Biosci. Biotech. Res. Comm. 6(2):
Estimation of diversity, abundance and composition of bacterial population in tropical lake sediments using terminal restriction fragment technique
Department of Zoology, University of Pune, MS, India
Soil bacteria carry out various ecological roles in the ecosystem. Diverse microbial community could be present in the soil. Very less knowledge is available regarding the association between soil structure and bacterial diversity. Soil bacteria are good indica- tors of soil health and soil fertility. The bacterial communities of soil/sediment of lake are affected by the environmental biotic, abiotic, natural and/or man made conditions. In the present study, bacterial diversity of soil/sediment of Sawanga Vithoba lake has been assessed by full form terminal restriction fragment length polymorphism in December and March. Bacterial diversity has been found to be rich, which ranged from 1 to 28 with an average number of restriction fragments 14.
KEY WORDS: TERMINAL RESTRICTION, FRAGMENT TECHNIQUE, MIC O IAL BIODIVE SITY, LAKE SEDIMENTS
Soil bacteria are vital components of the ecosystems and microbial community in the soil has been reported to be rela- tively diverse (Curtis et al., 2002; Robe et al., 2003), with the highest prokaryotic diversity compared to other environments (Van et al., 2006; Roesch et al., 2007). One gram of soil has been reported to contain up to 10 billion microorganisms and thousands of different species (Knietch et al., 2003). Soil bac- teria are genetically diverse and represent a major unexploited genetic resource (Whitman et al., 1998).
Diversity, abundance and composition of microbial commu- nities within soils are depth dependent. Changes in microbial community structure with soil depth are due to the response of
*Corresponding Author Received 20th October, 2013
Accepted after revision 31st December, 2013 BBRC Print ISSN:
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microbes to the
Microaggregates (2 to 20 μm) are considered to be the most favorable habitat for bacteria in most types of soil (Ranjard and Richaume, 2001), with a higher abundance of bacteria located in micropores (~2 μm) of the inner aggregate fraction (Hattori, 1988; Ranjard et al., 2000). Little is known regarding
the association between soil structure and bacterial diversity (Ranjard et al., 2000),
Microbial community composition may change with the location within
Anaerobic microsites within aggregates in aerobic soils may allow for a diverse range of aerobic- and
Soil bacteria are good indicators of soil health, soil fertility (Yao et al., 2000; O’Donnell et al., 2001) and ecosystem status in much more comprehensive way than physical or chemi- cal measures (Fierer and Jackson, 2006; Winding et al., 2005). Bacteria from specific taxa have been allied to definite eco- logical characteristics. For example, the presence of nitrogen fixers such as Rhizobia and Azotobacter and nitrifying bacteria
Bacteria are also responsive to man made activities such as agriculture, pesticide use and pollution (Deiglmayr et al., 2004; Klumpp et al., 2003). Soil microbial communities may alter their metabolic and genetic capability in response to changes in the environmental factors which result in rapid shifts in bacterial diversity within short period of time frame (Schmidt et al., 2007). Understanding soil microbial communities is therefore an ideal method to monitor the ecological changes occurring between seasons as well as over an extended period of time (Hill et al., 2000).
The variability of species richness and diversity index among enzyme digestions may be related to the fact that the same
(Marsh et al., 2000). The species of bacteria producing a par- ticular
In the resent communication, it is hypothesized that soil/ sediment bacterial communities of the Sawanga Vithoba lake do exhibit diversity and affect the ecological processes in the lake. The bacterial communities of soil/sediment of lake are affected by the environmental biotic, abiotic, natural and/or man made conditions. To my knowledge, no previous study has examined the bacterial diversity of the of soil/sediment of Sawanga Vithoba lake. It is hypothesized that the biogeo- graphical patterns exhibited by soil/sediment bacterial com- munities of lake could be fundamentally similar to the patterns observed with plant and animal taxa. Soil/sediment bacterial communities of lake could be a good predictor of the status of the particular ecosystem, metabolic processes going on inside that ecosystem and energy balance. In this study, soil/sedi- ment’s bacterial diversity were studied to monitor the ecologi- cal system in this region by
MATE IALS AND METHODS
For the present study, three stations were selected. Station two is west station, station three is north station and station four is south station. Station two is near the village (Sawanga Vithoba). Other stations of the lake are surrounded by veg- etations. Soil(sediment) samples from the lake bottom were collected as described in the soil kit (make: Prerana), samples were transformed to sterilized plastic or glass containers and transported to the laboratory. The bacterial analysis was per- formed in the months of December and March.
a. DNA isolation from environmental samples
For processing of soil samples 0.5g of soil was added to 2ml
The DNA was deproteinised thrice with
acetate and absolute ethanol. Dried DNA was dissolved in 30μl nuclease free water. Quality assessment of genomic DNA was performed by 1% agarose gel electrophoresis and estimated using aQubit^TM Fluorometer(Invitrogen,USA). DNA extrac- tion from positive control sample (E. Coli) was also performed to rule out the possibility of extraction failure. Similarly, a negative control (plain saline) was also subjected to extraction to establish the clean reagents.
b. PCR amplification of the 16S gene
The bacterial 16 S rRNA gene was amplified by PCR using the primer set
c. Restriction digestion and desalting of digested products
Following PCR, 10μl of PCR products were digested with 0.5 U of Taq I restriction enzymes (New England Biolabs) for 3 hours at 37°C in 20μl reaction volumes. Digests were separated on 3% agarose gels in 1X TBE buffer containing ethidium bro- mide, and visualized under UV light.
10μl of the digested DNA was desalted using the follow- ing procedure: 2.5μl of 125mM EDTA and 1/10 volume of 3M sodium Acetate pH 5.2 was added to 10μl of digested DNA. Further 2.5 volume of ice cold ethanol was added into the tubes and mixed well. Tubes were then centrifuged at 12000 RPM for 20 minutes at 18°C. Supernatant was removed being careful not to dislodge the pellet. Pellet was then washed with 60μl of 70% Ehtanol twice with centrifugation at 12000RPM for 20 minutes at 18°C temperature. Pellet was then dried at 37°C for 30 minutes.
d. Sample preparation and loading
e. Gene Mapper data analysis
Gene Mapper software based analysis was performed for frag- ment analysis after completion of the capillary electrophoresis. Output from automated sequencers is in the form of an elec- tropherogram, with peaks representing fluorescently labeled
tively. Software specific to each sequencing unit collects data from each run. The ABI 3730 capillary sequencer operates, Gene Mapper v3.5 (Applied Biosystem), which performs the functions of both GeneScan and Genotyper. Either data col- lection program provides researcher with several algorithms for sizing sample fragments by comparing their mobility with that of the size standard. Once data are processed and frag- ment lengths assigned, the data set is typically imported into a spreadsheet program, such as Microsoft Excel (Microsoft Corp., Redmond, WA). In the
RESULTS AND DISCUSSION
TABLE 1: Total number of terminal Restriction
Total No of
The bacterial richness is the types of fragments obtained and elucidated in the
is a north station of the lake. Total number of 50 different taxa (designated as allele in the supplementary data) were observed as per the size of the
The result of a
In the present study, bacterial diversity of soil/sediment of lake Sawanga (Vithoba) was assessed. The soil /sediment bac- teria were tried to identify upto phylum level by 16S rDNA. Employing the 16S rDNA gene for this purpose is proper due to the ubiquitous nature of the gene and extremely low mutation rate and its phylogenetic significance (Ludwig1999; Ludwig and Schleifer1994). RFLP analysis of the 16S rDNA was used
as an initial estimator of bacterial diversity. Indeed, it is well established that RFLP analysis of 16S rDNA can be used to study bacterial diversity (Moyer et al., 1994; Moyer, 1996).
In Table No. 4, the probable bacteria in the sediment/soil sample of the Sawanga Vithoba lake have been tested.
The composition and diversity of soil bacterial communities have a straight effect on many ecosystem processes (Schimel, 1995; Balser et al., 2002). Diversity of soil bacterial com- munities, soil microbial ecology, diversity of soil bacteria are affected by environmental biotic and abiotic factors (Buckley and Schmidt, 2002).
Ecological monitoring is crucial for assessing the environ- mental effects. There are previous studies on characterization of the dominant bacteria in environmental samples by using 16S rDNA molecular signatures . RFLP analysis of 16S rDNA can be used to study bacterial diversity. The bacterial diversity in soil is high (Dunbar et al., 2002; Tringe et al., 2005). More species could be found with greater sampling sizes. Thus, this study has been successful at establishing a baseline of bacte- rial diversity in soil/sediment of Sawanga (Vithoba) lake of Pune, India.
Special thanks to Director of
FIGURE 1: Bacterial analysis of Sawanga Vithoba lake sediment samples in ecember (winter) and in March (summer).
FIGURE 2: Electropherogram of the bacterial popu- lation for the North station in December.
FIGURE 3: Electropherogram of the bacterial population for the South station in December.
FIGURE 4: Electropherogram of the bacterial population for the station two (west station) in March.
FIGURE 5: Electropherogram of the bacterial pulation for the station three (north station) in March.
FIGURE 6: Electropherogram of the bacterial population for the station four(south station) in March.
TABLE 2: Probable bacterial communities in
TA LE 3: Probable bacterial communities in Sawanga-
Vithoba lake soil of station 2
2EF528288 acillus subtilis subsp.
3EF656455 acillus licheniformis
4GQ903382 Bacillus firmus
5GU097448 Rhizobium sp. R5.
6AY191846 Ralstonia sp. 12D.
7A 091196 Frateuria aurantia IFO3249.
8EF620455 Stenotrophomonas maltophilia.
9AB091201 Frateuria aurantia
10EU624430 Bacillus cereus
11GQ903396 Bacillus firmus
12EU248957 Geobacillus sp. H6a.
13GQ903410 Salimicrobium halophilum
14AJ301830 Enterococcus faecium (T) LMG 11423.
15Ay685145 Selenomonas ruminantium L6.
TABLE 4: Probable bacterial communities in Sawanga- Vithoba lake soil of station 3
1AF300975 Bacterium C16S.
2AM111055 Psychrobacter sp. 7317.
3AB188223 Isoptericola sp. TUT1252.
4AM111092 Pseudomonas sp. 8058.
5EF528288 Bacillus subtilis subsp. Subtilis
6EF656455 Bacillus licheniformis
7EF656456 Bacillus subtilis subsp. Subtilis
8GQ903382 Bacillus firmus
9AJ222546 Anaerobacter polyendosporus.
10Am167521 Streptomyces qinlingensis type
11GU097448 Rhizobium sp. R5.
12AY191846 Ralstonia sp. 12D.
13FM957478 Vibrio sp.
14AB091196 Frateuria aurantia IFO3249.
15EF620455 Stenotrophomonas maltophilia
16AB353074 Stenotrophomonas maltophilia MPU98.
17AB091201 Frateuria aurantia IFO13333.
18EU685825 Bacillus sp.
19EU624430 Bacillus cereus
20EU248957 Geobacillus sp. H6a.
21GQ903410 Salimicrobium halophilum
22DQ358727 Paenibacillus zanthoxyli Jh95.
23AJ301830 Enterococcus faecium (T) LMG 11423.
24Ay685145 Selenomonas ruminantium L6.
2568315145 Stridium botulinum 468 toxin type
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