Test And Optimization Urease Activity

3.1. Test And Optimization Urease Activity

Test and optimization activities carried out to determined the optimum activity biogrouting bacteria to produce urease. These enzymes will be produced to be applied on a laboratory scale. Bacterial isolates used in this study was isolate P3BG43 code. Based on previous research these isolates were from the collection of the Biotechnology Research Center LIPI Cibinong. According to [5], this P3BG43 isolates taken from altitude (± 4,000 m above sea level), which has low air pressure so that the ambient temperature was too low. Therefore, it was difficult bacteria grown tropics. This bacterium was sensitive to environmental change, temperature and medium. This condition indicated by the number of repetitions performed to adapt. Need 4-5 times of cultivation using medium NB, B4 (marine agar), and NA.

Based on previous research P3BG43 isolates identified as Oceanobacillus sp., with phenotypic characteristics include a rod-straight shape (size 0.3-2.2x1.2-7.0 m), motile with flagella lateral-type, heat-resistant form endospores (the amount of not more than one in one cell sporangia) and give a positive reaction on the urease test. Oceanobacillus sp. does not have the activity of extracellular enzymes when starch hydrolysis test, trybutirin and casein. Isolates P3BG43 been known to have the highest urease activity based methods Weatherburn (1967) were modified (Figure 1) [5].

Optimization of bacterial growth biogrout done by growing isolates in medium B4 and B4 modified using urine (urine B4). Then the cell density was measured using a spectrophotometer. B4 medium is the medium that contains the nutritional content of minerals and urea. Urea (NH ) CO containing ammonium which causes precipitation of calcite [17]. While the source of urea B4 medium replaced with urine (urine B4) containing ammonia (NH ) [18]. The content of ammonia in the urine causing incomplete reaction to precipitate carbonate. The existence of ammonia in a medium containing water (distilled water) causes a spontaneous reaction. Spontaneous reaction caused by the presence of water (H O) convert ammonia (NH ) into ammonium (NH ⁺) and carbon dioxide (CO ) will balance the chemical reactions become carbonic acid, bicarbonate ions and carbonate ions. The increase in pH due to hydroxyl ions formed from NH ⁺ production that exceeds the availability of Ca²⁺. This condition causes an alkaline environment so that the carbonates needed for calcite precipitation.

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

Optimization was done by conditioning the growth of bacteria on two types of medium, variations in pH range of 3-9 and at a temperature of 25 ° C and 29 ° C. The setting conditions of the medium, pH and temperature was done with the assumption that it still appropriate when applied biogrout products in the environment. Based on data analysis using the General Linear Model was known that at temperature of 25 °

C and pH 7 are the optimum condition biogrouting bacterial growth (Figure 1). P value less than 0.05 (p <0.05) which means pH and temperature affect the growth of bacteria biogrout.

Figure 1. Bacterial growth curve at pH 3-8 and a temperature of 25 ° C and 29 °

C in Medium B4 Urea

While the growth bacteria biogrout optimization test on the urine medium showed a less significant. The treatment was done up to 10x repetition. This was possible due to several factors such as pH range of urine and ammonia levels were not measured. According to Shafiee et al ., (2003) urine contains more salt waste from the body and

a little ammonia [19]. Based on the test, known biogrout bacteria can grow optimally in medium B4 urine pH 8 and a temperature of 25 ° C. Based on data analysis using the General Linear Model, the growth of bacteria in the urine B4 shows the p value less than 0.05 (p <0.05, ᾱ (alpha) of 5%.). The figure shows that the urine contained in the medium B4 significantly affect the growth of bacteria.

Sept em ber 8 th 9 – th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

Figure 2. The growth curve Bacteria in Urine B4 Medium

Urease activity was measured based on optimum conditions bacterial growth. Based on the analysis of data, the growth of bacteria known optimal time on 12th and 13th. The time assumed to be a potential to produce urease. Based on the measurement of enzyme activity using Bradford methods known 144 units / ml. While the measurement of enzyme activity using methods Weatherburn (1967) obtained urease activity of 203.32 units / ml.