Isolation And Characterization Srb Defense

Then comes man…

Isolation and Characterization of Sulfate-Reducing Bacteria Found in Himatugan River and Sapangdaku River Near Atlas Consolidated Mining and Development Corporation (ACMDC), Toledo City, Cebu Fiel G. Ybañez

Acid Mine Drainage • outflow of acidic water from metal or coal mines • most severe environmental problem facing the mining industry • consequence: loss of aquatic life

Acid Mine Drainage • exposure of metal sulfide minerals to moisture and air which generates acid > Autooxidation and biooxidation

• FeS2 + 3.5O2 + H2O  Fe2+ + 2SO42- + 2H+ • the acid can dissolve metals like lead, zinc, copper, arsenic, selenium, mercury and cadmium making the ecosystem unsuitable for habitation

• intrinsic capacity to produce alkalinity by microbial sulfate and iron reduction forming iron sulfides and utilization of H+ ions > Sulfate reducing bacteria (SRB) – oxidizes simple organic molecules using the sulfate ion as an electron acceptor producing H2S and HCO3>SRB need organic carbon source and growth substance for attachment

• however, rate of acid production exceeds the rate of acid neutralization

Objectives The primary objective of this study is to isolate and characterize and sulfate-reducing bacteria from surface waters found in Himatugan River, Malubog Lake, and Sapangdaku River in Toledo City, Cebu as possible bioremediators.

This study specifically aims to: 1.  isolate SRB and aerobic bacteria from the benthic regions of the study sites; 2. characterize the isolated SRB and aerobic bacteria affected by acid mine drainage from the study sites; 3.  determine the abundance of SRB and aerobic bacteria found in the study sites during the sample collection period;

4. and, to determine the relationship between pH, temperature, water velocity, sulfate concentration and iron concentration with abundance of SRB and aerobic bacteria.

Methodology

Himatugan River: Reference Site

Atlas draining site: source of Malubog Lake: Sangi: AMD, high diluted AMD, low downstream velocity velocity area

Field Sampli LAB ng Inoculati on

Sample collection with physico-chem parameters (ie flow rate, temperature, pH) Laboratory Analysis

for sulfate and iron concentration

Incubation

Abundance (CFUs and % growth

Isolation

Characteriza tion aerobic bacteria: Gram staining, cell shape, endospore formation, motility, colony morphology, catalase test, and starch hydrolysis SRB: Gram stain, motility, endospore formation, cell Analysis of Data (One-way shape and catalase test

ANOVA & Regression)

Results and Discussion

Average % Cover

Average Percentage Cover of SRB after 6-8 Days Incubation

SRB growth:

150 100 50

86

62

60

Himatugan River Atlas Malubog Lake

20

Sangi

0

Average CFUs/mL for Aerobic Bacteria at 36hrs Incubation for the Study Areas

Aerobic bacteria:

3 Atlas draining

4 Atlas draining site

14000 Average CFUs/mL

2 Sangi

2 Malubog Lake 3 Himatugan River

Study area

1 Himatugan River

1 Sangi

12000 10000

6.99E+03

8.14E+03

8000 6000 4000

Himatugan River

Atlas Drainage Site Malubog Lake

3.46E+03 1.78E+03

2000 0 Study Areas

Sangi

Average pH for the Study Areas 11 pH

10

10.07 9.37 9.4

9

Himatugan River 8.9

Atlas Malubog

8

Sangi Site

pH

temp velocity sulfate iron

 

Himatugan 10.07 25.89

Atlas

9.37 24.11

0.406

19 <0.02

0.52

1716

1.96

Malubog

9.4

26.4

0

279

1.87

Sangi

8.9

26.4

0.11

991

3.82

Inverse (weak) relationship Relationship betw een pH and SRB

Inverse relationship Relationship betw een pH and Aerobic CFUs

grow th

5000

% cover

CFUs/mL

10000 R2 = 0.9303

0 8.8

9

9.2

9.4

100 50

R2 = 0.552

0

9.6

8.8

9

9.2

pH

Relationship Betw een Velocity and SRB Grow th

% cover

% cover

100 R2 = 0.8704

0 23

24

25

26

tem perature in Celsius

Direct

9.6

pH

Relationship Betw een Temperature and SRB Grow th

50

9.4

27

100 50

R2 = 0.5476

0 0

0.2

0.4

Velocity (m /s)

Inverse (weak) relationship

0

% cover

Relationship Betw een Sulfate Concentration and SRB Grow th 100 50 0

R2 = 0.2828 0

500

1000

1500

sulfate concentration (m g/L) Inverse (weak) relationship

2000

A total of 14 kinds of SRB were isolated from the study sites. Himatugan River: 3 Atlas draining site: 1 Malubog Lake: 5 Sangi: 5

23 kinds of aerobic bacteria were isolated from the study areas. Himatugan River: 5 Atlas draining site: 4 Malubog Lake: 7 Sangi: 7

Desulfot Desul Desulf Unknown Unknown omacul fococ ovibrio 1 2 um cus

Gram stain

-

-

-

-

-

Motility

+

NA

+

+

+

Endospore

+

-

-

+

-

Cell shape

Rods

Catalase # traits similar

-

Sphe Rods rical -

Rods/sp Rods/sp heres heres + + 4

3–4

Desulfotom Desulfococ aculum cus/Desulfo vibrio

The most likely isolated SRB Desulfovibrio, Desulfotomaculum and Desulfococcus based on the tests performed. Desulfovibrio: gramnegative, nonsporeforming, anaerobic bacteria, 0.7µm consisting of actively motile curved rods that reduce sulfur compounds to hydrogen sulfide

Desulfotomaculum is a genus of endospore-forming, rodshaped bacteria of the family Bacillaceae, made up of anaerobic, gram-negative cells that reduce sulfates, sulfites, and other sulfur compounds. They are found in soil, water, and geothermal regions, and in the intestines of insects, and in the contents of animal rumens

Desulfococcus is a genus of anaerobic, gram-negative, nonspore-forming, spherical bacteria that reduce sulfate compounds to hydrogen sulfide, found in anaerobic sediments from fresh and marine waters and in sewage sludge

CONCLUSION 23 kinds of aerobic bacteria were isolated from the water samples. 5 from Himatugan river, 4 from Atlas draining site, 7 from the Malubog Lake, and 7 from Sangi Aerobic bacterial growth was primarily affected by pH.

14 kinds of SRB were isolated from the study sites. 3 kinds of SRB from Himatugan River, 1 from the Atlas draining site, 5 from Malubog lake, and 5 from Sangi SRB growth was greatly influenced by temperature, and a weak correlation was found between bacterial growth and velocity, pH, availability of sulfate in water

the most likely isolated SRB were Desulfovibrio, Desulfotomaculum and Desulfococcus.