Biocementation mediated by native microbes from Brahmaputra riverbank for mitigation of soil erodibility

Biostimulation of ureolytic communities

Enrichment

The native communities of the soils were successfully grown in the enrichment media (NB5U). The cultivated communities after two subcultures were serially diluted (10^–2 to 10^–6) and were spread with a sterile loop over Nutrient agar plates supplemented with 2% urea. Later, 36 morphologically distinct single colonies were obtained on the urea agar base plates based on visual observation.

Isolation, identification, and characterization of the ureolytic isolates

Out of 36 isolated bacteria, six isolates (BS1, BS2, BS3, BS4, LS1, and LS2) were selected after checking for the urease activity test on the urea agar base (UAB) plate. These selected isolates turned the color of the UAB plate from orange to pink within 12 h. The 16 s rRNA sequence revealed the isolates as relatives of Sporosarcina pasteurii (SP). The details of the identified isolates are provided by NCMR (details in Supplementary Table 1). The biochemical characterization (details in Supplementary Table 2) of the isolates revealed that all the isolates are Gram-positive. All the isolates were rod-shaped, endospore-forming, urease, and oxidase positive. All the isolates were not able to utilize the Lysine and ONPG, contrary to SP.

Further investigation of the isolated sequence was done via the NCBI database. The sequences were submitted to the GenBank database of the NCBI (National Center for Biotechnology Information) under the accession number MW024144 to MW024149. The BLAST analysis suggested that these strains are close relatives and indicate the possibility of being novel strains of the Sporosarcina family. We found that the isolate BS1 and BS2 had 96.62% (coverage 100%) and 96.22% (coverage 99%) identity with Sporosarcina siberinisis (NCBI accession number NR 134188). BS3 had 98.8% identity (coverage 97%) with Sporosarcina pasteurii (NCBI accession number NR 104923). BS4 and LS1 had 97.4% (coverage 99%) and 97.37% identity (coverage 100%) with Sporosarcina soli (NCBI accession number NR 043527). Contrarily, LS2 was found to be related to the Pseudogracilibacillus family. LS2 was observed to be closely related to Pseudogracilibacillus auburnensis P-207 with 97.06% identity (96% coverage). Based on these findings, the Phylogenetic tree was constructed with bootstrap (1000 replicates) considering the reference sequences obtained from the BLAST analysis, as shown in Fig. 3. The threshold criteria to differentiate two species is defined as 98.65% similarity score with the reference culture from databank⁴⁰, while another study has suggested that in case of similarity index is > 99%, the unknown isolate should be assigned to a species, and if the unknown isolates have similarity score between 95 to 99% to a reference sequence, the isolate should be assigned to the genus⁴¹. However, further investigation is suggested to conclude if the reported strains are novel or merely mutants of the reference strains of the databank. Similar observations were made at Graddy et al.²², where the majority of the isolated strains (47 out of 57) from bio-stimulation soil tanks were found to be strains of the Sporosarcina genus. It is worth noting that the soil enrichment media for stimulation was rich in urea, similar to Gomez et al.⁴² and Graddy et al.²², which is conditional stress for selective stimulation of ureolytic microorganisms. Moreover, the isolated strains were screened based on morphology and qualitative urease activity.

Evaluation of biocementation potential of the isolated strains

Growth and pH

The various parameters of the biocementation potential of the isolated strains in comparison with Sporosarcina pasteurii (SP) have been plotted in Fig. 4. The growth characteristics of the isolates in NBU media and pH during growth have been represented in Fig. 4a and b. The initial pH of the growth media is kept at 7.5. It was observed that the pH of the growth media rises to 9.5 within 24 h of growth, indicating that these strains favor an alkaline environment to grow similar to SP⁴³. All the isolates start growing when the pH of the media rises to 8.5 or above. Isolate LS2 was observed to have slower growth when compared with other isolates. This can be explained as LS2 belongs to different genera (Pseudogracilibacillus).

Specific urease activity

The specific urease activities of the isolates were found to be comparable with SP (shown in Fig. 4c). Based on the provided NBU media and growth condition, the specific urease activity of SP is found to be 173.44 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹, which is around 2.9 mM urea hydrolyzed min⁻¹ (OD₆₀₀)⁻¹. The specific urease activity of the isolate BS3 was observed to be maximum as 186.6 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹ during a growth period of 24 h and pH > 9. Consortia also demonstrated significant urease activity as 160 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹ at a growth period of 48 h. The maximum ureolytic activity in BS1 was observed after 72 h of growth with a value of 106.67 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹. Maximum specific urease activity of the isolate BS2, BS4, and LS1 was observed to be 160.2, 120, and 173.4 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹ respectively after a growth duration of 48 h. LS2 demonstrated the maximum specific urease activity of 146.4 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹. The observed order of specific urease activity at 24 h of growth period is BS3 > SP > Consortia > LS1 > BS2 > BS4 > LS2 > BS1. As the urease activity of the strains depends on the growth media, urea content, and environmental conditions such as pH and Temperature⁴⁴, we considered the conditions at the riverbank at the time of isolation, and the pH and temperature of the growth media were set at 7.5 and 37 degrees Celsius. The specific urease measured by the electrical conductivity method is reported to be between 3 to 9.7 mM urea hydrolyzed min⁻¹ (OD₆₀₀)⁻¹ in yeast-extract urea media at pH 7 and temperature 30 degrees Celsius⁴³. It is reported around 5 mM urea hydrolyzed min⁻¹ (OD₆₀₀)⁻¹ in the nutrient broth urea (2%) media at a temperature of 25 degrees Celsius⁴⁴. The comparative analysis of the urease activity (measured by electrical conductivity method) was done considering SP as positive control in this study. The maximum specific urease activities of all isolates were found to be in a range of 106.67 to 186.67 mM urea hydrolyzed h⁻¹ (OD₆₀₀)⁻¹ (1.78 to 3.11 mM urea hydrolyzed min⁻¹ OD₆₀₀^–1), which indicates that all of the isolated strains are capable of biocementation^43,45.

Calcium utilization and carbonate precipitation potential

It was experimentally observed that the depletion of the supplemented soluble calcium in the precipitation media (PM) was corresponding to the ureolytic activities of the isolated strains. Within 48 h of introducing 1% bacteria (OD600 = 1) in the precipitation media, the soluble calcium chloride (50 mM) was utilized to precipitate carbonate crystals, as illustrated in Fig. 4d. Within 12 h of the inoculation period, BS3 was able to utilize 75% of the supplied calcium, while SP was able to utilize only 62.5% of the soluble calcium. The order of the calcium utilization potential in the isolates was observed as BS3 ≥ LS2 > L.S.1 > Consortia > SP > BS4 > BS2 > BS1 during the inoculation period. Contrarily, LS2, despite being a comparatively slow urease-producing bacteria, was able to utilize calcium ions at par with other isolates. Negligible changes were observed in the soluble calcium concentration of the control group eliminating the possibility of abiotic precipitation.

The carbonate precipitation rate for each isolate (1% at OD₆₀₀ = 1) for the 50 mM cementation media is plotted in Fig. 4e. The isolate BS3 with maximum ureolytic activity (specific urease activity 186.6 mM urea hydrolyzed min⁻¹ OD₆₀₀^–1) precipitated the highest carbonate crystals after 96 h of the incubation period. BS3 precipitated 438 mg/100 ml of carbonate crystals, which is around 87.66% precipitation from the total supplied CaCl₂, while precipitation with SP was quantified as 389 mg/100 ml (78%). The precipitation in consortia was observed to be 407 mg/100 ml (81%), which is slightly higher than SP. Precipitation in other isolates was found to be significantly lower than isolate BS3. Isolate BS1and BS2 precipitated 334 mg/100 ml (67%) and 343 mg/100 ml (69%) of carbonate crystals respectively, whereas isolate LS1 and LS2 precipitated around 357 mg/100 (71%) ml of carbonate crystals each. Isolate BS4 precipitated minimum carbonate crystals 292 mg/100 ml (58%). No precipitation was observed in the negative control set. Low concentrations of bacterial cells (1%) were considered in this experiment to slow down the urea hydrolysis in order to differentiate the calcium utilization potential of the isolated strains. This approach was modified from Dhami et al.⁴⁶, and our results show agreement with their finding where 1% of SP cells depletes the 25 mM of CaCl₂ within 24 h. It was observed that all the isolates took approximately 48 h to deplete the 50 mM CaCl₂. The depletion of soluble calcium concentration was rapid in the initial 24 h in all the isolates. After 48 h, the residual soluble calcium was observed to be in the range of 2.5–5 mM in all the isolates (except BS1 and BS2), which might be due to loss of super-saturation caused by the unavailability of nutrient for bacterial cells to continue urea hydrolysis in the precipitation media^13,43,47. The mineralogy of precipitated carbonate polymorphs (calcite, aragonite, vaterite) and the residual calcium are also influenced by pH, temperature, saturation index, dissolved organic carbon concentration, and the Ca²⁺ /CO₃²⁻ratio along with the presence of metabolites in the precipitation media^13,47,48. As maximum precipitation was recovered with the isolate BS3, the isolate BS3 was selected for further investigation on soil improvement.

Microstructure analysis of the precipitates

The FESEM images of the carbonate crystal precipitated from BS3 were investigated further. The shape of the precipitated crystals was observed to be rhombohedral and trigonal (Fig. 5a). The average size of the crystals was observed in a range of 25 to 50 microns. The entrapped bacteria and rod-shaped bacterial imprints were identified (Fig. 5b), indicating that the bacteria acted as a nucleation site¹⁴. The smaller crystals were observed to coagulate in layers to develop larger calcite crystals. The entrapped bacteria were noticed on the grown and coagulated calcite crystal in Fig. 5c. After taking the FESEM image (Fig. 5a) of the precipitate, EDX analysis was conducted, and the elemental composition suggested an abundance of calcium, carbon, and oxygen, which indicates the presence of calcium carbonate crystals (Supplementary Fig. 1). XRD analysis was conducted to confirm the mineralogy of the precipitates, and the majority of the observed peaks of the XRD plot belonged to calcite, which is consistent with the observation of rhombohedral crystal shapes in the FESEM image. The XRD analysis also suggested an insignificant presence of aragonite in the precipitates.

Application of native communities on riverbank soil and its influence on soil strength

Needle penetration resistance of treated soil

The average NPI (N/mm) for different cases has been shown in Fig. 6a. No notable resistance was observed in the loose untreated sand (control) against the needle penetration. With one bio cementation cycle treatment, the consortia-treated soil sample (Consortia-BC1) demonstrated a higher value of NPI (5.15 N/mm) than SP-BC1 (4.19 N/mm) and BS3-BC1 (4.64 N/mm). The increase in the biocementation cycle treatment significantly improved the needle penetration resistance. Sample BS3-BC2 showed 116% improvement with the NPI value of 10.03 N/mm when compared to one cycle treated sample BS3-BC1. A similar trend was observed in the sample BS3-BC3 (NPI = 16.12 N/mm), which showed around 347% improvement in NPI when compared to sample BS3-BC1. From the needle penetration test, it was evident that the penetration resistance of treated soil improves significantly with the increased level of biocementation cycles, indirectly indicating an improvement in the soil erodibility resilience. Since non-uniformity is one of the undesired traits of MICP, a contour was plotted corresponding to the 25 points NPI, as shown in Fig. 6b. The contrasting color difference in the contours of the samples BS3-BC1, BS3-BC2, and BS-BC3 clearly demonstrates the stark difference in the strength of treated samples. The non-uniformity in the strength of treated soil crust of sample BS3-BC2 and BS3-BC3 can also be realized with the contrasting color gradient of the NPI contours.

Since the rate of penetration has an insignificant influence on the test results, the needle penetration test is recommended by the International Society of Rock Mechanics (ISRM) for quick, non-destructive testing of the strength of the stabilized soils and soft rocks⁴⁹. As a large number of tests can be conducted due to the small diameter of the needle without destroying the sample, the needle penetration test is a better alternative to evaluate the local grain bonding in the biocemented soil than bulk strength properties like unconfined compressive strength and calcite content. Another rationale for choosing needle penetration test over conventional soil strength evaluation tests was that a pocket type penetrometer could be developed with the configuration in the present study for non-destructive monitoring of the soil strength improvement with biocementation application in the field. The response of the needle penetration resistance in terms of nominal strain (ratio of penetration to rod diameter) also indicated that the measured responses are independent of needle diameter for a small range, i.e., 1 to 3 mm^49,50. A portable penetrometer of Maruto. Co. ltd. (needle maximum diameter 0.84 mm at 12 mm from the tip) have been correlated with high confidence value to conventional physicochemical parameters such as unconfined compressive strength (UCS), elasticity modulus, and elastic wave velocity in several studies⁵⁰. In our setup, we have utilized a similar configuration chenille 22 needle with (maximum diameter 0.86 mm at 9 mm from the tip) and a penetration rate of 15 mm/minute for measuring the strength properties of cemented soil. Adopting the UCS and NPI correlation suggested by Ulusay et al.⁵¹, the UCS of samples BS3-BC1, BS3-BC2, and BS3-BC3 are around 1.67 MPa, 3.4 Mpa, and 5.3 Mpa.

It is worth noting that in the needle penetration resistance tests, the boundary of the Petri dish can influence the test results. Therefore, trials were conducted and based on the findings, all penetrations were conducted at points at least five times the diameter of the needle away from the boundary to negate the influence of boundary conditions. The maximum penetration was conducted only up to 50% of the depth of prepared biocemented soil samples in the Petri dishes to avoid inference from the bottom of the Petri dish.

Erodibility test in the hydraulic flume

To investigate the influence of hydraulic current on different levels of biocementation, all the treated samples were exposed to hydraulic current gradually varying from gentle flow (0.06 m/s) to five times the critical velocity (0.75 m/s) in a 45-min duration test and soil mass loss percentage by the initial dry mass of the treated sample is presented as a measure of soil erodibility in Fig. 7. As expected, with an increase of biocementation cycles, i.e., calcite content, the soil erodibility reduced substantially. The initial dry weight of the samples control, BC1, BC2, and BC3, were measured as 398, 403, 406, and 410 g, respectively. Approximately 7.3% of calcite content resulted in a drastic reduction in erodibility (12% mass loss), while 56% soil mass loss was recorded for control (untreated sand). One biocementation cycle treatment (sample BS3-BC1) produced an average of 2.5% of calcite, reducing the soil loss to 31%. Sample BS3-BC2 with 4.93% calcite content resulted in 22% soil mass loss during the hydraulic flume test. It is worth noting that higher precipitation in the soil pores may hinder the flow of water in the soil matrix and increase the pore water pressure resulting in catastrophic failures. However, the MICP technique is reported to be a great tool to improve soil strength, maintaining an adequate hydraulic conductivity to prevent the build-up of the excess pore water pressure¹¹. Theoretically, the percentage pore volume filled with precipitates for samples BS3-BC1, BS3-BC2, and BS3-BC3 considering the observed calcite contents and pore volume (100 ml) is around 3.7, 7.14, and 11.08%. The influence of pore water pressure on erodibility has not been established in the present study, and it certainly is one of the exciting parameters to consider for future studies.

From the visual observation of the soil specimen after the flume test, it was evident that the soil particles start bonding with an increased level of bio cementation. A tough crust was formed on the top of BS3-BC2 and BS3-BC3 the samples, which got eroded with the fluvial current. Insignificant aggregation was observed in the sample BS3-BC1. However, with two and three cycles of biocementation treatment (BS3-BC2 and BS3-BC3), the biocemented soil particles (BCS) were evidently noticed (photos are shown in Supplementary Fig. 4).

Clarà Saracho et al.²⁷ addressed the erosion due to tangential flow (similar to river current) by treating the soil specimen with ten pore volume of low concentration of cementation media (0.02 M to 0.1 M) by injection strategy and tested the specimen in the flow velocity ranging from 0.035 to 0.185 m/sec for 120 min in a modified erosion function apparatus (EFA). The study concluded that the treatment with 0.08 M cementation media (calcite content varying from 1.2 to 4%) resulted in negligible erosion in the stated test conditions, and with the increase in MICP treatment, a shift in the erosion mode from particulate mode to block failure was observed indicating that with the increase in calcite content and needle penetration resistance, there might be a threshold for biocemented soil, where the soil erosion might be catastrophic due to block failure. However, in this study, a consistent decrease in soil erodibility is observed with the increase in needle penetration resistance. We found that 7.3% of calcite content was required to control the soil erodibility substantially in the test flow range (0.06 m/s to 0.75 m/s). A similar trend was observed by Kou et al.⁵² and Chung et al.⁵³, where consistent reduction in wave-induced erosion and rainfall-induced erosion was observed with an increase in needle penetration resistance for biocemented fine sand treated with the exogenous bacteria.

Another aspect to note in the context of the applied treatment is the produced ammonia which can be toxic for riparian flora and fauna¹⁵. From stochiometric calculations, for each biocementation cycle, the produced ammonia is evaluated as 8.5 kg per metric ton of soil treated, and for the best performing treatment approach, i.e., BS3-BC3, the ammonia generated is evaluated as 2.63% by weight of the retained soil. The acceptable limit of ammonia in the surface water was recommended as 17 mg per liter for acute exposure and 1.9 mg/l for chronic exposure for protecting the aquatic life in the freshwater as per the environmental protection agency⁵⁴. With the MICP technique, the threat of produced ammonia crossing the maximum acceptable quantity is highly plausible; however, for the field application, the ammonia generated can be reduced by reducing the quantity of reagents and increasing the period of applications. It is to be noted that the produced ammonia will also be subjected to dilution in the river stream. The average discharge of the Brahmaputra river is around 19.8 megaliters per second in the Assam valley³³. Therefore, the area of the riverbank to be biocemented must be decided judiciously with the context of the produced ammonia quantity and its possible dilution to non-toxic levels.

Microstructure and mineralogical analysis of the biocemented samples

To investigate the influence of different biocementation levels on the erodibility of the treated sand grains, FESEM imaging was conducted for light biocemented samples (BS3-BC1) and heavy biocemented samples (BS3-BC3). While precipitated crystals were observed to be growing on the grooves of sand grains in the light biocemented sample (BS3-BC1), bridging of sand grains with rhombohedral crystals was observed in the heavy biocemented sample (BS3-BC3), as shown in Fig. 8. The effective calcium carbonate bridging between sand grains increases the frictional and cohesive property of sand grains^55,56, leading to a substantial reduction in the erodibility of the soil. Bacterial imprints were observed in both cases, suggesting the precipitation to be biodgenic¹⁴. Further EDX analysis on a bridged sand grain (Supplementary Fig. 5) suggested an abundance of silicon and oxygen on the sand grains with a trace amount of chlorine and calcium. This indicates the presence of residual calcium chloride on silica grains. The EDX analysis on the grain bridge indicated the presence of calcium, carbon, and oxygen, suggesting CaCO₃ precipitation. XRD analysis on treated and untreated sand confirmed the precipitation of calcite. Most of the peaks correspond to quartz (silica). In the biocemented sand sample, a visible peak of calcite was observed at around 29 degrees of 2Ɵ (Details in Supplementary Fig. 6). Therefore, the incorporation of microbial calcite as a binding agent for loose grain silica soil was found to reduce the soil erodibility.

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