THE POTENTIAL USE OF TOFU-PROCESSING WASTEWATER AS BACTERIAL GROWTH MEDIA FOR SOIL STRUCTURE IMPROVEMENT BY BIOCLOGGING AND BIOCEMENTATION

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1 THE POTENTIAL USE OF TOFU-PROCESSING WASTEWATER AS BACTERIAL GROWTH MEDIA FOR SOIL STRUCTURE IMPROVEMENT BY BIOCLOGGING AND BIOCEMENTATION Adibtya Asyhari 1, Muhammad Rizatul Yunus 1, Ariska Desy Haryani 1, Maytri Handayani 1, Emma Yuliani 1 1 Brawijaya University, Engineering Faculty, Department of Water Resources Engineering, Malang, Indonesia adibtya.asyhari@gmail.com ABSTRACT In order to examine a potential use of tofu-processing wastewater as the bacterial growth media for soil structure improvement, we conducted a research by inoculating five non-pathogenic bacteria (Pseudomonas sp., Nitrobacter sp., Bacillus subtilis, Lactobacillus sakei, and Agrobacterium tumifaciens) into five soil samples. As a comparison, we used chemical medias and natural soil samples as control. After 30 d of inoculation, we examined the soil structure through constant head test, direct shear test, and Scanning Electrone Microscopy (SEM). Based on the constant head test, the sample with Agrobacterium tumifaciens inoculation has the lowest permeabily value by 53,62 % reduction from control. This value is higher than those with chemical media. Based on the direct shear test, the sample with Lactobacillus sakei inoculation has the highest shear strenght value by 102,74 % increase from control. This value is a bit lower than those with chemical media. From this study, we concluded that tofu-processing wastewater is effective to use as bacterial growth media for soil structure improvement using biogrouting. Keywords: tofu-processing-wastewater, bacteria, bioclogging, biocementation Introduction Soil structure is the physical characteristic of the soil that describes the spatial arrangement of soil particles that combine to form aggregates (7). The condition of the soil structure will directly affect the bearing capacity of the soil. In some cases low soil bearing capacity can lead to the structural failure. Conventional efforts commonly used to overcome these problems are the grouting. Grouting is one attempt to reduce the permeability of coarse-grained soil by injecting a micture of cement and chemicals into the ground. The use of chemicals in the process of grouting can cause negative effects to the environment. The experts have begun to develop new methods of improving soil structure by biogrouting. The advantages of biogrouting is environmentally safe and relatively inexpensive compared with the grouting process using chemicals. One of the competitive biogrouting developed to improve the stability of the soil is by adding bacteria into the ground (12). The idea of biogrouting was suggested as early as in the 1950s (8). In later years, supportive evidence was obtained by number of researchers (1,9,10,13). Exploiting bacteria in biogrouting process associated with exopolysaccharides produced by certain types of bacteria. Each bacterial cells synthesize exopolysaccharides with different compositions, chemistry, and physics. Exopolysaccharides have been defined as organic polymers of microbial origin, which in biofilm systems, are frequently responsible for binding cells and other particulate materials to each others and to the substrate (2). When inoculated in soils, filamentous organisms, such as fungi, actinomyces, and some algae, produce a mechanical binding effect (4). In soil, the production of polysaccharidebinding materials by bacteria can cause sand particles to adhere to one another and build aggregates (5). These aggregates determine the mechanical and physical properties of soil, such as water retention, movement of water, aeration, and temperature (3). Aggregate formation facilitates cultivation, drainage, and aeration; increases the moisture holding capacity of the soil; and reduces erosion. (4). Exopolysaccharides production by bacteria is influenced by the growth phase of bacteria, bacterial growth media, available nutrients, acidity, and temperature (14). In the process of bacteria inoculation in the soil, there will be three processes. They are bioclogging process where bacteria will produce a material that is capable of filling the pores of the soil to reduce

2 tha value of permeability, biocementation process where bacteria produces a material that is able to increase the shear strength of the soil, and the process of gas production through bacterial activity which can lower the risk of liquefaction in sandy soil (6). Tofu is a common food in Asian countries, and during its production large amounts of wastewaters are produced as it is a water-intensive process. About 60 kg of soybeans and 2.7 L of water are required to produce 80 kg of tofu which ends up with a large amount of wastewater up to 2.6 L (11). The wastewater is a serious environmental pollutant due to its high organic content comprising mainly of reducing sugars, sucrose, starch and volatile fatty acids. Owing to its non-cellulosic nature with high carbohydrate and proteins, tofu-processing wastewater has the potential use to be developed as a medium for bacterial growth (15). The use of tofuprocessing wastewater as bacterial growth media can minimize the cost required in the grouting process. Materials and Methods Soil Samples The soils were river sand obtained from Brantas River. Before conducting our research, we analyzed the structure of soil samples, namely: 1. Soil particles gradation 2. Spesific gravity 3. Porosity and void ration Microorganisms In this study, we used five non-pathogenic bacteria. The bacteria were obtained from Microbiology Laboratory, Medical Faculty, Brawijaya University. They were Pseudomonas sp., Nitrobacter sp., Bacillus subtilis, Lactobacillus sakei, and Agrobacterium tumifaciens. Bacterial growth media In this study, we used three kinds of bacterial growth media. They are tofu-processing wastewater, chemical, and natural soil sample without any addition of bacterial growth media. Tofu-processing wastewater Tofu-processing wastewater was obtained from a tofu-making facility in Malang City, Indonesia. Tofu-processing preparation process is explained as follows: 1. 1 L of tofu-processing wastewater was filtered out and the filtrate was divided into 5 pieces of flask. Each flask contained 200 ml. 2. The tofu-processing then was sterilized at a temperature of 100 o C for 1 h. Chemical media Chemical media preparation process is explained as follows: 1. 1 L of aquades was added by CaCl 2 (0,25 g), MgSO 4 (0,25 g), K 2 HPO 4 (2,5 g), urea (2,5 g), and sucrose (25 g). 2. The blended mixture then was divided into 5 pieces of flask. Each flask contained 200 ml. 3. Chemical media then was sterilized at a temperature of 100 o C for 1 h. Soil sample preparation with bacterial inoculation Soil sample preparation with bacterial inoculation is explained as follows: 1. The soil samples sieved with ASTM standard sieve No. 10, subsequently sterilized by heating at a temperature of 105 o C for 1 h. 2. Soil samples were divided into three sections, each weighing 12 kg with treatment as follows: Using tofu-processing Using chemical media Without any use of growth media 3. Each part was mixed with growth medium until blended and aged for one day. 4. Subsequently each treatment is further divided into six sections, each weighing 2 kg to be inoculated with the five types of bacteria and one part as a control. 5. The soil samples that have been mixed with the growth medium, then inoculated with a bacterial colony with 25 ml of concentration x 10 9 CFU, then left undisturbed for 30 d.

3 Permeability analysis using constant head The soil samples that had been inoculated with bacteria then were tested by constant head permeameter to investigate the value of soil permeability. The results obtained from this test then were compared to control to analyze its effectivity. Shear strenght analysis using direct shear Besides testing for permeability, the soil samples that had been inoculated with bacteria were also tested with direct shear to investigate the value of soil shear strenght. The results obtained from this test then were also compared to the value of shear streght from control sample to analyze its effectivity. Scanning Electron Microscopy Analysis In order to determine the visual appearance of the results of bacterial inoculation for soil structure improvement, soil samples with the lowest permeability and the highest shear strength values were analyzed using a scanning electron microscopy (SEM) FEI TM : Inspect-S50. SEM test aims to determine the presence of exopolysaccharides formed on soil samples. The test is performed on a soil sample that has the lowest permeability and the highest shear strength values after the inoculation for 30 d. Results and Discussions Soil samples analysis Table 1 summarizes relevant physical properties of these materials as well as Scanning Electrone Miscroscopy (SEM) image. Composition of tofu-processing wastewater Wu, T., Y., et al. reported that tofu-processing wastewater is a valuable source of nutrients in the form of carbohydrate and protein (15). In our study, protein content was shown to be high, but the carbohydrate content was quite low. The composition of tofu-processing wastewater is shown in Table 2. Relevant properties of tested soils Parameters Results D60 (mm) 0.50 D30 (mm) 0.24 D10 (mm) 0.10 Specific gravity 2.63 Porosity (%) Void ratio 0.90 SEM image Composition of tofu-processing wastewater Parameter Results Total sugars (%) 0.16 Carbohydrate (%) 0.07 Protein (%) 0.21 Soil permeability analysis TABLE 1 TABLE 2 The value of soil samples permeability after is shown in Table 3. The highest declined of permeability value occurred in soil samples inoculated with bacteria Agrobacterium tumifaciens with 53.62% reduction of the control soil samples, followed in particular order by the inoculation of bacteria Nitrobacter sp., Bacillus subtilis, Lactobacillus sakei, and Pseudomonas sp. This value is higher when compared with the inoculation of bacteria Agrobacterium tumifaciens on soil samples with chemical media shown in Table 4.

4 TABLE 3 The value of soil samples permeability after Bacteria Pseudomonas sp. The value of permeability The value of permeability in control sample Percentage of reduction (cm/dt) (cm/dt) (%) Nitrobacter sp B. subtilis L. sakei A. tumifaciens TABLE 4 The comparison of permeability value in soil samples inoculated with bacteria Agrobacterium tumifaciens in three kinds of bacterial growth media after inoculation for 30 d Bacterial growth media Tofu-processing The value of permeability Percentage of reduction (cm/dt) (%) Chemical media Without any growth media Soil shear streght analysis The value of soil samples shear strenght after is shown in Table 5. The highest increasing of shear strenght value occurred in soil samples inoculated with bacteria Lactobacilus sakei with the percentage of increased of 102,74% of the control soil samples, followed in particular order by the inoculation of bacteria Pseudomonas sp., Bacillus subtilis, Agrobacterium tumifaciens, and Nitrobacter sp.. This value is a bit lower compared with the inoculation of bacteria Lactobacillus sakei on soil samples with chemical media shown in Table 6. TABLE 5 The value of soil samples shear strenght after Bacteria Pseudomonas sp. Shear strenght Shear strenght in control sample Percentage of increased (kg/cm 2 ) (kg/cm 2 ) (%) Nitrobacter sp B. subtilis L. sakei A. tumifaciens TABEL 6 The comparison of the value of shear strenght in soil samples inoculated with bacteria Lactobacillus sakei in three kinds of bacterial growth media after inoculation for 30 d Bacterial growth media Tofu-processing Shear strenght Percentage of increased (kg/cm 2 ) (%) Chemical media Without any growth media Scanning Electron Microscopy Analysis Soil samples used for SEM test were chosen with consideration of the value of the lowest permeability and highest shear strength of the soil samples after inoculation for 30 d. Based on the constant head test, it was obtained that soil samples inoculated with bacteria Agrobacterium tumifaciens has the lowest permeability values. While from the direct shear test, it was obtained that soil samples inoculated with bacteria Lactobacillus sakei has the highest shear strength value. Both of these soil samples will be further analyzed.

5 Fig. 1. shows that Agrobacterium tumefaciens formed exopolysaccharides which serves as a cap for the pores of the sample. Exopolysaccharides formed particles stick to the walls of the soil which in turn will fill the pores of the soil through a process known as bioclogging. The test results are consistent with constant head test showed that soil samples inoculated with bacteria A. tumefaciens has the lowest value of soil permeability. Exopolysaccharides produced by Lactobacillus sakei has a role as an adhesive between the soil particles, it is then led to an increase in shear strength of this soil sample (Fig. 2). Inoculation by L. sakei is suitable to be used in the improvement of soil structure through biocementation. Conclusions From this study, we concluded that tofu-processing wastewater is effective to use as bacterial growth media for soil structure improvement by bioclogging dan biocementation.-processing tofu wastewater contains ingredients that can be used by bacteria to produce exopolysaccharides. The culture conditions developed are not expensive, because they do not require the addition of any nutrients to the medium. This strongly suggests that the currently used chemical media for biogrouting can be replaced by tofu-processing wastewater. Acknowledgements The authors wish to thank the Education Ministry of Indonesia for financial support through PKM. REFFERENCES Fig. 1. The results of scanning electron microscope with soil samples inoculated with Agrobacterium tumifaciens (left) 5K zoom (right) 10K zoom Fig. 2. The results of scanning electron microscope with soil samples inoculated with Lactobacillus sakei (left) 5K zoom (right) 10K zoom The scanning electron microscopy test results indicate that these findings are consistent with the expected, that inoculation of bacteria in the soil is able to produce exopolysaccharides used to improve the soil structure. 1. Avnimelech, Y., and Z. Nevo. (1964) Biological clogging of sands, Soil Sci., 98, Characklis, W. G., and Wilderer, P. A. (1989) Glossary. In: Characklis, W. G., Wilderer, P. A. (eds) Structure and function of biofilms, Wiley, Chichester, United Kingdom of Great Britain, pp Dickson, E. L., Rasiah, V., and Groenevelt, P. H. (1990) Comparison of four prewetting techniques in wet aggregate stability determination, Can. J. Soil Sci., 71, Geoghegan, M. J., and Brian, R. C. (1948) Aggregate formation in soil: Influence of some bacterial polysaccharides on the binding of soil particles, Biochem. J., 43, Godinho, A. L., and Saroj, B. (2009) Sand aggregation by exopolysaccharide-producing microbacterium arborescens-agsb, Current Microbiol., 58, Ivanov, V., and Chu, J. (2008) Applications of microorganisms to geotechnical engineering for bioclogging and biocementation of soil in situ, Environ Sci. Biotechnol., 7,

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