ecologi-

cal and economical perspectives, plant-associated biological

nitrogen fixation is highly desirable in non-leguminous

plants as well as leguminous plants29).

Sago palm (Metroxylon sagu) is one of the oldest tropical

plants exploited by humanity33) and its starch has been used

in Southeast Asia, including Papua New Guinea, Indonesia,

and Malaysia, and in Pacific Oceania16). These plants have

great potential as a source of food for people in the regions

in which they grow5). Sago is still an important crop with a

vital role as a subsistence food in some areas of Papua New

Guinea and Irian Jaya33). In East Malaysia and Indonesia,

industrial utilization has helped in the development of this

crop33). Sago palm grows naturally under semi-cultivated

conditions in vast tracts of lowland15). It has several charac-

teristics that make it a quite remarkable plant: it is well

adapted to swampy, acidic peat soils, where few other crops

survive, and it tolerates flooded or drought conditions43).

In our previous study40), nitrogen-fixing bacteria (NFB)

* Corresponding author. E-mail address: [email protected]; Tel. &

Fax: +81–42–388–7915.

SHRESTHA et al.60

were isolated from the different parts of sago palm and were

identified as Klebsiella pneumoniae, Klebsiella oxytoca,

Pantoea agglomerans, Enterobacter cloacae, and Bacillus

megaterium. Moreover, it was suggested that NFB utilized

exclusively simple carbon sources like glucose and sucrose,

and not complex carbon sources like starch, pectin, and

hemicellulose. Haahtela et al.10) also found that simple car-

bon sources are preferable for nitrogen-fixing activity by

NFB. In addition, Halsall et al.12) reported that straw (hemi-

cellulose is the major component) was not utilized as an

energy source for nitrogen fixation. Simple carbon sources

like glucose and sucrose, however, are not readily available

in natural microenvironments10) while the major carbohy-

drates of plants like cellulose, hemicellulose and pectin24)

are easily and readily available in natural microenviron-

ments for different microbial activities2). Besides, this sago

palm bears a typical characteristic, the storing of starch in

its trunk33).

Typically, numerous bacterial populations with different

characteristics coexist in a natural environment2). Hence,

microbial interactions providing simple carbon sources

from naturally existing polymers might be one way to

enhance nitrogen-fixing activity of NFB. Therefore, the

aims of the present study were to examine the nitrogen-

fixing activity of NFB in the presence of different types of

indigenous bacteria: (1) indigenous dominant bacteria, (2)

starch-degrading bacteria, (3) pectin-degrading bacteria,

and (4) hemicellulose-degrading bacteria.

The application of molecular biological methods has

greatly facilitated the study of natural bacterial communities

and the identification of functionally significant organisms

within them27,31). In this study, we confirmed the existence

of NFB isolated from root and starch of sago palm in natural

samples by comparing banding patterns from denaturing

gradient gel electrophoresis (DGGE) targeted at nifH

between the isolates and community DNA extracted from

root and starch samples.

Materials and Methods

Isolation and identification of bacteria

The sago palm parts (root, starch, midrib and trunk) were

collected during March 2005 from Hilusig (N10°38'80'',

E124°56'72''), Leyte, The Philippines. Roots, starch, trunk

and midrib were washed with tap water to remove all soil

particles and rinsed with sterile distilled water. The washed

roots and midribs were cut into 1 to 2 cm segments with

scissors, and 3 g (fresh basis) of sample was transferred into

a vial. The vials were capped with rubber stoppers, and

acetylene gas was injected into the headspace of the vial at a

final concentration of 10%. Experiments were done as one

replicate, in duplicate, or in triplicate, depending on the

availability of samples. Acetylene-reducing activity (ARA)

was quantified, using nitrogen-free Rennie medium36), 4 to 7

days after the injection of acetylene by injecting 0.5 ml of

the headspace into a gas chromatograph (GC-14B; Shi-

madzu, Kyoto, Japan) equipped with a flame ionization

detector and a column packed with Shincarbon S (3 mm×2

m, 60/80 mesh, Shimadzu GLC Center, Tokyo, Japan).

Nutrient agar medium was used for the isolation of indig-

enous dominant bacteria from sago root, midrib, and trunk.

For the isolation of starch-, pectin- and hemicellulose-

degrading bacteria, an enrichment culture was done using

an inorganic salt medium (1.5 g NaNH4HPO4. 4H2O, 1.0 g

KH2PO4, 0.2 g MgSO4, 1000 ml distilled water) supple-

mented with 2.5 g of starch, pectin, or hemicellulose, and

incubated at 27°C for 48 h without shaking, and then the

corresponding polymer-degrading bacteria were isolated

using the same media supplemented with agar (15 g L−1)

and the same conditions. For the enrichment culture of

hemicellulose- and pectin-degrading bacteria, 1 ml of seri-

ally diluted sample of root (1 g macerated root) was used

along with the same technique for isolating starch-degrad-

ing bacteria from starch samples (1 g macerated starch).

To estimate phylogenic positions of NFB and polymer

degraders, DNA was extracted from the purified bacterial

colonies by using the conventional method30). PCR primers

(27f; 5'-AGAGTTTGATCCTGGCTCAG-3' and (1378r; 5'-

TACAAGGCCCGGGAACG-3') universal to the domain

Bacteria18) were used to amplify the segment of the 16S

rRNA gene corresponding to nucleotide positions 27 to

1378 in terms of Escherichia coli numbering25). Then, direct

sequencing of the PCR products was done by Hitachi Sci-

ence Systems, Ltd. (Tokyo, Japan). Approximately 750-bp

fragments of the 16S rRNA gene were sequenced between

nucleotides 27 to 1378 from the reverse side. The sequences

obtained were examined using the DDBJ homology search

system BLAST (http://www.ddbj.nig.ac.jp/search/blast-

j.html). For phylogenetic analysis, the nucleotide sequences

were aligned using CLUSTAL W (http://www.ddbj.nig.

ac.jp/search/clustalw-j.html).

PCR-DGGE analysis of nifH genes from the pure cul-

tures and the root and starch of sago palm

The roots collected (ca. 2–3 g) were cut into pieces a few

millimeter long with scissors and in the case of starch, 2–3 g

was collected. Both the root and starch samples used for

DNA extraction was the same samples used for assaying

Microbial Consortium Enhance N-fixation 61

ARA activity and the isolation of NFB. DNA was extracted

from a portion (0.1 g) of each sample using a bead beater

(Bead Smash-12, WAKENYAKU, Kyoto, Japan) with 1 ml

of extraction buffer (0.1 M Tris-HCl (pH 8), 40 mM EDTA,

0.2 M NaCl, 20 g L−1 SDS). The supernatant was collected

and to it, 400 µl of 7.5 M NH4OAc was added. This mixture

was kept on ice for 5 minutes and centrifuged for 3 min at

13,200×g. To the supernatant, a 70% of volume of ethanol

was added and the mixture was kept at −80°C for 1 h, cen-

trifuged at 13,200×g for 10 min, washed with 70% ethanol,

and finally centrifuged and suspended in TE buffer.

The first PCR was performed with the forward primer

FGPH19 (5'-TACGGCAARGGTGGNATHG-3') and the re-

verse primer PoIR (5'-ATSGCCATCATYTCRCCGGA-3')6).

The amplification product was 429 bp. Then, the second

PCR was performed with the forward primer PoIF-GC (5'-

TGCGAYCCSAARGCBGACTC-3' with a GC clamp and

the reverse primer AQER (5'-GACGATGTAGATYTCCTG-

3')6). The amplification product was 360 bp including a GC

clamp sequence. The cycling conditions used were: 30 cy-

cles of denaturation at 94°C for 1 min, annealing for 1 min

at 55°C for the first and at 48°C for the second PCR, and

primer extension at 72°C for 2 min, with a final extension at

72°C for 5 min. The 2nd PCR products were then subjected

to DGGE.

The DGGE analysis was done by using a Bio-Rad

DCode, Universal Mutation Detection System (Bio-Rad

Laboratories, Hercules, Ca, USA). About 15–20 µl of PCR

product was loaded onto a 6% (w/v) polyacrylamide gel

with 40 to 70% gradients (urea and formamide). The elec-

trophoresis was run for 14 to 16 h at 60°C and 100 V. The

gel was then stained with a SYBR green I nucleic acid gel

stain (1:10000 dilution; Cambrex Bio Science, Rockland,

ME, USA) for 30 min in Tris-acetate EDTA buffer. DNA

from the isolated diazotrophs in this study, Klebsiella

pneumoniae, K. oxytoca, Pantoea agglomerans, and Enter-

obacter cloacae, was used as positive controls.

NFA with different carbon sources

Various carbohydrates like sucrose, glucose, mannitol,

starch, lactate, pectin, hemicellulose galactose, mannose,

cellobiose and galactouronic acid were used to estimate

suitable carbon sources for NFA. All the reagents were spe-

cial grade and purchased from Wako Chemicals, except

galactouronic acid which was from Sigma Co. Ltd. and cel-

lobiose which was from MP Biomedicals, LLC. In this

experiment, a 30-ml sterilized rubber capped vial, into

which 3 ml of semi-solid nitrogen-free Rennie medium

(0.3% agar) containing different individual carbohydrates at

a rate of 5 g L−1, was dispensed and used. Individual pure

bacterial colonies were first inoculated into 3 ml of sterile

distilled water to remove extra nutrients accompanied by

cells, and then a loopful of the bacterial suspension was

inoculated into each medium containing a single carbohy-

drate source. The vials were capped with rubber stoppers,

and acetylene gas was injected into the headspace of the vial

at a final concentration of 10% and incubated at 28°C for 48

h without shaking. The amount of ethylene in the headspace

was quantified by gas chromatography.

NFA in consortia

NFB were inoculated into semi-solid N-free Rennie

medium along with different types of bacteria, all of which

were isolated from the sago palm samples used for ARA

and isolation. Four experimental sets were designed in

which, 1) indigenous dominant bacteria, 2) starch-degrading

bacteria, 3) hemicellulose-degrading bacteria, and 4) pectin-

degrading bacteria were inoculated. In experiment 1, semi-

solid Rennie medium was used, while in experiments 2, 3,

and 4, starch, hemicellulose or pectin was used as a sole

carbon source, instead of mannitol, lactate, or glucose. In

experiment 1, a total of 50 indigenous bacteria were isolated

(25 from sago root, 10 from sago midrib, and 15 from sago

trunk) on simple nutrient agar medium and then separated

into different groups randomly, named as A, B, C, D and E

for root isolates; F and G for midrib isolates, and H, I and J

for trunk isolates and each group contained 5 strains. Exper-

iments were done in triplicate and the amount of ethylene in

the headspace was quantified after 3 to 4 days by gas chro-

matography.

In experiment 2, N-free starch broth medium was used to

estimate the population density of i) NFB, ii) Bacillus sp.

strain B1, and iii) Bacillus sp. strain B1+NFB and incubated

at 27°C for 48 h. From each broth culture, a set of serial

dilutions was prepared and then colony counts were made

on simple nutrient agar media by using a spread plate tech-

nique after incubation at 27°C for 48 h. In the mixed cul-

ture, the colonies of Bacillus sp. strain B1 and NFB were

separated on the basis of colony morphology.

NFA under reduced oxygen conditions and in the cell-

free filtrates of indigenous bacteria

A 30 ml sterilized rubber capped vial was used in this

experiment into which 3 ml of N-free Rennie medium was

dispensed. Then, individual pure bacterial colonies were

inoculated into 3 ml of sterile distilled water and a loopful

of bacterial suspension was inoculated into the medium.

The vials were capped with rubber stoppers and then about

SHRESTHA et al.62

18 ml (2 times of 9 ml) of air was withdrawn using a syringe

and replaced with the same amount of N2 gas and acetylene

gas was injected into the headspace of the vial at a final

concentration of 10% and incubated at 28°C for 48 h with-

out shaking and ARA was measured.

In order to obtain fresh cultures, all the indigenous domi-

nant bacteria isolated from, root, midrib, and trunk, were

sub-cultured on fresh nutrient agar media and incubated at

27°C for 24 h. Then the bacterial suspensions of each group

were prepared by inoculating the colonies in 5 ml of sterile

distilled water. Then the prepared suspensions of each

group of indigenous dominant bacteria were cultured in

semisolid nutrient broth at 27°C for 48 h and cell-free fil-

trate fluid was obtained by filtering the culture using a 0.2

µm filter. Thus collected filtrates were used to measure the

ARA of NFB.

Statistical analysis

Data were analyzed using a standard analysis of variance

(ANOVA) (Excel Toukei, 2002 for Windows).

Results

Isolation and identification of bacteria

16S rRNA gene sequencing showed that all of the NFB

with high levels of ARA belong to the family Enterobacte-

riaceae with Klebsiella oxytoca, K. pneumoniae, Entero-

bacter cloacae, and Pantoea agglomerans as their closest

relatives (Table 1 and Fig. 1). The nitrogenase activity of

the strains varied from 2.4 to 19 nmol C2H4 culture−1 h−1, the

lowest value being measured in Klebsiella sp. strain R3 and

the maximum, in Klebsiella sp. strain R4 (Table 2). Both of

these species were isolated from sago root (Table 1). All of

the partial sequences of the 16S rRNA gene determined in

NFB have been submitted to the DDBJ database under the

accession number AB270603 to AB270612.

Starch-degrading bacteria, Bacillus sp. strain B1 (acces-

sion number, AB270626), was isolated from sago starch;

pectin-degrading bacteria, Burkholderia sp. strain BT1

(AB271166) and Paenibacillus sp. strain P1 (AB271167),

and hemicellulose-degrading bacteria, Agrobacterium sp.

strain HMC1 (AB270627) and Flexibacter sp. strain HMC2

(AB270628), were isolated from sago root (Table 1).

Table 1. Bacterial strains isolated from sago palm in this study

Bacterial strains

isolated

Isolated

from

Accession number for

16S rRNA gene sequences

determined in this study

16S rRNA gene sequences comparison

Closest relative (accession number) % similarity

Nitrogen fixers

R1 Root AB270612 Pantoea agglomerans (AM184307) 100

R2 Root AB270610 Enterobacter cloacae (AY914097) 98.4

R3 Root AB270608 Klebsiella oxytoca (AY873801) 99.8

R4 Root AB270605 K. pnuemoniae (DQ379506) 100

S1 Starch AB270611 E. cloacae (DQ379506) 98.4

S2 Starch AB270603 K. pnuemoniae (DQ379506) 99.8

S3 Starch AB270607 P. agglomerans (AM184307) 99.8

M Midrib AB270606 P. agglomerans (AM184307) 100

T1 Trunk AB270604 K. pnuemoniae (DQ379506) 99.5

T2 Trunk AB270609 K. oxytoca (AY873801) 99.8

Starch-degraders

B1 Starch AB270626 Bacillus megaterium (DQ872156) 100

Pectin-degraders

BT1 Root AB271166 Burkholderia tropicalis (AY321307) 100

P1 Root AB271167 Paenibacillus sp. (AY728023) 99.4

Hemicellulose-degraders

HMC1 Root AB270627 Agrobacterium tumifaciens (DQ14505) 99.5

HMC2 Root AB270628 Flexibacter sancti (AB078068) 98.8


Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequence analysis of sago root, starch, midrib and trunk bacteria. Scale bar indicates 10%

sequence divergence. Strain names are preceded by their GenBank accession numbers and the date of sample collection is written in parenthe-

ses. DNA sequences of some control strains were obtained from the DDBJ database.

Table 2. Nitrogen-fixing activities (acetylene-reducing activity) of parts of sago palm plants collected from Hilsug, Leyte, Philippines in March

2005 and strains isolated from the plant

Plant part of isolationAcetylene-reducing activity

(nmol C2H4 g−1 day−1)

Strains isolated from

the corresponding plant part

Acetylene-reducing activity

(nmol C2H4 culture−1 h−1)

Root 26±14a) Pantoea sp. strain R1 15±9.2

Enterobacter sp. strain R2 5.3±8.1

Klebsiella sp. strain R3 2.5±0.4

Klebsiella sp. strain R4 19±0.1

Starch 210±21 Enterobacter sp. strain S1 11±1.4

Klebsiella sp. strain S2 14±3.1

Pantoea sp. strain S3 15±0.8

Midrib 66±22 Pantoea sp. strain M 12±3.2

Trunk 590±0.8 Klebsiella sp. strain T1 2.4±0.5

Klebsiella sp. strain T2 4.4±0.2

a) Mean values in triplicates.

SHRESTHA et al.64

PCR-DGGE analysis of nifH genes from the pure cul-

tures and the roots and starch

DGGE revealed the diversity of the nitrogen-fixing

microbial populations in sago root and sago starch (Fig. 2).

There were at least 5 prominent bands in the sago root

(Fig. 2, bands a, b, c, d and e) and in the starch sample

(Fig. 2. band g, h, i, j and k). The bands of 4 isolated nitro-

gen fixers were clearly separated (A, B, C and D). Although

the band corresponding to Klebsiella sp. strain R3 was not

observed in the root or starch sample, the bands d, e, and l

were identical to the positions of the remaining 3 reference

strains and thus were excised and sequenced (Fig. 2). The

sequences of bands “d”, “e” and “l” were identical to nifH

gene sequences (100% homology) of the reference strains

of bands A, B, and D, respectively (Fig. 3).

NFA under different carbon sources

Among all the carbon sources, almost all the NFB pre-

ferred glucose for high NFA and some preferred sucrose

(Table 3). The highest nitrogenase activity was found to be

9.1±0.9 nmol C2H4 culture−1 h−1 (Pantoea sp. strain M) with

glucose and 11±1.4 C2H4 culture−1 h−1 (Klebsiella sp. strain

T1) with sucrose. The use of other carbon sources like man-

nitol, lactate, cellobiose, galactose, mannose, and galactou-

ronic acid resulted in little or no nitrogenase activity and the

same results were obtained with the polymers like starch,

pectin and hemicellulose.

NFA with indigenous dominant bacteria

The consortium of NFB plus indigenous bacteria showed

increased NFA in all the cases: from 2.1–31 (single culture)

to 20–280 (dual culture) nmol C2H4 culture−1 h−1 (Table 4).

However, in Pantoea sp. strain M+group G and Klebsiella

sp. strain T2+group J, the ARA activity was not so

enhanced, as shown by the single culture of NFB. No ARA

activity was measured in any of the groups of indigenous

bacteria (data not shown).

NFA with starch-degrading bacteria

The ARA of individual cultures of Enterobacter sp. strain

S1, Klebsiella sp. strain S2 and Pantoea sp. strain S3 in

semi-solid N-free starch medium was 0.1, 1.5 and 0 nmol

C2H4 culture−1 h−1, respectively. Starch-degrading Bacillus

sp. strain B1 did not show any ARA. The consortium of

Bacillus sp. strain B1 and Enterobacter sp. strain S1, Kleb-

siella sp. strain S2, or Pantoea sp. strain S3 showed signifi-

cantly (P<0.01) enhanced NFA. Bacillus sp. strain B1 plus

Enterobacter sp. strain S1 showed the highest level of nitro-

genase activity, 270 nmol C2H4 culture−1 h−1, while Kleb-

siella sp. strain S2 and Pantoea sp. strain S3 showed 260

and 150 nmol C2H4 culture−1 h−1 respectively (Table 5).

Fig. 2. nifH DGGE banding patterns from sago palm root and starch sample. Lane 1 and 2 are sago root replications; Lane 3 and 4 are sago

starch replications. Marker “M” is positive control (A, Klebsiella sp. strain S2; B, Enterobacer sp. strain R2; C, Klebsiella sp. strain R3; D,

Pantoae sp. strain R1).


The population density of NFB (all NFB isolated in this

study) in individual cultures ranged from 0.2 to 2.3×106 cfu

ml−1 while in dual cultures with B. megaterium strain B1, it

increased in all cases by ca. 100 times, ranging from 0.1 to

1.4×108 cfu ml−1. The population density of Bacillus sp.

strain B1 in single cultures was 3.0×108 cfu ml−1 which was

1.5 times higher than that of Bacillus sp. strain B1 (1.4 to

2.0×108 cfu ml−1) in dual cultures (Table 5).

Fig. 3. Phylogenetic inference cluster analysis based on the nifH gene sequencing. Scale bar indicates 10% sequence divergence. GenBank

accession numbers of the nifH sequences are given in parentheses. DNA sequences of some control strains were obtained from the DDBJ

database.

Table 3. Effect of carbon sources on nitrogen-fixing activity (acetylene-reducing activity)

StrainAcetylene-reducing activity (nmol C2H4 culture−1 h−1)

Glua) Suc Man Lac Gal Galt Mann Cell Sta Pec Hem

Pantoea sp. strain R1 4.3±1.9 5.6±1.3 0.5±0.1 0.2±0.1 1.2±0.1 0 0 1.1±0.2 0.8±1.6 0.4±0.7 0

Enterobacter sp. strain R2 7.2±2.1 5.3±1.6 0.4±0.2 0.1±0.1 0.1±0.1 0 0.1±0.1 2.1±1.2 1.9±1.9 0 0

Klebsiella sp. strain R3 6.6±3.7 6.4±0.7 6.7±0.3 0.1±0.1 0 0 0 0 0.8±0.1 0 0

Klebsiella sp. strain R4 6.6±2.4 7.1±0.8 0.5±0.1 0 0 0 0 0 0 0.7±0.5 0.2±0.1

Enterobacter sp. strain S1 4.2±2.5 6.4±2.8 0.3±0.0 0.1±0.1 NT NT NT 0.2±0.1 0.8±0.9 0.2±0.2 0

Klebsiella sp. strain S2 8.9±3.6 6.7±5.6 0.1±0.3 0.3±0.3 NT NT NT 0 0.2±0.2 0 0

Pantoea sp. strain S3 6.7±3.5 4.4±2.5 0.2±0.2 0 NT NT NT 0 0 0 0

Pantoea sp. strain M 9.1±0.9 3.7±1.6 0.4±0.2 0.2±0.1 NT NT NT NT 0 0.1±0.3 0

Klebsiella sp. strain T1 4.8±1.3 11±1.4 0 0 NT NT NT NT 0.3±0.1 0 0.4±0.6

Klebsiella sp. strain T2 8.1±0.7 8.3±0.5 0.1±0.1 0 NT NT NT NT 0.1±0.1 0 0

a) Glu, Glucose; Suc, Sucrose; Man, Mannitol; Lac, Lactate; Gal, Galactose; Galt, Galactouronic acid; Mann, Mannose; Cell, Cellobiose; Sta,

Starch; Pec, Pectin; Hem, Hemicellulose

Mean values in triplicates

NT: Not tested.

SHRESTHA et al.66

NFA with hemicellulose-degrading bacteria

Individual cultures of hemicellulose-degrading Agrobac-

terium sp. strain HMC1 and Flexibacter sp. strain HMC2,

nitrogen-fixing Pantoea sp. strain R1, Enterobacter sp.

strain R2, Klebsiella sp. strain R3 and Klebsiella sp. strain

R4 in N-free semisolid hemicellulose medium showed

extremely low ARA (Table 6). In contrast, the nitrogenase

activity of the consortium of Agrobacterium sp. strain

HMC1 or Flexibacter sp. strain HMC2 and NFB, both types

of bacteria were isolated from the same sago root sample,

showed markedly stimulated NFA (Table 6). The highest

level of activity, 38 nmol C2H4 culture−1 h−1, was measured

in the co-culture of P. agglomerans strain R1 plus Flexi-

bacter sp. strain HMC2, followed by that of Pantoea sp.

strain R1 plus Agrobacterium sp. strain HMC1 (31 nmol

C2H4 culture−1 h−1). No ARA was measured on the single

inoculation of Agrobacterium sp. strain HMC1 and Flexi-

bacter sp. strain HMC2 in N-free Rennie medium (data not

shown).

NFA with pectin-degrading bacteria

The ARA of NFB (Pantoea sp. strain R1, Enterobacter

sp. strain R2, Klebsiella sp. strain R3, Klebsiella sp. strain

R4) in individual cultures was very weak (0–0.7 nmol C2H4

culture−1 h−1) or almost non-existence. NFB in the consor-

tium with pectin-degrading Burkholderia strain sp. BT1

(0.2–0.4 nmol C2H4 culture−1 h−1) and with Paenibacillus sp.

P1 strain (0.3–0.7 nmol C2H4 culture−1 h−1) didn’t show

stimulated ARA.

Table 4. ARA of co-cultures of nitrogen-fixing bacteria and non-nitrogen-fixing bacteria

Root isolatesAcetylene-reducing activity (nmol C2H4 culture−1 h−1)

Single +Group A +Group B +Group C +Group D +Group E

Pantoea sp. strain R1 17±14a) 180±23* 49±11 210±12* 230±25** 190±31*

Enterobacter sp. strain R2 2.1±1.4 120±12** 95±21** 130±6.5** 71±12** 42±12*

Klebsiella sp. strain R3 31±17 130±11** 120±1.2** 57±11* 81±16** 48±7.1

Klebsiella sp. strain R4 16±0.8 150±12** 81±16** 190±6.3** 280±15** 190±15**

Midrib isolate +Group F +Group G

Pantoea sp. strain M 21±6.1 67±11** 31±2.8

Trunk isolates +Group H +Group I +Group J

Klebsiella sp. strain T1 4.5±1.2 59±4.6** 92±13** 20±1.1

Klebsiella sp. strain T2 16±1.9 60±2.1** 99±2.8** 21±1.6

a) Mean values in triplicate

* Significant at P<0.05, ** significant at P<0.01

Each of the groups contained 5 non-nitrogen-fixing bacteria

Groups A, B, C, D and E were isolated from sago root

Groups F and G were isolated from sago midrib

Groups H, I and J were isolated from sago trunk

NT: Not tested.

Table 5. Effect of co-culture with a starch-degrading bacterium on nitrogen-fixing activity (acetylene-reducing activity) and population density

of NFB and Bacillus sp. strain B1

Strain


(nmol C2H4 culture−1 h−1)Population density (cfu ml−1) of

NFB NFB Bacillus sp. strain B1

Single culturewith Bacillus sp.

strain B1Single culture

with Bacillus sp.

strain B1with NFB

Enterobacter sp. strain S1 0.1±0.2 270±15** 0.2×106 1.4×108 2.0×108

Klebsiella sp. strain S2 1.5±0.1 260±6.5** 2.3×106 0.9×108 1.4×108

Pantoea sp. strain S3 0 150±4.5** 0.2×106 0.1×108 0.1×108

Bacillus sp. strain B1 0 3.0×108


NFA under reduced oxygen concentrations and in cell

filtrates of indigenous bacteria

Under normal atmospheric conditions, the ARA of NFB

varied from 25 to 44 nmol C2H4 culture−1 h−1 (Table 7). In

contrast, all of the NFB tested in this experiment showed

stimulated ARA under reduced oxygen concentrations. The

amount of oxygen in the reduced oxygen conditions was

268 µmol (equivalent to 67% of removed oxygen from the

vial) and the most ARA under such conditions was exhib-

ited by Klebsiella sp. strain R4 (120 nmol C2H4 culture−1 h−1).

In the other NFB listed in Table 7, increased NFA was

observed, ranging from 65 to 110 nmol C2H4 culture−1 h−1.

The ARA of NFB in the cell-free filtrate fluids of each

group of indigenous microbes was found not to be stimu-

lated (data not shown), compared to the ARA measured

under the reduced oxygen conditions.

Discussion

We previously reported the existence of NFB (K. pnue-

moniae, K. oxytoca, E. cloacae, and P. agglomerans) in dif-

ferent parts of sago palm40). In this study we again isolated

NFB belonging to the same species from sago samples. The

sequence similarity of our previously isolated NFB (Kleb-

siella sp. strain RD3, Klebsiella sp. strain RD4, Klebsiella

strain sp. RH2, Klebsiella sp. strain RH1, Enterobacter sp.

strain RA1 and Pantoea sp. strain RA2) and the strains iso-

lated in this study showed 99–100% homology among K.

pneumoniae strains, 98.4 to 100% among E. cloacace

strains and 100% among K. oxytoca strains and P. agglom-

erans strains. This suggested the generality of the identified

species colonizing sago palm.

In an attempt to confirm the existence of isolated NFB in

the plant tissues of sago palm, PCR-DGGE patterns target-

ing the nifH gene were compared between the isolated

strains and the community DNA samples from which the

strains were isolated. The presence of several dominant

bands directly reflected the existence of NFB in the sago

root and starch (Fig. 2). Out of four strains tested (K. pnue-

moniae, P. agglomerans, E. cloacae and K. oxytoca), identi-

cal band positions of the former three strains were detected

in the root and starch samples, suggesting that these NFB

may be colonizing the sago root and starch. The nifH

sequences from the excised DGGE bands (Fig. 2., band “d”,

“e” and “l”) were identical with those from pure culture

NFB (Fig. 2., band “A”, “B” and “D”), further supporting

that these NFB are truly colonizing the sago root and starch.

The occurrence of non-identical bands in DGGE (Fig. 2.

band “a”, “b” and “c” in sago root and “g” and “h” and “i”

in sago starch) as compared to the tested strains indicated

that there may exist some other NFB that were not isolated

by the culture-based approach in this study. PCR amplifi-

cation of nifH sequences, followed by their separation

through DGGE, has been used to examine the complexity

Table 6. Effect of co-culture with hemicellulose-degrading bacteria on nitrogen-fixing activity (acetylene-reducing activity)

Strain

Acetylene-reducing activity (nmol C2H4 culture−1 h−1)

Single cultureculture with:

Agrobacterium sp. strain HMC1 Flexibacter sp. strain HMC2

Pantoea sp. strain R1 0.1±0.5a) 31±15* 38±13*

Enterobacter sp. strain R2 0.1±0.1 28±14** 28±2.7**

Klebsiella sp. strain R3 0.1±0 16±13** 26±3.1**

Klebsiella sp. strain R4 0 30±13** 25±4.1**

a) Mean values in triplicate.

Table 7. Effect of oxygen concentration on nitrogen-fixing activity

(acetylene-reducing activity)

Strain


(nmol C2H4 culture−1 h−1)

Normal Reduced

Pantoea sp. strain R1 41±5.3a) 85±19*

Enterobacter sp. strain R2 26±17 97±8.4**

Klebsiella sp. strain R3 42±0.4 110±23*

Klebsiella sp. strain R4 25±0.5 120±4.4**

Enterobacter sp. strain S1 41±16 72±2.3*

Klebsiella sp. strain S2 33±15 65±3.6**

Pantoea sp. strain S3 41±0.7 81±8.7**

Pantoea sp. strain M 36±4.9 72±14**

Klebsiella sp. strain T1 40±16 95±2.7**

Klebsiella sp. strain T2 44±1.5 72±7.9**

a) Mean values in triplicate.

SHRESTHA et al.68

and stability of the diazotroph assemblage found in the

Spartina rhizosphere35). Several other studies4,6,7,34,38,41,45,47)

also showed that the analysis of partial nifH gene sequences

can provide information on the phylogeny and composition

of a diazotrophic community.

In our study, the nitrogenase activity of the consortium

of NFB with indigenous bacteria was remarkably increased

in all the groups of the consortium (Table 4). Many con-

sortium-based studies have suggested stimulated

ARA19,20,23,26,37,48). The evidence presented here also supports

the hypothesis that nitrogen fixation in a naturally occurring

bacterium is usually affected by interactions with other

microbes44). The reduction in the concentration of oxygen in

the culture medium by indigenous dominant bacteria could

result in anaerobic condition that could be preferred by

NFB. In contrast, it is known that the presence of growth

factor(s) in the cell filtrate of a bacterium can promote the

growth and function of the accompanying microbes42).

Therefore, the mechanisms whereby indigenous bacteria

enhance nitrogen fixation were estimated by measuring the

ARA of NFB (i) at a reduced oxygen level and (ii) in cell-

free filtrate fluids from a 48-h broth culture of indigenous

bacteria. Increased NFA at the reduced oxygen level (67%

reduction) suggested that the sago nitrogen fixers favor low

oxygen levels for high NFA (Table 7), like in the papers by

van de Broek et al.46) and Zlotnikov et al.48), who found that

most diazotrophs decreased their NFA under aerobic con-

ditions. Minamisawa et al.29) reported that Clostridium sp.

was able to grow and fix nitrogen after the accompanying

bacteria had eliminated the oxygen by respiration. No or

very weak ARA of NFB was detected in cell-free filtrates of

indigenous bacteria, suggesting that enhanced NFA through

the metabolites by indigenous bacteria may not be the case.

This result further suggested that sago NFB might favor low

oxygen levels for expressing high NFA. Haahtela et al.10)

reported that anaerobic conditions were required for maxi-

mum nitrogenase activity of facultatively anaerobic NFB

like K. pnuemoniae and E. agglomerans. It is known that

dinitrogen-fixing bacteria grow best in the presence of other

heterotrophic bacteria which may stimulate the nitrogen-fix-

ing bacteria and in turn receive required N compounds44).

Zlotnikov et al.48) showed a binary association of Bacillus

firmus E3 (promoter) and Klebsiella terrigena E6 (nitrogen

fixer), both of which were isolated from the rhizosphere of

Dactylus glomerata: 23.7 (single culture) to 111.2 (dual cul-

ture) nmol C2H4 culture−1 h−1. Holguin et al.20) reported that

the NFA that occurred in the rhizosphere of mangrove

plants was probably not the result of the individual nitro-

gen-fixing strains, but the sum of interactions between

members of the rhizosphere community.

In this study, a consortium of starch-degrading bacteria,

Bacillus sp. strain B1 plus NFB isolated from the sago

starch, showed markedly increased nitrogenase activity in

N-free medium containing starch as a sole C source (Table

5). This result suggested that Bacillus sp. strain B1

degraded starch into cellobiose or glucose, which could be

utilized by NFB for nitrogen fixation. In a different experi-

ment, we measured the ARA of NFB in glucose and cellobi-

ose. The activity varied from 4.2 to 9.1 and 0 to 2.1 nmol

C2H4 culture−1 h−1 in glucose and cellobiose, respectively

(Table 2), suggesting that glucose was preferably utilized by

NFB. The degradation of starch might have a beneficial

effect on the nitrogen fixation system of sago palm. This

was supported by the result that the population numbers of

NFB and Bacillus sp. strain B1 in single cultures were

increased by dual culture with Bacillus sp. strain B1 and

NFB, respectively.

The increased NFA in the consortium of hemicellulose-

degrading microbes, Agrobacterium sp. strain HMC1, and

Flexibacter sp. strain HMC2 plus NFB in hemicellulose-

based N-free medium, suggested that these microbes

degraded hemicellulose and released products like glucose,

galactose, mannose, and cellobiose which may be further

utilized by NFB. Flexibacter spp. are common soil and

freshwater saprophytes species, and none have been defined

as pathogens28). The genus Flexibacter spp. requires com-

plex media for good growth and digests polysachharides

like cellulose, agar, and chitin28). In our study, the consor-

tium of Agrobacterium sp. strain HMC1 and Flexibacter sp.

strain HMC2 plus NFB showed enhanced NFA when mea-

sured in semi-solid N-free hemicellulose media. That there

was no nitrogenase activity of pure NFB in hemicellulose

media also indicated that the hemicellulose was not utilized

by NFB alone for nitrogen fixation (Table 3). Hemicellulose

which constitutes the most abundant constituent of plants

especially in the roots24), is made up of water-insoluble

polymers consisting of various hexoses and pentoses17) and

can be degraded by bacteria, actinomycetes, and fungi24).

According to our study, the degradation of hemicellulose by

indigenous microbes existing in the natural habitat of sago

palm, could have a beneficial effect on NFA in sago palm.

The consortium of Cellvibrio sp. (hemicellulose-degrading

bacteria) plus Clostridium butyricum, studied in hemicellu-

lose medium as a form of crude xylan, showed a significant

increase in NFA21). Halsall et al.13) reported that straw

(hemicellulose major component) could not be utilized as

an energy source for nitrogen fixation by pure culture NFB,

but Halsall and Gibson11) found enhanced NFA using straw


as a carbon source in a consortium of Azospirillum spp.

(NFB) and Cellulomonas gelida.

No enhanced nitrogenase activity was observed in the

consortium of NFB plus Burkholderia sp. strain BT1 and

NFB plus Paenibacillus sp. strain P1, suggesting that col-

laborative nitrogen fixation using pectin might not occur in

the sago palm. Pectin is a complex of polymers present in

the cell wall of higher plants24) and the mucilage found in

root tips consists of polygalactoruonic acid, a pectin-like

mucus8). However, the study of carbon utilization ability

suggested that neither pectin and nor its monomer (polyga-

lactouronic acid) were utilized by NFB for nitrogen fixation

(Table 3). Hence, we might conclude from our laboratory

experiment that in the sago palm’s natural environment

there might be little possibility of utilizing pectin as a

substrate for NFA by NFB living in a consortium or as indi-

viduals. However, Khammas and Kaiser23) reported that

consortiums of different Azospirillum spp. with Bacillus

polymyxa or B. subtilis enabled the efficient utilization of

pectin as carbon and energy sources for nitrogen fixation.

In most previous studies about consortiums of NFB and

non-NFB, the origin and habitats were not clear9,11,13,14) and

therefore, whether beneficial interactions occur or not in

natural microhabitats remains unkown. In our study, all of

the NFA of different Enterobacteriaceae strains and of

different indigenous microbes used in the consortium study

was isolated from the same sago palm. Therefore, the bene-

ficial interactions between these microbes may occur in the

sago palm’s natural microhabitats.

Our overall results suggested the importance of bacterial

associations and this collaborative fixation of nitrogen by

NFB and indigenous bacteria could enable them to sustain

high levels of nitrogen acquisition in natural environments.

In this study, some of the genera of the indigenous nitrogen-

fixing bacteria associated with sago palm have not been iso-

lated (the bands a, b, c, h, i in Fig. 2). It is well known that

there are many uncultivable microorganisms in the natural

environment22,39). In addition, no efforts were made to iso-

late slow growing or anaerobic microorganisms in this

study, that could play an important role in some beneficial

microbial interactions22). Further study is, therefore, neces-

sary to evaluate their contribution to the nitrogen fixation

process in sago palm.

Acknowledgements

Archana Shrestha is the recipient of a scholarship from the

Jinnai International Study Exchange Program. We would like

to express special thanks to the sponsors of that program.

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Enhancement of Nitrogen-fixing Activity of