bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902379; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Detection of urease and carbonic anhydrase activity 2 using a rapid and economical field test to assess 3 microbially-induced carbonate precipitation 4 Fernando Medina Ferrer1*, Kathryn Hobart1,2, Jake V. Bailey1*. 5 1Department of Earth Sciences, College of Science & Engineering, University of Minnesota, 6 Twin Cities. John T. Tate Hall, 116 Church Street SE, Room 150, Minneapolis, Minnesota 7 55455, United States. 8 2Institute for Rock Magnetism, University of Minnesota, Twin Cities. John T. Tate Hall, 116 9 Church Street SE, Minneapolis, Minnesota 55455. 10 KEYWORDS: Calcium carbonate; carbon sequestration; MICP; CaCO3; biomineralization; 11 biocementation; biogrouting. 12 *Corresponding Authors: [email protected]; [email protected]. 13 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902379; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 14 ABSTRACT 15 Microbial precipitation of calcium carbonate has diverse engineering applications, from 16 building and soil restoration, to carbon sequestration. Urease-mediated ureolysis and CO2 17 (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate 18 minerals, yet many microbial community studies rely on marker gene or metagenomic 19 approaches that are unable to determine in situ activity. Here, we developed fast and cost- 20 effective tests for the field detection of urease and CA activity using pH-sensitive strips inside 21 microcentrifuge tubes that change color in response to the reaction products of urease (NH3) and 22 CA (CO2). Samples from a saline lake, a series of calcareous fens, and ferrous springs were 23 assayed in the field, finding relatively high urease activity in lake samples, whereas CA activity 24 was only detected in a ferrous spring. Incubations of lake microbes with urea resulted in 25 significantly higher CaCO3 precipitation compared to incubations with a urease inhibitor. 26 Therefore, the rapid assay indicated an on-site active metabolism potentially mediating carbonate 27 mineralization. Field urease and CA activity assays complement molecular approaches and 28 facilitate the search for carbonate-precipitating microbes and their in situ activity, which could be 29 applied toward agriculture, engineering and carbon sequestration technologies. 30 31 INTRODUCTION 32 Microbially-induced carbonate precipitation (MICP) has been explored as an alternative 33 solution for several engineering and environmental challenges, such as restoration of building, 34 monument, and concrete structures, soil consolidation, pollutant bioremediation and CO2 35 sequestration.1–4 Despite its relevance in the carbon cycle and its multiple applications, MICP is 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902379; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 36 difficult to assess directly in a given environment. Current environmental microbiota analyses 37 commonly characterize communities via 16S rRNA gene sequencing and metagenomic 38 sequencing, which fail to determine active metabolisms, unless challenging and labor-intense 39 transcriptomic or culture-based analysis are performed, which do not necessarily reflect in situ 40 activity. It is possible, however, to directly test the activity of certain enzymes in the field and 41 identify active metabolisms on-site. Here, we developed a method for the field detection of 42 carbonic anhydrase (CA; EC 4.2.1.1) and urease (EC 3.5.1.5) activity, two enzymes associated 43 with MICP. 44 Metabolisms such as photosynthesis, methane oxidation, nitrate reduction, bicarbonate 45 transport and ureolysis can locally increase carbonate saturation and promote MICP.4,5 Two of 46 these metabolisms rely on enzymes whose activity can be detected in the field: ureolysis and CO2 47 hydration via CA. Ureolysis, catalyzed by urease, allows microorganisms to use urea as a 6 – 48 nitrogen and carbon source. CA facilitates rapid carbon transport into the cell via CO2-HCO3 49 interconversion.7 Both enzymes generate carbonate anions and increase the pH as a product of 50 their activity. Urease generates ammonia and CO2 from urea (eq. 1), which coupled to ammonia 51 hydrolysis and CO2 hydration, catalyzed by CA (eq. 2), produces one mol of hydroxide and one 52 mol of bicarbonate per mol of urea (eq. 3): 53 (NH2)2CO(aq) + H2O(l) → CO2(g) + 2NH3(g) (1) – + 54 CO2(g) + H2O(l) ⇌ HCO3 (aq) + H (aq) (2) – + – 55 (NH2)2CO(aq) + 3H2O(l) → HCO3 (aq) + 2NH4 (aq) + OH (aq) (3) 56 Urease and CA have been widely associated with MICP in a variety of environments.4,8–10 57 Ureolytic microbes have been applied in the restoration of buildings, soil consolidation and 58 bioremediation,3,11–15 while CA has been proposed as an eco-friendly carbon sequestration 59 technology to mitigate global warming.2,16,17 Yet, urease and CA effects are commonly evaluated 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902379; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 60 via culture-based approaches, disregarding the activity of complex communities in situ. We 61 evaluated on-site urease and CA activities using a rapid test applied to samples from calcareous 62 fens, iron-rich springs, and a saline lake. In these settings, the environmental conditions may lead 63 to carbonate precipitation, however, the contribution of MICP is difficult to assess in the absence 64 of a field assay. By using a field test, we obtained a preliminary assessment of potential MICP, 65 which can be applied to the management of urease- and CA-based technologies. 66 67 MATERIALS AND METHODS 68 Rapid urease field test 69 Urease activity was evaluated by a modification of the rapid urease test for Helicobacter 70 pylori detection.18,19 The test consists of a sample-loading strip and an indicator strip (see a 71 detailed preparation protocol in the supporting information). Sample-loading strips were 72 prepared by immersing a cellulose paper in a fresh 0.6 M urea, 0.4 mM EDTA solution and 73 drying the paper at room temperature. Indicator strips were similarly prepared by impregnating a 74 paper with fresh 0.02% phenol red pH 6.0. Small strips of equal area (33 mm2) were cut from the 75 urea-impregnated paper and circles of 6.5 mm diameter cut from the indicator-impregnated 76 paper. Individual sample-loading strips were introduced in Seal-Rite 0.5 mL microcentrifuge 77 tubes and indicator strips were placed inside the seals of the caps (Figure 1A). Urease activity 78 was detected by adding standard solutions of urease from Canavalia ensiformis (Sigma-Aldrich, 79 St. Louis, Missouri, USA) or wet biofilm samples in direct contact with the sample-loading strip 80 before tightly closing the tube. A positive reaction was visualized by a color change from yellow 81 to red in the indicator strip after ~5 to 60 minutes of incubation at room/field temperature 82 (Figure 1B). Volatile ammonia released from urea (eq. 1) increases the pH in the indicator strip, 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902379; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 changing its color (Figure 1C). A negative control was prepared under the same conditions and 84 additionally adding 1 mM phenyl phosphorodiamidate (PPD)—a urease inhibitor—in the 85 soaking solution of sample-loading strips. A positive reaction without the inhibitor and a 86 negative reaction with PPD strongly indicate urease activity. 87 88 89 90 Figure 1. Urease and CA field tests. (A) Representation of a positive urease assay after adding a 91 biofilm sample in direct contact with the loading strip. (B) A switch to red in the tube cap 92 (indicator strip) indicates a positive urease reaction. If no urease is present or if a loading strip 93 with urease inhibitor (PPD) is assayed, the indicator is expected to remain yellow. (C) Color 94 transition of phenol red (pKa 7.4, pH range 6.4–8.0) used for urease tests. (D) CA assay scheme 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902379; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 95 after adding a sample in the tube with bicarbonate solution. (E) Faster development of a yellow 96 color in the indicator than a negative control (no sample added or using a CA inhibitor) indicates 97 positive CA activity.