Microbially induced carbonate precipitation (MICP) occurs as a by-product of microbial activities in the environment (
Urease activity is widely distributed in the environment, and urease is produced by a wide range of microbes (Anbu
The major cost associated with ureolysis-driven MICP applications is the production of ureolytic microbes (Cheng and Cord-Ruwisch, 2013; Whiffin, 2004). The cost of sterilizing the cultivation medium is in the range of US$0.46–0.66 per l of medium, which is comparatively higher than the cost of producing a yeast extract-based medium for culture of
Here, we report the enrichment and production of ureolytic microbes from a soil sample by using a down-flow hanging sponge (DHS) bioreactor system. This bioreactor system was originally developed as a low-tech biofilm-type sewage treatment technology (Agrawal
Enrichment samples squeezed from even-numbered sponge cubes (counting from the top) were used to determine urease activity and biomass concentration and for fluorescence
Potential urease activity was determined using a conductivity method as reported by Cheng and Cord-Ruwisch (2013), with slight modification. In brief, the relative conductivity changes in 10 ml of urea solution (1.5 M final concentration) containing 1 ml of enrichment culture at 25°C was measured using a conductivity meter (LAQUAtwin B-771; HORIBA) at 3−15 min intervals. The rate increase in conductivity was converted to potential urease activity as described previously (Cheng and Cord-Ruwisch, 2013; Whiffin, 2004). One unit (U) of potential urease activity was defined as the enzyme activity that hydrolyzes 1.0 μmol of urea per minute at 25°C. All assays were performed in triplicate.
Biomass concentrations were recorded as mg of dry weight per ml of volume. The dry weight of the biomass was determined after drying samples to a constant weight at 105°C. Subsampled enrichment cultures used for the dry weight determination were washed twice with sterilized distilled water before drying. All assays were performed in triplicate.
The tubes were incubated at 25°C for 24 h with shaking (at 120 rpm). Negative controls, without the enrichment, (n = 3) were used to detect any non-biological CaCO3 precipitation. After the incubation period, precipitates were filtered with Whatman filter paper No. 1 (GE Healthcare UK Ltd., Buckinghamshire, UK) and washed with distilled-deionized water. Then, the filters were dried at 105°C for 24 h and were weighed. The weight of precipitates (
where,
To reveal the mineralogy and surface morphology of the precipitates, a representative sample from the CaCO3 precipitation test was subjected to X-ray diffraction (XRD) and scanning electron microscope (SEM) analyses. The XRD analysis was performed on a SmartLab (Rigaku, Tokyo, Japan) with Cu Kα radiation operated at 40 kV and 30 mA. Prior to the XRD analysis, the collected precipitates were powdered with a mortar and pestle. The XRD data were collected on the scale of diffraction angle (2θ) = 5°−90°. The XRD analysis was performed by the Industrial Technology Center of Wakayama Prefecture (Wakayama, Japan). The SEM analysis was performed with a JSM-6510 (JEOL Ltd., Tokyo, Japan) operated at an accelerating voltage of 5 kV. The collected precipitates used for the SEM analysis were sputter coated with platinum prior to the analysis.
To confirm the enrichment and production of ureolytic microbes in the DHS bioreactor system, the potential urease activity of enrichment samples retrieved from the sponge cubes was determined. Although no obvious potential urease activity (
In the initial phase 1 (day 0−23), the yeast extract concentration in the supplied medium was set at 1 g/l. The average potential urease activity observed in this phase was 2.9 U/ml (Fig. 2). Since the bioreactor system used in this study was a closed system, we expected that oxygen could not be effectively supplied to any aerobic ureolytic microbes that colonized the sponge cubes. In fact, the negative ORP values of the bioreactor effluents (Fig. 3) suggested depletion of the dissolved oxygen in the supplied medium as the medium flowed down into the sponge cubes. Therefore, from day 24 to 130
A positive correlation (coefficient of determination
Our data demonstrate that the low-tech DHS bioreactor system described in this study has potential for the enrichment and production of ureolytic microbes under non-sterile conditions. In addition, the ureolytic enrichment obtained from the bioreactor system showed effective CaCO3 precipitation capacity