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
Malubog
Sangi: of
Lake:
AMD,
diluted AMD,
downstream high
low
area velocity
velocity
 Field Sample collection
with physico-chem
Sampli parameters (ie flow
LAB ng rate, temperature,
pH)
Laboratory Analysis
Inoculati for sulfate and iron
on concentration

Incubation

Abundance (CFUs
Isolation
and % growth
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 Percentage Cover of SRB after 6-8 Days
Incubation
SRB growth:
150
Average % Cover

62 86 Himatugan River 1 Sangi

100 60
Atlas
Malubog Lake
2 Malubog
50
20
Sangi Lake
0
3 Himatugan
Study area
River
Average CFUs/mL for Aerobic Bacteria at 36hrs
Incubation for the Study Areas 4 Atlas
Aerobic draining site
bacteria: 14000
12000
Average CFUs/mL

1 Himatugan 10000
8.14E+03 6.99E+03 Himatugan River

River 8000 Atlas Drainage Site

Malubog Lake
6000 3.46E+03
Sangi
2 Sangi 4000 1.78E+03
2000

3 Atlas draining 0
Study Areas
 Average pH for the Study Areas

11
10.07
Himatugan River
10 9.37 9.4
8.9 Atlas
pH

9
Malubog
8 Sangi
Site
 pH temp velocity sulfate iron
 

Himatugan 10.07 25.89 0.406 19 <0.02

Atlas 9.37 24.11 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 relationship Inverse (weak)
Relationship betw een pH and Aerobic CFUs relationship
Relationship betw een pH and SRB
grow th
10000
100
CFUs/mL

% cover
5000
R2 = 0.9303 50
R2 = 0.552
0 0
8.8 9 9.2 9.4 9.6 8.8 9 9.2 9.4 9.6
pH pH

Relationship Betw een Temperature and Relationship Betw een Velocity and SRB
SRB Grow th Grow th

100 100
% cover
% cover

50 50
R2 = 0.8704 R2 = 0.5476
0 0
23 24 25 26 27 0 0.2 0.4 0
tem perature in Celsius Velocity (m /s)

Direct Inverse (weak)

relationship
 Relationship Betw een Sulfate
Concentration and SRB Grow th

100
% cover

50
R2 = 0.2828
0
0 500 1000 1500 2000
sulfate concentration (m g/L)

Inverse (weak)
relationship
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 Sphe Rods Rods/sp Rods/sp
rical heres heres
Catalase - - - + +
# traits 4 3–4
similar Desulfotom Desulfococ
aculum cus/Desulfo
vibrio
The most likely isolated SRB
Desulfovibrio, Desulfotomaculum and
Desulfococcus based on the tests
performed.
Desulfovibrio: gram-
negative, nonspore-
forming, anaerobic
bacteria, 0.7µm consisting
of actively motile curved
rods that reduce sulfur
compounds to hydrogen
sulfide
 Desulfotomaculum is a genus
of endospore-forming, rod-
shaped 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.