Optimization of HCO3- production reflect to CaCO3 precipitation for selfhealing by Bacillus Sphaericus Norfaniza Mokhtar1, a ,Zhameir Shafiq Mohd Ilias2,b, Husnul Azan Tajarudin3,c*, Megat Azmi Megat Johari4,d a
[email protected],
[email protected],
[email protected],
[email protected] 1,4
School of Civil Engineering, Universiti Sains Malaysia, Malaysia
2,3
School of Industrial Technology, Universiti Sains Malaysia, Malaysia
3
Cluster of Solid Waste Management, Engineering Campus,Universiti Sains Malaysia, Malaysia
Keywords: Bacillus Sphaericus, urease activity, microbial mineral precipitation, HCO3Abstract Bacteria are able to perform metabolic activities which promote the precipitation of calcium carbonate in the form of calcite. Bacillus Sphaericus was used in this study, which is an ureolytic bacteria that can precipitate calcium carbonate in its environment by the decomposition of urea into ammonium and carbonate. The bacterial degradation of urea basically increases the pH and promotes the microbial deposition of carbonate as calcium carbonate. In this research, the capability of bacteria to influence the formation of HCO3- by the production of urease enzyme was investigated. Results of growth rate and characteristics of bacteria showed that 20g/L of urea concentration was able to provide a good environment for bacteria with sufficient amount of nutrient to survive. The formation of HCO3- was parallel with NH3 production where the formation of HCO3- increased slowly as the ammonia production decreased. Urea degradation with suitable concentration of urea by 20g/L may form high HCO3- compared to 25g/L urea concentration. The results from the experimental works indicated that the optimal urea concentration was 20g/L.
Introduction Microorganisms are able to influence the precipitation of carbonaceous minerals, specifically calcium carbonate (CaCO3) in a number of ways. Examples of such metabolic processes include photosynthesis, organic acid utilization and urea hydrolysis [1]. Urea hydrolysis presents a common microbial process, which causes mineral precipitation in natural environments and can be used in biotechnological applications [2-4]. For example, microbial cell surfaces act as heterogeneous crystal nucleation sites in super-saturated CaCO3 solutions. Alternatively, microbes can alter the saturation state of under-saturated solutions, thereby also catalyzing mineral precipitation. CaCO3 is rather a straightforward chemical principle process that concerns in four key factors namely the concentration of dissolved inorganic carbon, pH, concentration of calcium ions (Ca2+) and the presence of crystal nucleation site [5]. Several microbial metabolic processes directly influence the first three factors, while the physical and chemical characteristics of bacteria cells render than ideal crystal nucleation sites [6]. Previous research has shown that Bacillus Sphaericus bacteria are able to precipitate CaCO3 on their cell constituents and in their own environment which catalyzes the hydrolysis of urea into ammonium and carbonate [3]. The bacterial degradation of urea basically increases the pH and promotes the microbial deposition of CaCO3 in a calcium environment. Through this process, the bacterial cell is coated with a layer of CaCO3. Enzymatic hydrolysis of urea, dissimilitory reduction of NO3- SO43- and ammonification of amino acids cause pH rise that, in turn, shifts the biocarbonatecarbonate equilibrium to produce more CO32- and, ultimately, to precipitate CaCO3 if free Ca2+ is present. The aims of this research are to investigate the influence of urea concentration and amount
of HCO3- formed which is the important element of CaCO3 production, on the efficiency of the bio deposition treatment by examined the increasing of bacteria concentrations and various concentration of urea in growth medium.
Materials and Methods Bacteria and growth conditions The bacterial strains used in the experiments were Bacillus Sphaericus LMG 22557 (Belgian Coordinated Collection of Microorganisms, Ghent). Based on previous research, Bacillus Sphaericus was found to be able to precipitate CaCO3 in a simple and controllable way [3], long survival time [4] and has a high urease activity [2]. Living cells were grown in the sterile growth medium consisting yeast extract and different concentrations of urea. The yeast extract medium was first autoclaved for 20 min at 120°C and the sterilized urea solution by means of filtration through a sterile 0.45µM Milipore filter, was added. Final concentrations of yeast extract and urea in growth medium were both 20g/L. In all experiments, Bacillus Sphaericus cultures were obtained after subsequent culturing from a -80°C stock culture. The cultures were incubated at 28°C on a shaker at 120rpm for 24h and were re-suspended in saline solution (NaCl, 8.5g/L).
Determination of the optimal conditions for bio-precipitation The concentration of bacteria and urea will greatly affect the amount of HCO 3- formed. A series of tests were performed to investigate the optimal bacterial and urea concentrations. The ability of bacteria to remain viable and sustained in high urease activity will also be tested. Batches of 100 mL bacterial solutions (109 cell/mL) were added into a sterilized schott bottle. The bottles were then closed tightly and put in the incubator at 28°C. Bacteria in each bottle were inoculated into 900 mL of deposition medium (the deposition medium was used for bacteria to form HCO3-), various concentration of urea solution and yeast extract. Yeast extract (20g/L) was used to serve as a nutrient for the bacteria. The detailed information about the experimental arrangement can be seen in Table 1. The media were then put on the shaker (28°C, 100 rpm). The ureolytic activity of the bacteria was indicated by the amount of urea decomposed by the bacteria in the deposition medium for every hour by calculating the total ammonium nitrogen (TAN)[7]. One mole of urea (CO(NH2)2 produced 2 moles of NH4+. The amount of NH4+ can indicate the amount of urea decomposed. Table 1: Composition of growth media Series U0,Y20 U5,Y20 U15,Y20 U20,Y20 U25,Y25
Concentration of bacteria [cells/mL] 109 109 109 109 109
Urea [g/L] 0 10 15 20 25
Yeast [g/L] 20 20 20 20 20
Production of HCO3Alkalinity is important for many uses and treatments of natural waters and wastewaters. This is due to many surface waters contain the element of carbonate, bicarbonate and hydroxide content, so alkalinity has been the guidance or an indicator of the concentration of these elements. Furthermore, the measured values from this alkalinity method may be influenced by the presence of other elements such as borates, silicates, phosphorus or other elements. Normally, alkalinity measurements are used to interpret and control the water and wastewater treatment processes. Therefore, for these methods, 50 ml of sample was titrated with 0.02N Sulphuric Acid (H2SO4), and the alkalinity of the sample was monitored by using the pH probe. The endpoints of these methods,
since pH at the equivalence point of the titration have been determined by the concentration of carbon dioxide, showed the content of carbonate in the sample. Based on the titration, the production of carbonate can be calculated by using the following formula: (1) 𝐴 𝑥 𝑁 𝑥50000 𝐴𝑙𝑘𝑎𝑙𝑖𝑛𝑖𝑡𝑦, 𝑚𝑔 𝐻𝐶𝑂3− /𝐿 =
50𝑚𝐿 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
where as 𝐴 𝑖𝑠 𝑚𝐿 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑎𝑐𝑖𝑑 𝑢𝑠𝑒𝑑 and 𝑁 𝑖𝑠 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑎𝑐𝑖𝑑
Total ammonium nitrogen and optical density measurement The production of ammonia by various concentrations of urea was measured as a function of growth in liquid deposition medium. After 10% (v/v) inoculation with an overnight culture, sample was taken for every hour for optical density (OD) and total ammonium nitrogen (TAN) measurements. In order to assess the growth, the OD was measured with a spectrophotometer at 580nm. TAN concentrations were measured calorimetrically by the method used by Nessler [7]. The specific urea degradation rate (SUD) is defined as the ratio of ammonium production (AP) per unit of bacterial growth in a given time (t) and is given by the following formula: 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑢𝑟𝑒𝑎 𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛, (𝑆𝑈𝐷) =
𝐴𝑃 (𝑔 𝑎𝑚𝑚𝑜𝑛𝑖𝑢𝑚 𝑥 1−1 ) [𝑂𝐷 𝑐𝑒𝑙𝑙𝑠]𝑥 𝑡(ℎ)
(2)
where as 𝐴𝑃 𝑖𝑠 𝑎𝑚𝑚𝑜𝑛𝑖𝑎 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛, 𝑂𝐷 𝑖𝑠 𝑜𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 and 𝑡 = 𝑡𝑖𝑚𝑒
Result and Discussion Growth rate and characteristics of bacteria in various urea concentrations The growth profile (Fig.1) was studied up to 12h. It was observed from the graph that the concentration of bacteria increased due to time consuming and concentration of urea. The maximum growth observed for concentration of bacteria was 9.79 x 109 cells/mL in urea concentration of 20g/L. Table 2, summarizes the result of kinetic growth of bacteria regarding the various concentration of urea. Since Fig. 1 presented the growth of bacteria that was indirectly correlated with time, it is imperative to examine the kinetics of HCO3- formed as well as its relationship to cell growth. The specific rate (µ) for various concentration of urea was calculated. The µ is one of the parameters that was considered in optimizing the suitable urea concentration. From the experimental result, the µ for 20g/L of urea concentration showed the highest value of 0.278 (h-1), followed by urea concentration of 10g/L, 5g/L and 25g/L. It has been proved that high concentration of urea cannot give a suitable condition for the culture due to the high content of urea that contributed to saturated condition, unable to produce carbonate. Lower concentration of bacteria gave the lowest specific growth rate due to lack of nutrient during the growth process. Doubling time, (td) is the time taken for the number of cells to double, with shorter doubling times implying more rapid growth. By referring to the result of td, 20g/L of urea concentration showed the shorter td of 2.493h, and urea concentration of 0g/l needed 20.382h to double their cells. It showed that 20g/L of urea concentration was the most suitable concentration for growth medium as it may enhance an ultimate environment for the urea degradation process compared to 0g/L that will definitely show that Bacillus Sphearicus needs to struggle to grow and needs longest time to be produced in the culture. It is proved by the bacteria concentration for 20g/L of urea concentration. Bacteria concentration is the last parameter measured in order to optimize the suitable urea concentration. Urea concentration of 5g/L and 20g/L showed higher growth yields which were 9.96 x 109 cells/mL and 9.79 x 109 cells/mL, respectively. The bacteria concentration was observed for 10 hours, which consistently increased due to time. Even 5g/L of urea concentration showed higher
results. Other characteristics should be considered in order to achieve the optimum condition which provides a good environment for the bacteria such as the amount of nutrients that will affect the survival of bacteria for a long time. Based on the three parameters observed, 20g/L urea concentration was the optimal urea concentration that will be used in further research.
Fig 1. Growth profile of various concentration of urea Table 2: Kinetics growth of bacteria in various concentration of urea µ [h-1]
Urea Concentration [g/L] 0 5 15 20 25
Doubling time, td [h] 20.382 3.686 3.536 2.493 4.175
0.034 0.188 0.196 0.278 0.166
Concentration of bacteria [cells/mL] 3.44 x 109 9.96 x 109 6.46 x 109 9.79 x 109 4.00 x 109
Production of HCO3- vs NH3 in various urea concentration As presented in Fig.2, the formation of HCO3- was correlated directly to the production of ammonia due to various urea concentration. The specific rate of HCO3- formed became relatively high at the time the rate was increased considerably. However, the specific rate for ammonia production steadily decreased with the increase of HCO3- formed and cell concentration due to the increasing time and pH, and it went to high concentration of urea. It is hypothesized that when the cell concentration is low, every cell would be responsible for serving as a nucleation site of HCO 3formation and producing ammonia to increase the pH in their immediate surroundings, and the more urea concentration leads to high formation of HCO3-. This hypothesis was proved in the basic calcocarbonic system. During the microbial urease activity, 1 mole of urea is hydrolysed intracellularly to 1 mole of ammonia and 1 mole of carbamate (Eq. 3), which occurs spontaneously to form an additional 1 mole of ammonia and carbonic acid (Eq. 4) [8]. CO(NH2)2 + H2O
NH2COOH + NH3
(3)
NH2COOH + H2O
NH3 + H2CO3
(4)
These products were subsequently equilibrated in water to form bicarbonate and 2 moles of ammonium and hydroxide ions (Eqs. 5 and 6) H2CO32NH3 + 2H2O
HCO3- + H+ 2NH4+ + 2OH-
(5) (6)
The latter 2 reactions increase pH, which in turn shifts the bicarbonate equilibrium, resulting in the formation of carbonate ions (Eq. 7) and ultimately, to precipitate CaCO3 if free Ca2+ is present.
HCO3- + H+ + 2NH4+ + 2OH-
CO32- + 2NH4+ + 2H2O
(7)
CaCO3 precipitation is controlled by intracellular calcium metabolism, instead of by changing carbonate ions concentration. Second carbonate nucleation takes place on cell walls due to ion exchange through the cell membrance or due to promotion by negatively charged specific cell wall functional groups that adsorb Ca2+ ions. However, for this research Ca2+ source was not included.
Fig 2. HCO3- and NH3 production due to various concentration of urea Urea Degradation The amount of urea degradation in each series of various urea concentration was measured every hour (as shown in Fig 3). It can be seen in the 0 g/L urea concentration, no urea was degraded. The more urea used, the higher the amount of urea hydrolysed. Urea concentration of 20g/L and 25g/L generated the highest amount of urea degradation by 0.769 g/L/h and 0.817 g/L/h, respectively. Typically, bacteria that contain higher urea degradation rate will show a high affinity for urea. However, the concentration of urea cannot be too high because the rate of decomposed urea decreased as the concentration of urea increased, meaning that there will be more urea left in the highest concentration of urea, 25 g/L. In addition, at high urea concentrations, level of ammonium will be lower due to the insufficient urease activity of the microbial population. Concentration of urea at 20 g/L showed a smooth decrease of the urea concentration which literally increase the amount of the urea consumption compared to other concentration of urea. This urea degradation will lead to the production of ammonia and carbonate which increased the pH and carbonate concentration in the bacterial concentration [9]. Thus, it can be seen that 20 g/L urea concentration is the optimum concentration of the bacteria as it produced the highest amount of carbonate, lowest rate of ammonia production and higher degradation of urea compared to other concentrations.
Fig 3. The specific urea degradation Conclusion
Based on the studies of growth rate and characteristics of bacteria, it showed that 20g/L urea concentration proved to be the best concentration related to the combination of three elements which were µ (0.287 h-1), td (2.49h) and cell concentration of 9.79 x 109 cells/mL, that were able to provide a good environment for bacteria with sufficient amount of nutrient to survive. Urea degradation for 25g/L urea concentration was higher than the 20g/L of concentration. Nevertheless, by considering the production of HCO3- and NH3 as the major indicator for optimization has proved that the suitable urea concentration of 20g/L may form high HCO3- compared to 25g/L urea concentration and the production of HCO3- increased slowly as the ammonia production decreased. There were some ureas remained left and will act later to enhance the concrete durability. Table 3 presents the summary of parameters considered for the optimization of HCO3- production and can conclude that 20g/L urea concentration has been selected as optimal urea concentration and will be applied in further research for self-healing of bio-cement. Table 3: Parameter for optimization of HCO3- production Urea concentration [g/L] 20
Doubling time, td [h] 2.493
Concentration of bacteria [cells/mL] 9.79 x 109
HCO3[mg/L] 412.546
NH3 [mg/L] 1.002
Urea degradation [g/L/h] 0.769
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