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


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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


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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.


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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.


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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


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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


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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


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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).


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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.


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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.


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.

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).


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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


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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







SA.08.6 Bacillus lentus AB021189.1 1535 98

SA.08.11 In progress In progress In In progress


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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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16 Bacterial Carbonate Puspita Lisdiyanti

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