Field detection of urease and carbonic anhydrase activity using rapid and economical tests to assess microbially induced carbonate precipitation

Abstract

Microbial precipitation of calcium carbonate is a widespread environmental phenomenon that has diverse engineering applications, from building and soil restoration to carbon sequestration. Urease-mediated ureolysis and CO 2 (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many environmental microbial community studies rely on marker gene or metagenomic approaches that are unable to determine in situ activity. Here, we developed fast and cost-effective tests for the field detection of urease and CA activity using pH-sensitive strips inside microcentrifuge tubes that change colour in response to the reaction products of urease (NH 3) and CA (CO 2). The urease assay proved sensitive and useful in the field to detect in situ activity in biofilms from a saline lake, a series of calcareous fens, and ferrous springs, finding relatively high urease activity in lake samples. Incubations of lake microbes with urea resulted in significantly higher CaCO 3 precipitation compared to incubations with a urease inhibitor, showing that the rapid assay indicated an on-site active metabolism potentially mediating carbonate precipitation. The CA assay, however, showed less sensitivity compared to the urease test. While its sensitivity limits its utility, the assay may still be useful as a preliminary indicator given the paucity of other means for detecting CA activity in the field. Field urease, and potentially CA, activity assays complement molecular approaches and facilitate the search for carbonate-precipitating microbes and their in situ activity, which could be applied toward agriculture, engineering and carbon sequestration technologies.

Potential role for microbial ureolysis in the rapid formation of carbonate tufa mounds

“…Both reactions potentially increase the pH at the biofilm microenvironment—thus, promoting carbonate precipitation—by generating ammonia, in the case of urease, or by increasing the rate of CO 2 degassing, in the case of CA. Using an enzymatic field detection assay (Medina Ferrer et al, 2020), we found in situ active urease and CA in surficial mats from subaqueous tufas (Figure 8a,b; Figure S6). Preliminary assessments in the field showed multiple urease‐positive tufa biofilms in October 2018, which were re‐evaluated and semi‐quantified after a second sampling campaign in August 2020 (Figure 8a).…”

“…The carbonate samples for microscopic analysis were crushed, homogenized, stained with DAPI (4′,6‐diamidino‐2‐phenylindole), and imaged on an Olympus BX41 microscope (Olympus Corporation). In addition, surficial biofilm samples were also taken in October 2018 from 14 subaqueous tufas and in August 2020 from two subaqueous tufas and from green filaments in the lake water collected adjacent to, but not directly on tufas, and analyzed on‐site to determine urease and carbonic anhydrase (CA) activity (see Section 2.5) using the method described by Medina Ferrer et al (2020).…”

“…To determine whether the enzymes urease and carbonic anhydrase (CA) were active in situ , surficial biofilms from submerged tufas were field tested using the assay described by Medina Ferrer et al (2020). The tests consist of a pH‐sensitive indicator strip (a cellulose paper impregnated with a 0.02% phenol red pH 6.0 solution for the urease assay, or impregnated with a 6.5 m M Na 2 CO 3 , 0.01% metacresol purple, 50% glycerol for the CA assay) situated in the cap of a microcentrifuge tube, and the substrate of the respective enzyme (a cellulose paper impregnated with a 0.6 M urea solution as urease substrate, or a cold 1 M NaHCO 3 solution as CA substrate) located in the main body of the tube.…”

“…A positive reaction was indicated by a color change from yellow to red in the indicator strip of the urease assay (produced by a pH increase after NH 3(g) release in the reaction tube), or by a faster color change from purple to yellow (produced by a pH decrease after CO 2(g) release in the reaction tube) compared with a negative control for the CA assay. Negative control assays were evaluated under the same conditions, but additionally adding a specific inhibitor of urease, phenyl phosphorodiamidate (PPD; Alfa Aesar; Kafarski & Talma, 2018), or a specific inhibitor of CA, acetazolamide (Sigma‐Aldrich; Zimmerman et al, 2004), to the corresponding enzyme substrate as previously described (Medina Ferrer et al, 2020). The progression of the enzymatic reactions was followed by semi‐quantifying the hues of the indicator strips from photographs taken in the field (Section 2.6).…”

“…In situ urease and CA assays were evaluated by semi‐quantifying their hue changes over time from photographs taken in the field as previously described (Medina Ferrer et al, 2020). Average hues were calculated from RGB measurements over a standard circle area of the assay color indicator using the NIH imagej 1.49v software.…”