DOI: http://dx.doi.org/10.15226/2475-4714/3/1/00133
Keywords: Haloalkaliphilic bacteria; rhizosphere; 16S rRNA gene; Dichanthium annulatum; hydrolytic enzymes
The rhizosphere of halophytes harbors an impressive array of halophilic and alkaliphilic microorganisms. Poly extremophilic organisms have ability to tolerate two or more extreme conditions, such as haloalkaliphiles, halothermophiles and alkalithermophiles [5]. Haloalkaliphiles are organisms that require high salinity (3-30%) and an alkaline pH (pH 9-13) for their growth [6]. These organisms have been isolated and characterized from a number of environments such as saline- sodic lakes, acid mines, hypersaline saline soils, salt mines, marine environments and salt marshes [7, 8]. Haloalkaliphiles usually use small organic molecules (osmolytes, e.g., ectoine, betaine and proline) and intracellular enzymes (α-galactosidase) to maintain their osmotic balance and pH ranges near neutral to survive under extreme saline and basic environments [9, 10]
Haloalkaliphiles have a wide range of applications in biodefense, bioenzymes and biofuel production [11, 12]. These organisms provide a good source of novel alkaliphilic and halophilic enzymes such as proteases, gelatinase, amylases, lipases, cellulases and xylanases [13, 14]. Enzymes isolated and characterized from haloalkaliphiles have ability to function properly even at high pH and salinity. These enzymes can be used in industrial applications such as detergent industry, food stuffs, paper and pulp and pharmaceuticals industry [15, 16].
Though a number of studies have been reported on the isolation of haloalkaliphiles from different environments but this study is the first report on characterization of haloalkaliphilic bacteria from the rhizosphere of Dichanthium annulatum (halophyte) collected from Khewra Salt Mines, Pakistan. In the present study, haloalkaliphilic bacteria were isolated from rhizospheric soil and roots and identified on the basis of 16S rRNA gene sequence analysis. Selected haloalkaliphilic bacterial strains were further characterized for their biotechnological potential and ability to produce different industrially important enzymes (cellulases, proteases, amylases, xylanases and lipases).
Sequences of 16S rRNA gene were assembled and analyzed with the help of Chromus Lite 2.01 sequence analysis software (Technelysium Pty Ltd. Australia). The gene sequences were compared to those deposited in the GenBank nucleotide database using the NCBI BLAST program. Sequences were aligned using Clustal X 2.1 program and phylogenetic tree was constructed using neighbor-joining method. Bootstrap confidence analysis was performed on 1000 replicates to determine the reliability of the distance tree topologies obtained [26]. The evolutionary distances were computed using the Neighbor-joining method [27]. Phylogenetic analyses were conducted in MEGA7 [28]. There were a total of 1434 positions in the final dataset. Sequence of 16S rRNA gene from Micrococcus luteus was sued as outgroup. Bacterial strains identified in this study were submitted in GenBank under the accession numbers MH489029-MH489050.
Bacterial isolates were grown in the presence of different salt concentrations (3-12% NaCl), pH ranges of 4-12 and temperature ranges of 4-42°C by using HaP broth medium. Isolates were cultured in 250 ml flasks at 30°C with continuous rotatory agitation at 150 rpm for 72 h (hours) [29]. During incubation, bacterial growth in terms of optical density (OD 600) was measured after different time intervals (3, 6, 12, 24, 48 and 72 h).
Parameters |
D. annulatum 1 |
D. annulatum 2 |
D. annulatum 3 |
D. annulatum 4 |
pH |
8.11a |
8.29ab |
8.56b |
8.35ab |
EC1:1 (dS/m) |
4.14ab |
3.77a |
4.19ab |
4.65b |
Moisture (%) |
25.83ab |
24.15a |
27.32b |
25.52ab |
Temperature (°C) |
29.23a |
32.52b |
31.01ab |
30.82ab |
Texture class |
Silty loam |
Silty loam |
Silty loam |
Silty loam |
OM (g.Kg-1) |
35.77b |
33.15a |
34.55ab |
32.59a |
P (mg.kg-1) |
3.99ab |
3.26a |
3.82ab |
3.59ab |
K (mg.kg-1) |
0.76a |
0.58b |
0.65b |
0.49a |
Ca (mg.kg-1) |
1.70b |
1.67b |
1.51a |
1.48a |
Mg (mg.kg-1) |
1.28b |
1.15a |
1.26b |
1.19a |
NO-3 (mg.kg-1) |
12.76b |
13.12b |
10.21a |
10.87a |
H+Al (mg.kg-1) |
67.55b |
59.32a |
61.24a |
65.87b |
V (mg.kg-1) |
4.13b |
3.87a |
4.18b |
3.76a |
CEC (mg.dm-3) |
68.45a |
73.61b |
72.73b |
67.78a |
SAR |
10.24b |
13.51a |
11.15a |
12.42b |
Isolate code |
Organism identified |
Accession No. |
Closest type strain in NCBI data base |
Sequence length(bp) |
Sequence similarity (%) |
PGRS1 |
Exiguobacterium |
MH489029 |
E. mexicanum DSM 6208 (JF505980) |
1406 |
99 |
PGRS2 |
Bacillus |
MH489030 |
B. pseudofirmus ATCC 700159 (NR_026137) |
1425 |
100 |
PGRS3 |
Exiguobacterium |
MH489031 |
E. mexicanum DSM 16483 (JF505982) |
1365 |
99 |
PGRS5 |
Kocuria |
MH489032 |
K. rosea ATCC 186 (KM114943) |
1412 |
99 |
PGRS6 |
Oceanobacillus |
MH489033 |
O. oncorhynchi DSM 16557 (KJ145755) |
1305 |
100 |
PGRS7 |
Bacillus |
MH489034 |
B. cohnii DSM 6307 (JF689927) |
1465 |
100 |
PGRS9 |
Bacillus |
MH489035 |
B. alcalophilus JCM 5262 (NR_036894) |
1345 |
99 |
PGRS10 |
Bacillus |
MH489036 |
B. polygoni NCIMB 14282 (NR_041571) |
1513 |
99 |
PGRS11 |
Enterococcus |
MH489037 |
E. durans ATCC 19432 (NR_036922) |
1362 |
99 |
PGRS12 |
Virgibacillus |
MH489038 |
V. halodenitrificans DSM 10037 (HG931337) |
1473 |
100 |
PGRP2 |
Citricoccus |
MH489039 |
C. alkalitolerans KCTC 19012 (KF322100) |
1434 |
99 |
PGRP3 |
Bacillus |
MH489040 |
B. alcalophilus ATCC 27647 (NR_036889) |
1421 |
99 |
PGRP4 |
Kocuria |
MH489041 |
K. polaris CIP 107764 (KF876845) |
1464 |
99 |
PGRP6 |
Bacillus |
MH489042 |
B. halodurans NRRL B-3881 (HQ446864) |
1384 |
99 |
PGRP7 |
Bacillus |
MH489043 |
B. alkalinitrilicus DSM 22532 (NR_044204) |
1298 |
99 |
PGHP1 |
Exiguobacterium |
MH489044 |
E. mexicanum DSM 16483 (JF505982) |
1469 |
99 |
PGHP2 |
Bacillus |
MH489045 |
B. clarkii DSM 8720 (KY849416) |
1405 |
99 |
PGHP4 |
Citricoccus |
MH489046 |
C. alkalitolerans DSM 15665 (KF322104) |
1478 |
99 |
PGHP5 |
Staphylococcus |
MH489047 |
S. equorum ATCC 43958 (AB975354) |
1394 |
100 |
PGHP6 |
Micrococcus |
MH489048 |
M. luteus CCM 169 (KJ843153) |
1443 |
99 |
PGHP8 |
Bacillus |
MH489049 |
B. pseudofirmus DSM 8715 (NR_026139) |
1356 |
99 |
PGHP9 |
Kocuria |
MH489050 |
K. rosea DSM 11630 (KF177263) |
1429 |
99 |
Bacterial strains |
Protease |
Lipase |
Amylase |
Cellulase |
Gelatinase |
Xylanase |
PGRS1 |
- |
++ |
- |
- |
+ |
++ |
PGRS2 |
+++ |
- |
++ |
+ |
++ |
- |
PGRS3 |
++ |
+ |
- |
- |
+ |
++ |
PGRS5 |
- |
+ |
+++ |
- |
++ |
+ |
PGRS6 |
++ |
++ |
++ |
++ |
++ |
- |
PGRS7 |
+++ |
+ |
- |
++ |
- |
++ |
PGRS9 |
- |
++ |
++ |
+ |
+++ |
- |
PGRS10 |
++ |
+ |
++ |
++ |
++ |
+ |
PGRS11 |
++ |
- |
- |
- |
+++ |
- |
PGRS12 |
+++ |
+ |
- |
++ |
+ |
++ |
PGRP2 |
- |
+ |
++ |
++ |
- |
+ |
PGRP3 |
++ |
++ |
++ |
+ |
+++ |
+ |
PGRP4 |
+ |
+ |
+ |
- |
+ |
- |
PGRP6 |
++ |
++ |
++ |
++ |
- |
+ |
PGRP7 |
+++ |
++ |
+ |
+ |
+++ |
- |
PGHP1 |
+ |
+ |
- |
- |
+ |
++ |
PGHP2 |
++ |
++ |
+ |
++ |
++ |
++ |
PGHP4 |
- |
+ |
++ |
+ |
- |
- |
PGHP5 |
+ |
+ |
+ |
+ |
- |
+ |
PGHP6 |
++ |
++ |
++ |
- |
- |
+++ |
PGHP8 |
++ |
++ |
+ |
++ |
++ |
- |
PGHP9 |
- |
+ |
++ |
- |
+ |
++ |
A total of 22 haloalkaliphilic bacterial strains have been identified from the rhizosphere and roots of D. annulatum. Phylogenetic analysis showed that these isolates were related to nine different bacterial genera Bacillus, Exiguobacterium, Kocuria, Citricoccus, Staphylococcus, Enterococcus, Oceanobacillus, Virgibacillus and Micrococcus (Table 2). Previous studies on the isolation of haloalkaliphilic bacterial strains from Soda Lake Magadi (Kenya) showed that Bacillus, Exiguobacterium and Halomonas were the dominant genera identified from these environments [5, 37]. The abundance of Gram positive bacteria (Bacillus, Exiguobacterium, Kocuria, and Staphylococcus) is attributed to their cell wall and endospore formation in Bacillus enable them to survive in hypersaline and saline-sodic environments. Members of Actinobacteria Kocuria, Citricoccus and Micrococcus identified in this study have been previously reported from hypersaline soil of halophytes and Texcoco Lake [38, 39].
Bacterial isolates characterized in this study were alkaliphilic and moderately halophilic in nature. More than 87% strains were able to grow at pH more 10, salt concentrations 5-10% and temperature 28-42°C. Previous studies also reported that alkaliphiles, moderately halophiles and mesophiles are more abundant as compare to extremely halophilic and thermophilic bacteria in different soils [38, 40]. Halophilic strains from the groups, Virgibacillus and Oceanobacillus show optimum growth at salt concentration 5-10% NaCl and 28-37 °OC [41].
Most of the bacterial strains showed ability to degrade different organic compounds such as carbohydrates, lipids, proteins and gelatin. More than 90% bacterial strains showed lipase activity, 73% bacterial strains showed proteolytic activity, 72% strains had ability to degrade carbohydrates, 73% strains showed positive results for gelatinase activity, 64% strains had ability to degrade cellulose and 63% strains showed xylanase activity (Table 3). Alkaliphilic, halophilic and mesophilic bacteria isolated from different saline and saline-sodic environments showed their ability to produce different industrially important enzymes such as amylases, proteases, lipases, gelatinase and xylanase [9, 11]. Enzymes produced by haloalkaliphilic bacteria have structural and catalytic properties to function properly even at high salinity, pH and temperature [42]. Lipase and protease producing alkaliphilic and halophilic bacteria have been previously isolated marine environment and food sources such as fish sauce [37, 43]. Halophilic bacterial strains related to Bacillus and Oceanobacillus are known to be a good source of different hydrolytic enzymes such as α-amylases, lipase, protease and xylanases [14, 42]. Members of Actinobacteria Citricoccus, Kocuria and Micrococcus have been well known for production of lipases, cellulases, amylases and gelatinase [39, 44]. Haloalkaliphilic cellulases and xylanases have been produced by different alkaliphilic and halophilic bacteria such as Kocuria, Bacillus and Staphylococcus [45]. Members of Exiguobacterium have been isolated from hypersaline tropical soils. These bacteria are able to grow at high pH and considered as good source of alkaliphilic enzymes such as proteases, lipases, cellulases and gelatinase [42, 46].
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