16 Bacterial Carbonate Puspita Lisdiyanti
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Transcript of 16 Bacterial Carbonate Puspita Lisdiyanti
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
204
BACTERIAL CARBONATE PRECIPITATION FOR BIOGROUTING
Puspita Lisdiyanti1, Eko Suyanto1, Shanti Ratnakomala1, Fahrurrozi1, Miranti Nurindah Sari1, Niken Financia Gusmawati2
1Research Center for Biotechnology, Indonesian Insitute of Science
Jl. Raya Jakarta Bogor km. 46, Cibinong 16911, Indonesia 2Research Center for Marine Technology, Ministry of Marine Affairs and Fisheries
Jl. Pasir Putih, Ancol, Indonesia
Email : [email protected] dan [email protected]
ABSTRCT The isolation and identification of bacterial carbonate precipitation and characterization of urease enzyme produced by preferred bacteria were conducted. Urea as the carbon source was employed in enrichment method. The formation of crystalline calcite was observed by light microscope. The urease enzyme activity was determined by Weatherburn method. The molecular identification of isolates was analysed by determination of 16S rRNA gene. As results, 21 bacteria from Papua, Yogyakarta, and Sulawesi areas showed calcite formation in the medium with urea as a carbon source. Each of isolates was capable to produce urease. Molecular identification of isolates that had high urease activity was in progress. As a reference strain, Sporosarcina pasteurii DSMZ 33T was used. Keywords: bacterial induced carbonatae precipitation, urease, biogrouting
INTRODUCTION
Grout is a construction’s material that typically consists of a mixture of cement water
and sand, and is used in construction. This construction material can also be used to
improve soil structure due to the deposition of this minerals which can alter the character
of soil geomorphology. In the construction industry, the process of injection of
construction material is known as the grouting. Generally, the process of grouting for the
purpose of designing was done chemically by using silica (waterglass). Silica is quickly
to settle when mixed with a metal solution or bicarboxylic acid. This rapid reaction is
considered as the weakness of chemical grouting, because it can only be applied at the
injection point near the ground. Furthermore, the chemical grouting process requires
high injection pressures so that it can create an unstable and low permeability of soil.
Currently, biogrouting, a process that transforms soil or sand into calcarenite or
sandstone by calcium carbonate precipitation bacteria has been developed with
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
205
mechanisms based on the mediation of carbonate precipitation. The main advantage of
biogrouting is the provision of substrate which can be moved in an inactive form into
areas far from the point of injection. Subsequently, the substrate can be converted by
bacteria into an active form. Biogrouting process is a process that simulates the process
of diagenesis, such as the transformation of sand grains of sand into rock
(calcarenite/sandstone). Naturally, this process may take up to millions of years. The
bacteria are used to accelerate the process in situ (DeJong et al., 2006; Lee, 3003).
Calcite (CaCO3) resulted from the precipitation of carbonate is a mineral that is
widely distributed on earth and found in rocks as marble, sand stone in the waters or on
land (Hammes and Verstraete, 2002). Precipitation (deposition) of calcite at least was
determined by 3 parameters: (1) the concentration of calcium, 2) carbonate
concentration, and (3) pH environment and the availability of nucleation sites (Hammes
et al., 2003a&b; Hammes and Verstraete, 2002). Carbonate precipitation can
theoretically occur in natural environments by increasing the concentration of calcium
and/or carbonate in solution or by lowering the solubility of calcium and/or carbonate.
Therefore, the source of microorganisms for biogrouting should be ideally
resistant or tolerant to high concentrations of urea and calcium. Microorganisms also
have to produce urease in high activity. Urease-producing microorganisms can be
classified into 2 groups based on the response to ammonium: (1) urease enzyme activity
is suppressed by the presence of ammonium such as the type of Pseudomonas
aeruginosa, Alcaligenes autrophus, Bacillus megaterium (Kaltwasser et al., 1972) and
Klebsiella aerogenes (Friedrich and Magasanik, 1997) and (2) urease enzyme activity is
not affected by ammonium i.e Sporosarcina pasteurii (Bacillus pasteurii), Proteus
vulgaris, Helicobacter pylori. In biogrouting process, because the high concentration of
urea is hydrolyzed, then, the suitable group of bacteria for use is the group whose
enzyme activity is not suppressed by ammonium. At this time, the genus Sporosarcina
(Bacillus) have been applied to biogrouting process because they have a high urease
activity and are not pathogenic (Fujita et al., 2000; Mobley et al., 1995).
In this paper, isolation, screening, and identification of bacteria to be used as
biogrouting were conducted. Expectation from this study, bacterial isolates and mastery
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
206
of technology superior handling biogrouting to coastal erosion would be attained. The
study of the diversity of bacteria that play roles in the process biogrouting has rarely
been done in Indonesia. This technology is possible to be used in strengthening the
structure of the soil in coastal areas in preventing coastal erosion, foundation of the
repair, reclamation, dredging and even consolidating the soil as building material. The
purpose of this study is to obtain bacterial isolates for biogrouting with high urease
activity that is able to generate carbonate precipitation.
MATERIALS AND METHODS
Isolation and purification
Samples (soils, sands, marine water, and rocks) were taken from 7 location in 3
provinces of Indonesia, which were, Grasberg in Papua; Selarong cave and Parang Tritis
coastal in Yogyakarta; Bantimurung National Park, Rotterdam castle, Lae-Lae island
and Samalona island in Southeast Sulawesi. The medium for the isolation of urease-
producing bacteria is B4 medium with the following composition: 3 g of nutrient broth,
20 g of urea, 2.12 g of NaHCO3, 10 g of NH4Cl, 4.41g of CaCl2·2H2O in 1L distilled
water, and 15 g of agar was added if needed. The cultures/plates were incubated at
ambient temperature for 5 days. Soil and rock samples were ground before use.
Purification using fourway streak method was then performed to obtain pure bacterial
isolates (Cappuccino and Sherman, 2005). Bacterial colonies which have crystalline-
forming in the medium, were observed after 5 and 10 days of cultivation with a light
microscope.
Screening of urease enzyme producing bacteria
The screening was carried out by growing the isolates in the urease test medium broth
using the method of Hammes et al. (2003b). The reaction was observed after being
incubated in 30ºC for 3 days. Bacterial isolates which have urease activity would
perform the color changes of liquid medium from yellow to fuchsia pink.
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
207
Urease activity test
The quantitative test of urease activity was conducted by the following method: isolates
were grown in production medium with the composition of 20 g of yeast extract, 10 g of
NH4Cl and 10 µM of NiCl2 in 1L distilled water, incubated with agitation at 150 rpm at
ambient temperature (30ºC) for 72 hours. Urease activity was measured using the
method of Weatherburn (1967) with some modifications as follow, Na2HPO4
hypochlorite was used in an alkaline solution instead of NaOH and the time of color
formation was changed from 20 minutes to 30 minutes. Reactions were carried out in
test tubes containing 100 µL of sample, 500 µL of 50 mM urea and 500 µL of 100 mM
KH2PO4 buffer (pH 8.0) so that the total volume was 1.1 ml. The reaction’s mixture was
incubated in a water bath with the temperature of 37ºC for 30 minutes. This reaction was
stopped by transferring 50 µl of reaction mixture into tubes containing 500 µl solution of
phenol-sodium nitroprusside. Alkaline hypochlorite solution 500 µl was added to the
tube and incubated at ambient temperature for 30 minutes. Then the optical density (OD)
was measured with a spectrophotometer at wavelength of 630 nm and compared with
standard curve (NH4)2SO4. One unit of enzyme activity is the amount of enzyme
required to liberate 1 µmol NH3 from urea per minute under standard assay.
DNA extraction
Total DNA of the bacteria was extracted using an InstaGene Matrix Kit (BioRad). One-
day old bacterial colonies ere added to microcentrifuge tube with 1.0 mL of sterile water
in order to get suspension of bacteria. The suspension then was centrifuged at 10,000-
12,000 rpm for 1 min, the supernatants were decanted, and the pellets were resuspended
with 50 l InstaGene matrix. The bacterial suspension was then incubated at 56C for
15-30 min, vortexed for 10 sec, incubated at 100C for 8 min, vortexed again for 10 sec,
and then centrifuged at 10,000-12,000 rpm for 2-3 min. The supernatants containing the
DNA of bacteria was stored at -20C before use.
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
208
Amplification of 16S rRNA genes
Amplification of 16S rRNA genes was performed using Polymerase Chain Reaction
(PCR) and by using the primers 9F (5'-AGRGTTTGATCMTGGCTCAG-3') and 1492R
(1492R: 5'-TACGGYTACCTTGTTAYGACTT-3') (Position-base sequence numbering
based on Escherichia coli numbering system, accession number V00348, Brosius et al.,
1981). The PCR reaction conditions are 95ºC, 2 min (1 cycle); 95ºC, 30 seconds, 65ºC, 1
minute, 72ºC, 2 min (10 cycles); 95ºC, 30 seconds , 55ºC, 1 minute, 72ºC, 2 min (30
cycles); and 72ºC, 2 min (1 cycle). Purification of PCR was performed using a kit
Pregman. Initial denturation (96oC for 5 minutes), Denaturation (96oC for 0.3 min), and
Annealing (55oC for 0.3 minutes).
DNA sequencing and Phylogenetic Tree Construction
Sequencing of 16S rRNA gene was analyzed by using automatic machine type ABI 310
DNA sequencer in PT. Genetika Science, Indonesia. DNA sequence information from
the sequence data base to track likeness with GeneBank/DDBJ/EMBL based on BLAST
(Altschul et al., 1997). Sequences were aligned using ClustalX program (Thompson et
al., 1994). The neighbour-joining (NJ) method was used to construct all phylogenetic
trees.
RESULTS AND DISCUSSION
The present study showed a possibility that bacterial carbonate precipitation isolated can
be exploited as a biologically induced mineralization and carbonatogenesis.
Carbonatogenesis, bacterially induced precipitation of calcium carbonate is an
established tool for the in situ restoration of building and monuments or biogrouting
(Castanier et al., 1999; Stocks-Fisher et al., 1999). We collected various natural habitats
including soil, sands, water, and rocks. As described in research methods, soil, sands,
water, and rocks that had been crushed, gradually diluted, and grown in isolation
medium, were observed under the microscope for the colonies’s appearance. If the
observed colony produced crystal, a single colony was picked up, grown in isolation
medium, and purified to obtain pure culture. As results, totally, 146 isolates were seen as
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
209
crystalline-forming in medium, Their crystal formations were carbonate mineral, it is a
good example of biologically induced mineralization (BIM) (Lee, 2003).
Table 1. Number of bacteria with the ability of crystalline-forming isolated from Papua.
Year Sampling code Location Sample Number of bacteria
2010
P1 Grasberg Soil 0
P2 Grasberg Soil 0
P3 Grasberg Soil 46
P4 Grasberg Sands 0
P5 Grasberg Sands 19
P6 Grasberg Soil 14
P7 Grasberg Soil 0
Total 79
Table 2. Number of bacteria with the ability of crystalline-forming isolated from
Yogyakarta.
Year Sampling
code Location Sample Number of bacteria
2010
Y1 Selarong cave Rock 0
Y2 Selarong cave Rock 2
Y3 Parangtritis coast Sand 1
Y4 Parangtritis coast Sand 10
Y5 Parangtritis coast Water 9
Y6 Parangtritis coast Rock 9
Y7 Parangtritis coast Rock 8
Total 39
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
210
Table 3. Number of bacteria with the ability of crystalline-forming isolated from
Southeast Sulawesi.
Year Sampling code Location Sample Number of bacteria
2010
S1 Batu cave, BNP Water 0
S2 Batu cave, BNP Rock 0
S3 Batu cave, BNP Soil 0
S4 Mimpi cave, BNP Water 0
S5 Mimpi cave, BNP Rock 0
S6 Mimpi cave, BNP Soil 0
S7 Mimpi cave, BNP Water 0
S8 Mimpi cave, BNP Rock 0
S9 Mimpi cave, BNP Soil 8
S10 BNP Water 0
S11 Pangkap Water 0
S12 Rotterdam castle Rock 0
S13 Rotterdam castle Soil 0
S14 Lae-Lae island coast Water 0
S15 Lae-Lae island coast Soil 0
S16 Samalona island coast Water 14
S17 Samalona island coast Soil 0
S18 Samalona island coast Rock 0
S19 Samalona island coast Soil 0
S20 Samalona island coast Rock 6
Total 28
The precipitate was always formed within the bacterial colonies on the agar
surface, which was also captured bacteria within the crystal structure (Figure 1). Four
basic morphologically type of crystal were sperulite with fibrous surface texture,
rhombohedral, spherical vaterite, and trianguler. This could have been a result of the
colony growth rate and/or actual urease activity, which thus, influenced the rate of
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
211
supply of chemical species required for precipitation (Sondi and Matijevic, 2001).
Alternatively, crystal growth can be inhibited or altered by the adsorption of proteins,
organic matter, or inorganic component to specific crystallographic planes of the
growing crystal (Rivadeneyra et al., 1998).
Figure 1. Morphological differences in calsite crystal within bacterial colonies of
bacterial carbonate precipitation grown on semisolid medium. It was produced by
intracellular urease enzyme activity in bacteria. The types of crystal, a) sperulite with
fibrous surface texture (2.1.4), b) rhombohedral type (P3BG43), c) spherical vaterite
(SA.08.6) , and d) trianguler type (3.2.2) (magnitude, 20x).
Screening of bacterial biogrouting conducted was to determine the ability of
bacterial isolates in the urease enzyme activity. This was done by growing the 146
isolates in liquid medium. Screening results showed that 4 isolates from Papua, 10
isolates from Yogyakarta, and 7 isolates from Sulawesi showed positive behavior on
urease test. Urease test was used for selected bacteria expressing a gene for the enzyme
urease. The hydrolysis of urea by the enzyme urease produces ammonia and carbon
dioxide which has the net effect by increasing the pH of the medium and causing the
phenol red indicator to become fuchsia pink (Figure 2).
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
212
Figure 2. Screening for bacterial carbonate precipitation. The hydrolysis of urea by the
urease enzyme activity causing color change of liquid medium from yellow to fuchsia
pink.
Determination of urease enzyme activity of positive urease test indicated that
the isolates had a higher ability of urease activity compared to reference strain for
biogrouting (Sporosarcina pasteurii DSMZ 33T). This was due to higher concentrations
of ammonium produced by microbes biogrouting from Indonesia. Isolate P3BG21,
P3BG24, P3BG41, P3BG43 had higher activity than the control isolates DSMZ 33T. As
for the Indonesian isolates, isolates with P3BG43 code had the highest concentration
compared to other isolates from Indonesia which ammonium concentration reached
95.778 mM (Figure 3) with the urease activity was 374.94 U/ml.
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
213
Figure 3. Ammonium concentration and urease activity for each of isolates from Papua
Ammonium was produced variously for each of isolates depending on isolates
type. Generally, there was a correlation between the enzyme activity and ammonium
concentration (Figure 3, 4, and 5). Bacterial carbonate precipitation from Yogyakarta
was also measured for urease activity. As the results, the four higher isolates had urease
activity as followed : 295.58 U/mL (2.1.4), 282.40 U/mL (2.3.4), 261.69 U/mL (4.2.2),
251.35 U/mL (3.1.4), respectively (Figure 4).
Figure 4. Ammonium concentration and urease activity for each of isolates from
Yogyakarta.
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
214
.
Figure 5. Ammonium concentration and urease activity for each of isolates from
southeast Sulawesi.
The genes sequences suggested that structural subunit of the enzyme were
coded by three contiguous genes, ureA, ureB and ureC. According to the nomenclature
of Mobley and Hausinger, the genes ureA, ureB, and ureC code for subunit γ, β and α,
respectively (c). This enzyme showed that it was not an extracellular expresses in any of
the isolates (Hammes et al., 2003b).
The identification resulted to determine the type of bacteria. To identify
bacterial biogrout to species level, molecular-based molecular identification was carried
out. Thus, it could identify the type (genus or species) of microbial accurately. Activities
that had been done on this first phase was the isolation of DNA from the microbial
genome biogrout, 16S rRNA gene was amplified using the machine Polymerase Chain
Reaction (PCR), and purified 16S rRNA gene. Subsequently, a base sequence
determination was employed using the sequencer tool. Length of bases of the amplified
16S rRNA gene was 1500 base pairs long (Figure 6).
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
215
Figure 6 The result of DNA electroforesis for 16S rRNA, from left to right showing the
bands of DNA of each isolates: 1) Marker, 2) 4.1.1, 3) 3.1.4, 4) 2.1.4, 5) 3.1.6, 6) 4.1.4,
7) 4.2.2, 8) 2.3.4, 9) 3.1.2, 10) 3.2.2, 11) 4.1.5.
All of the 21 bacterial carbonate precipitation were dominated by Bacillus
genera. Generally, characterization on the bacterial carbonate precipitation has been
examined as alkalophile bacteria (pH 7-9), Gram-positive (Lee, 2003). Isolates of
bacteria from Papua location gave 2 genera that were Staphylococcus and
Oceanobacillus.
Table 4. Identification of selected bacterial carbonate precipitation from Papua based on
analysis of 16S rRNA gene.
Isolat
ID BLAST Search Result
Accession
number
Query
length
Homology
(%)
P3BG21 Staphylococcus haemolyticus
JCSC1435 AP006716.1 1675 86
P3BG24 Oceanobacillus profundus strain
CL-MP28 DQ386635.1 1505 98
P3BG41 Oceanobacillus sp. BSi20641 EU330342.1 1594 92
P3BG43 Oceanobacillus sp. YIM DH3 DQ358670.1 1554 94
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
216
Tables 5. Identification of selected bacterial carbonate precipitation from Yogyakarta on
analysis of 16S rRNA gene.
Isolat
ID BLAST Search Result
Accession
number
Query
length
Homology
(%)
2.1.4 Bacillus pichinotyi strain RS2 AF519464.1 1495 97
2.3.4 Bacillus sp. strain WCC 4585 FN995266.1 1493 96
3.1.2 Bacillus sp. WB7 HQ224952.1 1492 98
3.1.4 Bacillus sp. WB7 HQ224952.1 1501 97
3.1.6 Bacillus sp. WB7 HQ224952.1 1493 97
3.2.2 Schineria sp. CHNDP40 DQ337535.1 1489 97
4.1.1 Sporosarcina luteola AB473560.1 1487 99
4.1.4 Bacillus sp. WB7 HQ224952.1 1519 89
4.1.5 Bacillus sp. WB7 HQ224952.1 1524 92
4.2.2 Bacillus sp. WB7 HQ224952.1 1495 97
Tables 6. Identification of selected bacterial carbonate precipitation from South Sulawesi
on analysis of 16S rRNA gene.
Isolat ID BLAST Search Result Accession
number
Query
length
Homology
(%)
BT03.1 In progress In progress In
progress In progress
BT03.5 In progress In progress In
progress In progress
BT03.7 In progress In progress In
progress In progress
BT03.8 In progress In progress In
progress In progress
SA.08.6 Bacillus lentus AB021189.1 1535 98
SA.08.11 In progress In progress In In progress
Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011
217
progress
SB.09.6 Bacillus lentus AB021189.1 1535 99
Figure 7. Neighbour-Joining dendrogram derived from 16S rRNA gene sequences of the
bacterial carbonate precipitation.
ACKNOWLEDGEMENT
We would like thank to Kompetitif grant from Indonesian Institute of Sciences
for the financial support of this research. We would also like to thank to PT. Freeport
Indonesia for providing the samples from Grasberg, Papua.
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