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This study was conducted to isolate an acid-producing, alkaliphilic bacterium to reduce the
alkalinity of cement industry waste (cement kiln dust). Gram-positive isolate KG1 grew well
at pH values of 612, temperatures of 2850 C, and NaCl concentrations of 016% and thus
characteristics of the KG1 isolate were consistent with those of the genus Bacillus, and the
Arajo
highest level of 16S rRNA gene sequence similarity was found with Bacillus halodurans strain
Keywords:
from other phylogenetic neighbors belonging to alkaliphilic Bacillus species, the isolated
was further screened for its potential to reduce the pH of an alkaline medium. Phenotypic
DSM 497 (94.7%). On the basis of its phenotypic characteristics and genotypic distinctiveness
Alkaliphiles
strain was designated B. halodurans strain KG1, with GenBank accession number JQ307184
Alkalinity reduction
(= NCIM 5439). Isolate KG1 reduced the alkalinity (by 83.64%) and the chloride content (by
Bacillus sp.
86.96%) of cement kiln dust and showed a potential to be used in the cement industry for a
variety of applications.
2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is
an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Microorganisms have been isolated from a variety of environments but are most abundant in moderate environments.
Several environments can be considered extreme, including
environments with a low or high pH, a low or high temperature, a high salinity, the presence of organic solvents, heavy
metals, etc.1 The microorganisms inhabiting extreme environment are termed extremophiles. Extremophiles have gained
considerable momentum in research due to their ability to
Corresponding author. Permanent address: School of Energy and Environment, Thapar University, Patiala, Punjab 147004, India.
E-mail: anitarajor@yahoo.com (A. Rajor).
http://dx.doi.org/10.1016/j.bjm.2015.11.001
1517-8382/ 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
CKD control
CaCO3 (C)
MgO (Mg)
Al2 O3 (Al)
SiO2 (Si)
SO3 (S)
K2 O (MAD 10 feldspar)
Ca (wollastonite)
Fe2 O3
CuO
ZnO
Equivalent alkali
(Na2 O + K2 O)
21.56 (100)
0.69 (100)
2.38 (100)
13.17 (100)
1.13 (100)
1.12 (100)
55.78 (100)
2.62 (100)
0.89 (100)
0.66 (100)
0.74 (100)
39.40 (+82.75)
0.58 (15.94)
2.19 (7.98)
10.97 (16.70)
0.82 (27.43)
0.59 (47.32)
48.19 (13.61)
2.41 (8.01)
0.80 (10.11)
0.64 (3.03)
0.39 (47.30)
Statistical analysis
Statistical analysis of the data (t-test and analysis of variance)
was performed using the GraphPad Prism (version 5) software,
and the signicance level was set at p < 0.05.
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
14
100
1500
Alkalinity
Enrichment medium
Chloride
Minimal medium
mg/L CaCO 3
pH
12
10
1000
500
100
16.36
13.04
0.0
2.0
3.0
4.0
5.0
of buffer systems such as phosphate buffer11,31 and carbonate buffer11,30 to increase pH of the growth medium, but no
work has been reported on the use of KClNaOH buffer as a
buffer system for increasing pH of the medium. The pH of the
KClNaOH buffer system is around 13, so it can be used for
preparing media with a high pH value such as 12.
Morphological, biochemical and phylogenetic characterization revealed that all the isolates were related to members
of the genus Bacillus, and thus they were designated as Bacillus
sp. strains KG1, KG4 and KG5. Strain KG1 was selected on the
basis of its potential to neutralize the alkaline medium and
reduce alkalinity and chloride levels more efciently than the
other two strains.
Acid production by bacterial strain KG1 resulted in a significant (p < 0.0001) reduction of alkalinity in both minimal and
enrichment media (Fig. 1). In the minimal medium, an overall
change in 3.95 pH units was recorded after 3 days compared to
4th and 5th day of treatment. A similar trend of decreasing pH
during 3 days of incubation was also observed in the enriched
medium. This reduction of pH supported the results of acid
production by Bacillus sp. strain KG1. Sucrose was the sole
carbon source in the minimal medium. It was reported to be
efciently utilized by various Bacillus strains, resulting in a
decrease in the pH of the medium.1 The change in pH could be
attributed to the production of organic acids in the medium by
enzymes, and these results were in close agreement with the
ndings of Paavilainen et al.32 Since the minimal medium is
an economical and dened medium compared to enrichment
media, it was used for subculturing of the isolate in this study.
According to the guidelines of several environmental agencies, the pH of industrial wastes should be neutral before their
discharge to the environment as this is an important parameter to control surface and groundwater pollution. The removal
of the CKD alkalinity using alkaliphilic bacterial strains is
an eco-friendly method and was a challenging task for this
study, since no study has been performed until now to treat
alkaline solid industrial wastes with biological organisms.
Fig. 2 represents the alkalinity reduction of cement kiln dust
after treatment with strain KG1 at different cell densities
(OD 0.61.0). The alkalinity of the control CKD leachate was
Control
0.6
0.7
0.8
0.9
1.0
O.D. of culture
Fig. 2 Alkalinity and chloride reduction in CKD as
inuenced by the cell density of Bacillus halodurans strain
KG1. The numbers above the bars represent the
percentages of alkalinity and chloride, respectively, in the
treated CKD samples compared to the untreated CKD
control. The differences are statistically signicant (p < 0.05)
as found by the t-test at 95% condence limits.
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
Ca
K
Ca
O Mg
C Zn Si
Fe
K
Al
Zn
Fe
Cu
Fe
Cu
8
1 mm
10
12
14
Electron Image 1
16
18
20
keV
B
Ca
Ca
O
K
Si
Al
Mg
1 mm
Fe
2
4
6
0
Full scale 11361 cts cursor: 0.000
10
12
14
Electron Image 1
16
18
20
keV
Fig. 3 SEM and EDX analysis of the control CKD (A) and bacterial strain KG1-treated CKD (B). The reduction in the K peak
height reects a decrease in the K2 O content in B compared to A.
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
B
500
C
CO
D/A
C
CO
D
400
300
Intensity
400
Intensity
C
500
C 2S
C
200
C A
300
C
S
C 3S
200
Sg
Ar
100
Sg
Q/A
CH
100
0
10
15
20
25
30
35
40
45
50
55
60
10
15
20
25
30
35
40
45
50
55
60
Fig. 4 Diffraction pattern, along with crystalline phases, in the control CKD (A) and bacterial-treated CKD (B). C, calcite
(CaCO3 ); D, dolomite (CaMg(CO3 )2 ); A, anhydrite (CaSO4 ); CO, calcium oxide (CaO); Sg, syngenite (K2 Ca(SO4 )2 ); Ar, arcanite
(K2 SO4 ); C2 S, dicalcium silicate; and C3 S, tricalcium silicate.
increases the alkalinity of CKD. Reduction in the chloride content also marks an important parameter to study, as its high
concentration in groundwater through leachate affects the
taste of groundwater and increases its alkalinity. Excess chloride causes cardiovascular diseases. A high chloride content
corrodes steel and imparts permanent hardness to groundwater. In agriculture, chloride increases salinity of soil through
irrigation. In this study, chloride was reduced by approximately 86.96%, which shows that CKD can safely be reused
and disposed off to landll after bacterial treatment. Thus, the
use of a biological tool for the neutralization of alkaline solid
wastes is a better alternative to conventional chemical methods. The growth of the bacterium on the minimal medium also
shows its economic effectiveness as a cheap growth medium,
compared to ready-to-prepare enriched growth media such as
nutrient broth, etc.
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
Table 2 Characteristics of Bacillus halodurans KG1 and some related Bacillus species.
Morphological, biochemical
and physiological
characteristics
Bacillus okuhidensisa , b
Gram staining
+ve
+ve
Cell shape
Colony shape
Colony color
Small rods
Irregular
White opaque
Rod
Filamentous
White
Growth at:
45 C
50 C
pH 5
pH 10
pH 11
pH 12
Optimum pH
+
+
+
+
+
+
89
+
+
ND
V
ND
9.0
+
+
ND
+
+
ND
10.5
Tolerance of NaCl
5%
7%
10%
12%
16%
20%
Methyl Red test
VogesProskauer test
Citrate utilization test
Starch hydrolysis test
Gelatin test
Catalase test
Lipase test
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ND
ND
ND
ND
+
+
ND
ND
+
+
+
ND
ND
ND
+
+
ND
ND
Carbohydrate utilization
Glucose
Sucrose
Lactose
Mannitol
Xylose
Fructose
Maltose
+
+
+
+
+
+
+
+
+
+
ND
+
+
+
ND
ND
+
+
+
ND
ND
Characteristics are scored as: +, positive reaction; , negative reaction; w, weak reaction; v, variable; ND, no data. For all growth assays: +, growth;
, no growth; w/+, weak growth. Superscript after the taxa denotes the reference from where the datum is obtained.
a
b
Vargas et al.33
Li et al.34
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19
54
94 65
0.02
Fig. 5 A neighbor-joining phylogenetic tree showing the relationships of bacterial strain KG1 and the type strains of
closely related Bacillus species, based on 16S rRNA gene sequences. The GenBank accession numbers are given in
parentheses. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at the branch
points. Bar, 0.02 nucleotide substitutions per site.
Conclusions
Cement kiln dust, a waste byproduct, is generated during the
manufacturing of cement clinker. During clinker production in
a kiln, volatile components such as sulfates, alkalis (K2 O and
Na2 O), chlorides, etc., are drawn toward CKD and make it alkaline, thus restrict CKD reuse in a cement kiln. Huge amounts
of CKD are dumped in landll, which, due to leaching, leads to
pollution of groundwater. Biological treatment using B. halodurans strain KG1 for the removal of alkalinity and chloride from
CKD is a better alternative to chemical methods in terms of
cost effectiveness, efciency and non-toxicity.
Conicts of interest
The authors declare no conicts of interest.
references
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 19