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The name and affiliation of authors have been removed for double blind peer review purpose. Abstract Karst regions in Indonesia have the uniqueness of the landscape and biodiversity. The karst is formed by the dissolution of rocks and the precipitation of mineral. In the cave, there are ornaments of stalactite and stalagmite which are formed by the process of mineral precipitation.

We have isolated, screened, and identified the soil bacterium from cave environment (Lysinibacillus macroides). A potential of this microorganism is as bio-calcification on bio-concrete. We investigated proportions and the properties of concrete mixtures containing lightweight aggregate and volcanic ash impregnated with bacteria. A comparison study was made with concrete cylinders subjected to compressive strength tests with and without the bacteria.

It found that the strength of bio-concrete decreased less than 10.56% for 28 days cured specimens. This study showed that the effects of bacteria on porous aggregate are not considerable. Therefore, this method is effective to repair in the micro crack. Keywords: Self-healing, concrete, bacteria, volcanic ash. Introduction Calcium carbonate is a mineral that represents a large portion of carbon reservoir in the earth.

Microorganisms are important active and passive promoters of redox reactions that can influence the precipitation of minerals, including calcium carbonate [1]. Carbonate precipitation is a by-product of microbial activities that plays an important role in the cementation of natural systems, such as caves, soils, sediments, aquifers, and open-water areas. Some researcher used microbial carbonate precipitation as metal remediation, carbon sequestration, enhanced oil recovery and construction restoration. [2].

Considerable research on calcite precipitation by bacteria has been investigated by using ureolytic bacteria [3],[4]. The ureolytic bacteria hydrolyzed a urea into ammonium and carbonate ion. The result of the hydrolysis increased the pH and calcium carbonate precipitation. In the nature, biotic urease activity is a widespread phenomenon and includes the actions of bacteria. Urease is an enzyme that hydrolyzes a urea and creates a calcite in alkaline environment.

Bio-mineralization phenomenon is observed in cave isolates, showing their abilities to precipitate and dissolve calcium carbonate [5]. Bio-calcification has been proposed as an alternative and environmentally-friendly technique to develop self-healing cementitious materials system in recent years [6]. The process of calcite formation was resulted from metabolic activities bacteria which fill up the cracks in the concrete matrix. Research leading to bio-calcification and its ability to self-healing has introduced many methods and applications [7],[8].

One of the self-healing method is impregnation the healing agent that got into to porous aggregate with bacterial spores, calcium and nutrient. These materials were added to concrete. [9]. Utilization of these concepts in concrete leads to the potential invention of a new environment-friendly material called bio-concrete. Indonesia has the uniqueness of the landscape and biodiversity, one of them is karst cave in Gunung Kidul, Yogyakarta.

Soil bacteria is correlated highly with characteristics of the karst ecosystems as represented by their different karst geochemical environments and vegetation [10]. The study of bacterial diversity in the bio-concrete rarely had worked in Indonesia. The purposes of this study are to obtain and to evaluate bacterial isolates for bio-concrete with high urease activity. The present work deals with the comparative study of the compressive strength of concrete subjected to compressive strength tests with and without the bacteria.

Methods Bacterial sources Isolation and purification Soil and stalagmites samples were took from Jomblang Cave in Gunung Kidul, Yogyakarta, Indonesia. The medium for the isolation of bacteria was selective medium with the following compositions: 20 g of urea, 3 g of nutrient broth, 10 g of NH4Cl, 2.12 g of NaHCO3, 4.41 g of CaCl22H2O in 1 L distilled water, and 15 g of agar [11]. The culture was incubated at the room temperature (28C) for 3 days. The method of streak plate purification was conducted to obtain pure bacterial isolates. Figure 1 below shows the stalagmite where the sample was taken.

/ Figure 1. Stalagmite Screening The screening was carried out by growing the isolates in the urease test medium broth using Hammes method [11]. The media of urease activity could observed after incubation at 28C during 1 to 3 days. Bacterial isolates that have the urease activity will change the color of the medium from yellow to fuchsia pink. The urease activity was determined by measuring the amount of ammonia released from urea according to the phenol-hypochlorite assay method [12].

The 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 at a temperature of 37C for 30 minutes. This reaction was stopped by transferring 50 L of the action mixture into tubes containing 500 L solution of phenol-sodium nitroprusside. Alkaline hypochlorite solution 500 L were added to the tube, then incubated at ambient temperature for 30 minutes.

The optical density was measured with a spectrophotometer at ? = 630 nm and compared with a standard curve (NH4)2SO4 Bacterial Identification All strains of bacteria that used in this research were gram positive, endospore forming and urease positive. The pure cultures of bacteria that have the highest urease activity were used for molecular identification. Identification was carried out using molecular analysis based on 16S rDNA fragments in bacteria.

Bacterial DNA isolation was performed using Polymerase Chain Reaction (PCR) method [13]. Amplification of 16S rDNA fragments was performed using GoTaq (Promega) with primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') [[14]]. PEG precipitation method was used to purify PCR, after that it continued by cycle sequencing [15]. The result of sequencing cycles was purified using ethanol purification method.

Nitrogen base sequence was analyzed using automated DNA sequencer (ABI PRISM 3130 Genetic Analyzer). The datas of sequencing result were processed by Bioedit program. 16S rDNA sequence homology program searched using Basic Local Alignment Search Tool Nucleotide (BlastN) on the website of the National Center for Biotechnology Information (NCBI). Materials Cement Portland Pozzolana Cement (PPC), which is available in the local market, was used. The cement used has been tested for various properties as per SNI 15-0302-2004. Coarse and fine aggregates All aggregates shall conform to SNI 03-1750-1990.

Fine aggregates; used the natural river sand, were washed and screened to eliminate unwanted deleterious material and oversize particle. Natural river sand, according to SNI 03-1968-1990 is zone II with the specific gravity 2.64. This study used the crushed rocks aggregate as coarse aggregate with 20 mm in diameter and 2.65 for the specific gravity. Lightweight expanded clay aggregates Hydroton was used in this study as replacement the coarse aggregates.

It was circle which the maximum diameter 10 mm and no pass sieve no 4 (4.75 mm). In the other hand, the specific gravity of Hydroton is 0.84. The crushed Hydroton was dried at 115C for 12 hours, then cooling it to room temperature. Some of these aggregates were subsequently brought under partial vacuum, after which a 150 mM calcium lactate solution was added until all the aggregates were submerged. The aggregates were dried at a temperature of 30C. Volcanic ash The material which was from Mount Kelud eruption in 2014 (particle size 0.053 mm and the specific gravity of 2.26) has been used as a medium for bacterial spore.

Volcanic ash was dried overnight at a temperature of about 115C, followed by cooling to room temperature. An amount of these particles was brought under partial vacuum, after which 1 mL of 1.2 x 105 cells mL-1 spores suspension was added per 17 g of ash particles. The particles were dried at a temperature of 30C. Figure 2 below shows the pore structure in volcanic ash. Microorganism Cells cultured and grown in the laboratory for 24 hours.

The medium composition required for growth of culture was Peptone: 5 g L-1, NaCl: 5 g L-1and Yeast extract: 3 g L-1. / Figure 2. Kelud ash observed by SEM Specimens and mix proportions Compressive strength was tested according to ASTM C31/C31M. According to ASTM C31/C31M and AASHTO T 23, the utilization of small concrete cylinders (diameter: 100 mm; high: 200 mm) is allowed as long as the size of the aggregate does not exceed 1/3 of the cylinder's diameter.

This research has investigated proportions and the properties of concrete mixtures containing hydroton and volcanic ash impregnated with bacteria. Table 1 show the mix proportion of concrete and various specimen designs. The addition of volcanic ash in concrete was 1.5% by weight of cement. The corresponding identification marks were labeled over the concrete surface. All specimens were water cured for 28 days then tested in the compression machine. The specimens were immersed in water for curing until the compressive strength testing at 28 days.

The compressive strength of each specimen was calculated as the mean value of six specimens. Table 1 Quantities of materials per cubic meter of concrete Sample Cement (kg) Fine aggregates (kg) Coarse aggregates (kg) Hydroton (kg) Volcanic ash (kg) W/C Volcanic ash contain cells  A1 337.08 710.76 1066.09 - 5.06 0.65 No  A2 337.08 710.76 1066.09 - 5.06 0.65 Yes  B1 336.25 709.01 974.85 30.06 5.04 0.65 No  B2 336.25 709.01 974.85 30.06 5.04 0.65 Yes  C1 335.42 707.27 884.05 59.97 5.03 0.65 No  C2 335.42 707.27 884.05 59.97 5.03

0.65 Yes  D1 333.78 703.81 703.78 119.35 5.01 0.65 No  D2 333.78 703.81 703.78 119.35 5.01 0.65 Yes  Results and discussion Microorganism Urease test All the isolates of bacteria were tested for urease activity. The discoloration of the media was from yellow to bright pink (fuchsia). This colour indicated there was the urease activity in the media. Figure 3 below shows the color change in screeening media. / Figure 3.

Screening for bacterial calcification Urease activity The ability to precipitate calcite is directly related to the amount of the enzyme urease produced by the bacteria. The activity of urease happened at 30 minutes after the incubation at 28C for Isolate-LMS5. The urease activity of Isolate-LMS5 was 8.94 gmL-1minute-1. Figure 4 below shows urease activity for each of isolate. Molecular identification Phylogenetic analysis positions determined by 16S rRNA sequence of Isolate-LMS5 showed 99% similarity with Lysinibacillus macroides strain CS26, compared to the sequence of DNA in National Center for Biotechnology (NCBI) Gen Bank database, with Max score: 2499; Total score: 2499; Query coverage: 100%; E-value 0.0; Max identities: 1368/1374 (99%); Gaps: 5/1374 (0%). This result confirms that our Isolate-LMS5 corresponds to Lysinibacillus macroides. / Figure 4.

Urease activity for each of isolates Bio-concrete Properties of concrete The properties investigated include density, compressive strength, and water absorption. The compressive strength of concrete tested in compression testing machine at 28 days for control concrete and for bio-concrete. The comparison study of compressive strength was made using specimens with and without the bacteria. The test results for concrete properties are shown in Table 2.

Table 2 Propreties of concrete specimens Specimens Density (kg/m3) Absorption (%)  Compressive strength (N/mm2) Loss in strength (%)  A1 2199 7.30 15.22 -  A2 2096 10.09 13.61 10.56  B1 2096 8.25 15.00 -  B2 2061 11.85 13.72 8.57  C1 2007 8.38 12.75 -  C2 2006 11.20 11.90 6.72  D1 1961 10.23 11.90 -  D2 1907 12.36 11.25 5.41   The result showed that hydroton as coarse aggregate increased the volume of pores and reduced the density.

The bacteria agent into volcano ash affected slightly reducing of the compressive strength. The compressive strength of the concrete with bacteria was 10.56% lower than the control. This result was the same with the result of Tziviloglou et al. [9]. It indicated the bacteria agent effected the characteristic of cement especially in the hardening process of concrete. The utilization of healing agent has not been able to reduce the natural porosity in the concrete.

As metabolically active, the bacteria consume the oxygen although there is not enough available oxygen after curing for 28 days in the pores of the concrete. The pH of fresh concrete is usually between 11 and 13. The temperature of fresh concrete can go up to 70C. After the drying of concrete, there is not enough water. Therefore, the selected bacteria needs to exhibit high resistance against high pH, temperature, and serious limitation of water. The suitability of the bacteria applied in concrete as self-healing agent clearly relates to their capacity to form spores [16].

Microscope observation The bacteria will not precipitate calcite in the pores of the concrete as long as they are in normal condition (there is no crack). Calcite precipitation will fill in the crack after the concrete was under water for 60 days. Recovery cracks that occur triggered by water and supported by oxygen content. Figure 5 below shows that the crystal precipitation process was able to seal the cracks less than 0.3 mm. / Figure 5.

Crack healing SEM observation Scanning electron microscopy of calcium carbonate crystals precipitated shows calcite produced in a concrete crack in different stages of formation with morphologies of platelets and rhombohedral. The specificity of crystal growth was knowns primarily because of differences in bacterial genera. The crystal growth could be inhibited or altered by the adsorption of proteins, organic matter, or inorganic components to specific crystallographic planes of the growing crystal [17]. Figure 6 below shows the crystal morphology of calcite. / Figure 6.

Calcite precipitate Conclusion The utilization of loaded bacteria onto porous aggregate (volcanic ash) is another way of protecting cells from inappropriate conditions and a suitable way in terms of maintenance costs. In another hand, the effect of this method resulted in a decrease in strength of less than 10.56% for 28 days cured specimens. This study shows that the effects of the bacteria on a porous aggregate are not considerable. This method is effective to repaire micro crack less than 0.3 mm. In additional, there was needed the further reseach to increase the compressive strength.

Acknowledgement We would like thanks to the Jomblang Cave Management who allowed us to take the sample and the Head of Research Center for Biomaterials, Indonesian Institute of Sciences who support us in this research program.