3.1 Biostimulation of ureolytic communities
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.
3.1.2 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 hours. The biochemical characterization (details in supplementary data, 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 Sporosarcina pasteurii (SP). The 16s rRNA sequence revealed the isolates as close relatives of SP. The details of the identified isolates provided by NCMR (details in supplementary data, table 1).
A 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 revealed that these strains are 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 figure 3. Similar observations were made at Graddy et al.30, where the majority of the isolated strains (47 out of 57) from bio-stimulation soil tank 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.44 and Graddy et al.30, which is conditional stress for selective stimulation of ureolytic microorganisms. Moreover, the isolated strains were screened based on the morphology and qualitative urease activity.
3.2 Evaluation of biocementation potential of the isolated strains
3.2.1 Growth and pH
The various parameters of the biocementation potential of the isolated strains in comparison with Sporosarcina pasteurii (SP) have been plotted in figure 4. The growth characteristics of the isolates in NBU media and pH during growth have been represented in figures 4 (a) and 4 (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 hours of growth, indicating that these strains favor an alkaline environment to grow similar to SP 45. 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 (Psuedogracillibacillus).
3.2.2 Specific urease activity
The specific urease activities of the isolates were found to be comparable with SP (shown in figure 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 hours and pH > 9. Consortia also demonstrated significant urease activity as 160 mM urea hydrolyzed h-1 (OD600)-1 at a growth period of 48 hours. The maximum ureolytic activity in BS1 was observed after 72 hours 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 hours. 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 hours 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 Temperature46, 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 degree Celsius 45. 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 Celsius46. 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 32,45.
3.2.3 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. It took 48 hours for the 1% bacteria (OD600=1) to approximately utilize all the soluble calcium chloride (50 mM) in the precipitation media (PM) to precipitate carbonate crystals (Figure 4d). Within 12 hours 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 figure 4 (e). 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 hours 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/100ml (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/100ml (67%) and 343 mg/100ml (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.47, and our results show agreement with their finding where 1% of SP cells depletes the 25 mM of CaCl2 within 24 hours. It was observed that all the isolates took approximately 48 hours to deplete the 50 mM CaCl2. The depletion of soluble calcium concentration was rapid in the initial 24 hours in all the isolates. After 48 hours, 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 nutrients or super-saturation condition in the precipitation media 45. The carbonate precipitation and calcium utilization by the ureolytic cells are also impacted by the metabolites during the growth and metabolic activities. As maximum precipitation was recovered with the isolate BS3, the isolate BS3 was selected for further soil treatment.
3.2.4 Microstructure analysis of the precipitates
The FESEM images of the carbonate crystal precipitated from BS3 was investigated further. The shape of the precipitated crystals was observed to be rhombohedral and trigonal (Figure 5 a). 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 (Figure 5b), indicating that the bacteria acted as a nucleation site 19. 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 figure 5 (c). After taking the FESEM image (figure 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 (details provided supplementary data, figure 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.
3.3 Application of native communities on riverbank soil and its influence on soil strength
3.3.1 Needle penetration resistance of treated soil
The average NPI (N/mm) for different cases has been shown in Figure 6 (a). With one bio cementation cycle treatment, the consortia treated soil sample (Consortia-BC1) demonstrated 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 soil properties improve significantly with the increased level of biocementation cycles, indirectly indicating an improvement in the soil erosion 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 figure 6 (b). 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 insignificant influence on the test results48, the needle penetration test is recommended for quick testing of the strength of the stabilized soils and soft rocks. 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. 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 mm49,50. A portable penetrometer of Maruto. Co. ltd. (needle maximum diameter 0.84 mm at 12mm 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 studies48,49. 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.48, the UCS of samples BS3-BC1, BS3-BC2, and BS3-BC3 are around 1.67 MPa, 3.4 Mpa, and 5.3 Mpa.
3.3.2 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 of the critical erosion velocity (0.75 m/s) in a 45-minute duration test and were compared with control (untreated soil). The results of the erosion tests are plotted in figure 7. As expected, with an increase of biocementation cycles, i.e., calcite content, the fluvial erosion reduced substantially. Approximately 7.3% of calcite content resulted in negligible erosion (12% mass loss) when compared to the control, i.e., untreated sand (56% mass loss). 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 flume erosion test.
From the visual observation of the soil specimen after the erosion 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 data, figure 4).
Clarà Saracho et al.35 addressed the erosion due to tangential flow (similar to river current) by treating the soil specimen with for 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 minutes in a modified erosion function apparatus (EFA). The study concluded that the treatment with 0.08M cementation media (calcite content varying from 1.2 to 4%) resulted in negligible erosion in the stated test conditions. In this study, we have considered spraying of bio-cementation media (0.5 M), assuming the convenience of field application. We found that 7.3% of calcite content was required to control the soil erosion substantially in the test flow range (0.06 m/s to 0.75 m/s). The partial loss of soil occurred as the top layer of the sample was not well bonded due to the recurring disturbances induced by spraying.
3.3.3 Microstructure of the biocemented samples
To investigate the influence of different biocementation levels on the microstructure of the treated sand grains, FESEM imaging was conducted for light biocemented samples (BS3-BC1) and heavy biocemented samples (BS3-BC3). While calcite 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 figure 8. The effective calcite bridging between sand grains increases the frictional and cohesive property of sand grains51,52, which improves the erosion resilience of the soil. Bacterial imprints were observed in both cases. Further EDX analysis on a bridged sand grain (supplementary data, figure 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 data, figure 6).