Calcite Without Urea: An Asparaginase-Driven Bio-Grout for Sand (Li et al., 2015)

Most work on Microbially Induced Calcite Precipitation (MICP) relies on one enzyme, urease, and inherits its by-product: a lot of ammonia. A 2015 paper in Scientific Reports by Mengmeng Li, Qing-Long Fu, Qiuzhuo Zhang, Varenyam Achal and Satoru Kawasaki asks whether a different enzyme could do the same job. The team used the bacterium Bacillus megaterium and the enzyme asparaginase, which breaks down the amino acid asparagine, to cement small columns of quartz sand. They then compared the result with a urease-based grout. The authors describe this as, to their knowledge, the first detailed study of asparaginase rather than urease for MICP.

The problem it targets

The paper raises two objections to the standard route described on Ureolysis-Driven Carbonate Precipitation. First, ammonia: the authors call it poisonous at high concentrations, note that its smell makes treated sites unpleasant to work in, and say it harms health and the environment once it rises above safety thresholds. Second, urea itself: they call it a secondary pollutant and argue that because the fertiliser industry also needs it, using it for MICP may not be economical. Their proposed replacement substrate is commercially available asparagine.

How the alternative is said to work

According to the paper, asparaginase splits asparagine into aspartate and releases ammonium. The products are then proposed to equilibrate in water to alanine and bicarbonate. The resulting rise in pH pushes dissolved carbon towards carbonate, and calcium carbonate precipitates around the cells once soluble calcium is present. The authors place the process within ammonification, in which bacteria remove amino groups from amino acids, rather than ureolysis. They are candid that the alanine-and-bicarbonate step is only a "probable reaction" that needs more work, including checking whether another enzyme takes part.

So the route still produces ammonium. The paper's claim is that it produces less, not none.

What was done

The cultures grew in a nutrient broth with 0.2 M asparagine and 40 mM calcium chloride added. Locally collected quartz sand (d10 = 150 μm, d90 = 300 μm) was packed to 10 cm in 60 mL plastic syringes, 3.0 cm across and 11.0 cm long. Bacteria were pumped up through each column for 10 hours, followed by fresh asparagine medium. A week later the columns were dried at 60 °C for 48 h and removed from their moulds. The team tested strength, measured permeability with a falling-head test, and characterised the precipitate by electron microscopy with elemental analysis (SEM-EDS), X-ray diffraction, X-ray computed tomography and thermogravimetric analysis. A control column with medium but no bacteria was also tested.

What was measured

Enzyme activity and pH. Asparaginase activity ranged from 25.1 to 40.6 U/ml over seven days. It peaked at 144 h, when the culture reached pH 9.55, and then fell back slightly. Urease peaked earlier, at 120 h, at 592 U/ml and pH 10.8. For context the paper cites other urease figures: 412 U/ml for S. pasteurii and 650 U/ml for B. megaterium SS3. On this basis the paper concludes that asparaginase releases "significantly less ammonia".

Strength and sealing. The asparaginase grout came close to the urease grout on both strength and permeability. The bacteria-free control failed completely, which the authors put down to the lack of calcite.

Mineral identity. X-ray diffraction found calcite as the only mineral in the precipitate. The paper contrasts this with urease-driven MICP, which often also yields the other Calcium Carbonate (Calcite, CaCO₃) forms aragonite and vaterite. It says further study is needed to explain why only calcite formed.

Distribution. Tomography showed a non-uniform interior, with the top cross-section denser than the bottom. Thermogravimetric analysis found calcite content falling from the top layer to the bottom. Because B. megaterium is aerobic, the authors say more precipitation is expected near the top of the column. They also suggest that early calcite in the upper layers might limit how far bacteria travel, leaving less calcite further down.

What the evidence does and does not show

The study shows that, in a few small laboratory columns, an asparaginase-active strain produced a calcite-cemented sand of similar strength and permeability to a urease-based one. Several limits follow from the paper itself.

• The ammonia comparison is a rate comparison. One unit is defined as the enzyme liberating 1 μmole of ammonia per minute. The two assays also used different conditions: 0.04 M L-asparagine for asparaginase, and nutrient broth with 2% urea and 25 mM CaCl₂ for urease. The paper does not report the total ammonia or ammonium released from, or left in, the cemented columns. That is the quantity examined at metre scale in Rinsing Out the Ammonium: A 3.7-Metre Test of By-product Removal After Ureolytic Bio-Cementation (Lee et al., 2019).
• The urease side is thinly described. The methods explain how urease activity was assayed but give little detail on how the urease grout columns were prepared.
• No replication or statistics are reported. The text gives single values with no sample counts or error ranges, despite calling the difference significant.
• The mechanism is partly hypothetical, as the authors acknowledge.
• Uneven cementation was observed within a 10 cm column, a recurring problem in Bio-Cementation.
• Cost and scale are open. The process used commercial asparagine, and the authors say organic sources need investigating to make it economical. No field or large-scale trial is reported.

The work was funded by the National Natural Science Foundation of China and East China Normal University.

Further reading

• Is Biocement Actually Green? A 2021 Life Cycle Assessment of Six Microbial Routes to Calcium Carbonate (Porter et al.): a life cycle assessment comparing several metabolic routes to biocement, useful context for judging whether a change of substrate improves the environmental balance.
• Enzyme-Induced Carbonate Precipitation (EICP): the cell-free, urease-based variant.