Proceedings National Symposium on Ecohydrology Jakarta, March 24, 2011

BACTERIAL CARBONATE PRECIPITATION FOR BIOGROUTING Puspita Lisdiyanti1, Eko Suyanto 1, Shanti Ratnakomala1, Fahrurrozi1, Miranti Nurindah Sari1, Niken Financia Gusmawati2
Research Center for Biotechnology, Indonesian Insitute of Science Jl. Raya Jakarta Bogor km. 46, Cibinong 16911, Indonesia 2 Research Center for Marine Technology, Ministry of Marine Affairs and Fisheries Jl. Pasir Putih, Ancol, Indonesia Email : puspita.lisdiyanti@lipi.go.id dan p_lisdiyanti@yahoo.com 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 33 T was used. Keywords: bacterial induced carbonatae precipitation, urease, biogrouting
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INTRODUCTION Grout is a constructions 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 ureaseproducing 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 CaCl22H2O 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 crystallineforming 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 30C 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 M of NiCl2 in 1L distilled water, incubated with agitation at 150 rpm at ambient temperature (30C) 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 L of sample, 500 L of 50 mM urea and 500 L of 100 mM KH2PO4 buffer (pH 8.0) so that the total volume was 1.1 ml. The reactions mixture was incubated in a water bath with the temperature of 37C for 30 minutes. This reaction was stopped by transferring 50 l of reaction mixture into tubes containing 500 l solution of phenol-sodium nitroprusside. Alkaline hypochlorite solution 500 l 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 mol NH3 from urea per minute under standard assay.

DNA extraction Total DNA of the bacteria was extracted using an InstaGene Matrix Kit (BioRad). Oneday 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 10,00012,000 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 95C, 2 min (1 cycle); 95C, 30 seconds, 65C, 1 minute, 72C, 2 min (10 cycles); 95C, 30 seconds , 55C, 1 minute, 72C, 2 min (30 cycles); and 72C, 2 min (1 cycle). Purification of PCR was performed using a kit Pregman. Initial denturation (96 oC 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 coloniess 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 P1 P2 P3 2010 P4 P5 P6 P7 Total Grasberg Grasberg Grasberg Grasberg Grasberg Grasberg Grasberg Sample Soil Soil Soil Sands Sands Soil Soil Number of bacteria 0 0 46 0 19 14 0 79

Table 2. Number of bacteria with the ability of crystalline-forming isolated from Yogyakarta. Year Sampling code Y1 Y2 Y3 2010 Y4 Y5 Y6 Y7 Total Location Selarong cave Selarong cave Parangtritis coast Parangtritis coast Parangtritis coast Parangtritis coast Parangtritis coast Sample Rock Rock Sand Sand Water Rock Rock Number of bacteria 0 2 1 10 9 9 8 39

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Table 3. Number of bacteria with the ability of crystalline-forming isolated from Southeast Sulawesi. Year Sampling code Location S1 S2 S3 S4 S5 S6 S7 S8 S9 2010 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 Total Batu cave, BNP Batu cave, BNP Batu cave, BNP Mimpi cave, BNP Mimpi cave, BNP Mimpi cave, BNP Mimpi cave, BNP Mimpi cave, BNP Mimpi cave, BNP BNP Pangkap Rotterdam castle Rotterdam castle Lae-Lae island coast Lae-Lae island coast Samalona island coast Samalona island coast Samalona island coast Samalona island coast Samalona island coast Sample Water Rock Soil Water Rock Soil Water Rock Soil Water Water Rock Soil Water Soil Water Soil Rock Soil Rock Number of bacteria 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 14 0 0 0 6 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 33 T). 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 , and , 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 P3BG21 BLAST Search Result Staphylococcus JCSC1435 Oceanobacillus profundus strain CL-MP28 haemolyticus Accession number AP006716.1 Query length 1675 Homology (%) 86

P3BG24

DQ386635.1 EU330342.1 DQ358670.1

1505 1594 1554

98 92 94

P3BG41 Oceanobacillus sp. BSi20641 P3BG43 Oceanobacillus sp. YIM DH3

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Tables 5. Identification of selected bacterial carbonate precipitation from Yogyakarta on analysis of 16S rRNA gene. Isolat ID 2.1.4 2.3.4 3.1.2 3.1.4 3.1.6 3.2.2 4.1.1 4.1.4 4.1.5 4.2.2 BLAST Search Result Bacillus pichinotyi strain RS2 Bacillus sp. strain WCC 4585 Bacillus sp. WB7 Bacillus sp. WB7 Bacillus sp. WB7 Schineria sp. CHNDP40 Sporosarcina luteola Bacillus sp. WB7 Bacillus sp. WB7 Bacillus sp. WB7 Accession number AF519464.1 FN995266.1 HQ224952.1 HQ224952.1 HQ224952.1 DQ337535.1 AB473560.1 HQ224952.1 HQ224952.1 HQ224952.1 Query length 1495 1493 1492 1501 1493 1489 1487 1519 1524 1495 Homology (%) 97 96 98 97 97 97 99 89 92 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 In progress Query length In progress In progress In progress In progress 1535 In Homology (%) In progress

BT03.1

In progress

BT03.5

In progress

In progress

In progress

BT03.7

In progress

In progress

In progress

BT03.8 SA.08.6

In progress Bacillus lentus

In progress AB021189.1 In progress

In progress 98 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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