Biocementation Mediated By Stimulated Ureolytic Microbes From Brahmaputra Riverbank For Mitigation of Soil Erosion

Riverbank erosion is a global problem with signicant socio-economic impacts. Microbially induced carbonate precipitation (MICP) has recently emerged as a promising technology for improving the mechanical properties of soils. The presented study investigates the potential of native calcifying bacterial communities of the Brahmaputra riverbank for the rst time via biostimulation and explores its effect on the mitigation of soil erosion. The ureolytic and calcium carbonate cementation ability of the enriched cultures were investigated with reference to the standard calcifying culture of Sporosarcina pasteurii (ATCC 11859). 16S rRNA analysis revealed Firmicutes to be the most predominant calcifying class with Sporosarcina pasteurii and Pseudogracilibacillus auburnensis as the prevalent strains. The morphological and mineralogical characterization of carbonate crystals conrmed the calcite precipitation potential of these communities. The erosion resistance of soil treated with native calcifying communities was examined via needle penetration and lab-scale ume erosion test. We found a substantial reduction in soil erosion in the biocemented sample with calcite content of 7.3% and needle penetration index of 16 N/mm. We report cementation potential of biostimulated ureolytic cultures for a cost-competitive and environmentally-conscious alternative to current erosion mitigation practices.


Introduction
The banks along the worlds' mega-rivers are susceptible to land degradation due to severe soil erosion 1 .
The natural causes of soil erosion include frequent changes in hydrological conditions, including strong river currents, intense rainfall, and climate change 2,3 . The land degradation imparts enormous socioeconomical vulnerability to the residents of riparian zone 4 . Human interventions such as deforestation and non-engineered construction are further worsening the stability of the vulnerable riverbanks 5 . In India, severe land degradation issues are confronted at the banks of Brahmaputra River, one of the top ten river by discharge in the world 6 .
The existing riverbank erosion control practices are inclined towards rigid structures such as aprons, gabions, and check dams, which can irretrievably damage the riparian ecology by producing the imbalance in sediment in ow and out ow 5,7 . These structures may drastically impact the features of the river channel and induce oods downstream 5,8 . Alternatively, riverbank erosion can be controlled by improving the riverbank soil properties by chemical grouting. However, synthetic grout material, including micro-ne cement, epoxy, and silicates has been reported to be toxic for the geo-environment 9,10 and, therefore, can negatively impact the ora, fauna, and crop productivity of the soil of the riparian zone.
One of the options as a sustainable erosion mitigation technique is vegetation along the bank. However, vegetation and their effect on the erodibility of soil are di cult to comprehend and engineer due to their transient life cycle and complex root structures which depend on the vegetation type, nutrition present in the soil, and the surrounding climate [11][12][13][14] . Therefore, an alternative riverbank erosion mitigation strategy with minimum intervention to the riparian ecology is urgently needed. Nature has been forming sustainable cement for millions of years, as seen in the case of corals, beach rocks, anthills, and cave speleothems 15,16 . Within this context, microbial induced calcite precipitation (MICP) is proposed by several studies as a potential tool for ecologically sustainable ground improvement technology 17,18 . The principle of MICP is to utilize the ureolytic bacteria to hydrolyze urea (NH 2 -CO-NH 2 ), as shown in equation 1. In the presence of any ionic calcium source, calcite is precipitated in the soil pore network to bind the cohesion-less granular soil particles 19,20 as summarized in equation (2).
There are certain advantages of MICP over existing grouting practices, such as easy permeation through soil media due to the water-like viscosity of the cementation solution and comparatively negotiable in uence on the geo-environment 21 . The limitations associated with its prospective eld application are ammonium production as a byproduct, non-uniformity of precipitation, and transport of healthy ureolytic bacteria in large quantities to the site 18,22 . While the potential strategies to negate a higher concentration of ammonium ions and non-uniformity are being investigated comprehensively [23][24][25][26][27][28] , the transport of calcifying bacterial culture to the desired site is largely unaddressed. The presence of native ureolytic bacteria and their enrichment on-site can tackle the challenge of bacteria transport 29,30 .
There are limited studies on soil erosion mitigation with MICP; however, most of these studies are based on the bio-augmentation approach with foreign bacteria Sporosarcina pasteurii (ATCC 11859). Salifu et al. 31 reported the effect of tidal cycles on different soil slope angles treated with 18 cycles of MICP treatment solution of 0.7 M cementation solution 31 . The study reported that calcite lled in 9.9% pore volume was effective in controlling soil erosion against 30 tidal cycles on the steep slope (53°). Jiang and Soga 32 investigated seepage-induced internal erosion for gravel-sand mix in a novel designed device considering Sporosarcina pasteurii (ATCC 6452) and reported cementation concentration higher than 0·4 M reduce the erosion to a negligible level 32 . Wang et al. 33 investigated hydraulic ume and erosion function apparatus (EFA) based study on erosion considering PVA-based modi ed cementation solution and observed non-uniform calcite precipitation with 1 M cementation solution via surface percolation strategy and suggested spraying of biocementation as an alternative application strategy. Later, Jiang et al. 34 investigated rainfall-induced erosion simulations for various treated soil 0.2,1 and 2.0 M by spraying of cementation solution four times and observed that 0.2 and 1.0 Molar cementation solution treated soils were substantially more resistant against rainfall-induced erosion when compared to 2 M treated soil. One of the recent studies by Clarà Saracho et al. 35 investigated the in uence of tangential erosion by utilizing erosion function apparatus (EFA) and reported that treatment of soil sample with one pore volume of 0.08 M biocementation solution for 10 days brought values down to a negligible level for a tangential ow of 0 to 0.185 m/s 35 .
As soil is rich in microbial diversity with approximately 10 9 -10 12 microorganisms per kilogram of soil nearby the ground surface 36 , any supplemented foreign bacteria has to compete with the native microorganisms for their survival in the new environment 29,30,37 . Hence, utilizing the indigenous microorganisms over the bio-augmentation approach for soil improvement has de nite advantages such as minimal intervention to native biodiversity and reducing the cost of bacterial transport.
With this background, we investigated the erosion mitigation potential of the native bacterial communities of the riverbank site in the Assam valley of the Brahmaputra River by conducting (a) Enrichment of ureolytic calcium carbonate precipitating microbial communities, (b) Isolation and identi cation of the ureolytic bacteria from the natural riverbank site, (c). Comparison of their biocementation potential in terms of the urease activity and calcium utilization rate with Sporosarcina pasteurii (SP), (d). Evaluation of soil strength improvement by the needle penetration test, and (e). Investigation on the in uence of incremental cementation level on the soil erodibility with a laboratory scale hydraulic ume erosion test.

Experimental Summary
Soil samples were collected from the Brahmaputra riverbank (26°10'50" N; 91°41'26" E) near the Indian Institute of Technology, Guwahati, Assam campus as shown in gure 1. The Brahmaputra river is an anabranching trans-boundary mega-river owing in the south -Asian countries, including China, India, and Bangladesh 38 . Severe erosion with a rate of 80 square kilometers per year is reported at the Brahmaputra riverbank in the Assam valley along with the demolition of 2500 villages and 18 towns, impacting the livelihood of half a million people 39 . During the twentieth century , a total of 2358 square kilometer area from the Brahmaputra riverbank was eroded, and 1490 square kilometer of the area was accreted as ll, resulting in approximate 868 square kilometers of net land degradation in the Assam valley along the 630 km length of the Brahmaputra river 40 . Another study reported a loss of 35.5% loss of the land in the world's largest river island, " Majuli," situated in the upper reach of the Brahmaputra river in Assam 6 . Therefore, the protection of these banks is paramount for social, economic, and environmental sustainability. For comparison, another soil sample was collected from a nearby natural vegetative slope (26°11'05" N; 91°41'32" E) inside the Indian Institute of Technology, Guwahati campus in the vicinity of the riverbank. The topsoil (1 cm) was removed and the soil beneath was collected in a sterile tube from both sites and kept in an ice bucket for isolation purposes. The soil was collected separately for geotechnical classi cation. The evaluated engineering properties of soil following ASTM standard methods for soil classi cation is shown in table 1 [41][42][43] .  . 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 Speci c urease activity The speci c urease activities of the isolates were found to be comparable with SP (shown in gure 4c). Based on the provided NBU media and growth condition, the speci c 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 speci c 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 signi cant 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 speci c 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 speci c urease activity of 146.4 mM urea hydrolyzed h-1 (OD600)-1. The observed order of speci c 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 speci c 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 speci c urease activities of all isolates were found to be in a range of 106 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 identi ed (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 gure 5 (c). After taking the FESEM image ( gure 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, gure 1). XRD analysis was conducted to con rm the mineralogy of the precipitates, and the majority of the observed peaks of the XRD plot belonged to calcite, which is and BS3-BC3 can also be realized with the contrasting color gradient of the NPI contours. Since the rate of penetration has insigni cant in uence 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 uncon ned 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 con dence value to conventional physicochemical parameters such as uncon ned compressive strength (UCS), elasticity modulus, and elastic wave velocity in several studies48,49. In our setup, we have utilized a similar con guration chenille 22 needle with (maximum diameter 0.86 mm at 9 mm from the tip) and a penetration rate of 15

Conclusions
Biostimulation of ureolytic carbonate precipitating communities from Brahmaputra riverbank soil was successfully conducted in this study. The majority of the isolates belonged to Sporosarcina genera highlighting the prevalence of these strains. One of the isolates (BS3) was found to have marginally better biocementation potential than the conventionally used Sporosarcina pasteurii (ATCC 11859) in terms of speci c urease activity and calcium utilization. This implicates that the enrichment of native bacteria has de nite advantages, along with reduced bacterial transport costs. This study provides promising evidence of the potential of native ureolytic communities from riverbank sites in their ability to mitigate soil erosion. Further, with needle penetration resistance tests, we were able to quantify the improvement of soil strength with the number of biocementation cycles, i.e., calcite content. However, the distribution of needle penetration resistance was found to be non-uniform, indicating the poor distribution of the precipitated calcite with spraying strategy. The assessment of soil erodibility was conducted in the hydraulic ume, and it was observed that with an increase in the calcite content in treated soil specimens, the eroded soil mass loss decreased substantially. With microstructural analysis (FESEM, EDX, and XRD), the morphology and the mineralogical composition of precipitated crystals were investigated. The calcite crystal growth in the sand grooves and bridging of sand grains was observed as the cause of increased soil strength in the biocemented soil samples. In conclusion, native bacterial communities were found to be effective in mitigating soil erosion.
The river ecology is very sensitive to environmental changes, and the practiced rigid-structures to mitigate the riverbank erosion are observed to be harmful in the long run 5 . In this study, we proposed MICP treatment as a potential eco-friendly erosion control technique with native bacteria so that there is a minimum intervention to the river ecology. However, there are numerous concerns for its eld application. The alkalinity of growth media and ammonia generation are major concerns for the river ecology and geo-environment. There is a high possibility of ammonia being diluted to negligible levels at the riverbanks; however, this can be threatening to the ora and fauna of the riparian zone. Therefore, a detailed investigation of ammonia dilution or removal strategies will be required, which is not addressed in our study. As non-uniform soil improvement was observed with spraying, a systematic approach for uniform distribution of calcite with a low concentration of cementation media and continuous application by spraying or injection is recommended. There are chances of the cementation media getting diluted in the saturated zones of the riverbanks. Therefore, it is essential to consider the application of the MICP for erosion control only in the low ood seasons to vulnerable riverbanks. A detailed investigation of MICP treatment of the riverbank soil, based on their suction characteristics, will be critically useful to design the eld application strategy. Further investigations can be conducted on actual eld site conditions for a longer duration of time to investigate the potential of this technology in the long term.

Enrichment of ureolytic communities from the collected soils
Around 10 g of soil from the riverbank and natural slope was procured to the laboratory in sterile containers. One gram of the collected soil was inoculated into the 100 ml enrichment media containing 13 g/l of Nutrient Broth (NB) and 5% Urea in a shaking incubator at 37º C and 120 rpm for ve days. This was followed by two subcultures of the enriched communities into new enrichment media. Con rmation of the ureolytic capability of these communities was veri ed by inoculating the bacterial culture into urea agar base (Christensen, 1946) Petri dishes wherein change in color was recorded within 12 hours. The details of each of the media (sterile) are reported in Table 2.

Isolation, identi cation, and characterization of the isolates
After the enrichment of ureolytic communities, we attempted to isolate the e cient ureolytic cultures from the consortia. For this, the enriched consortia were serially diluted and inoculated on NBU agar plates and single colonies were obtained. These colonies were then screened for qualitative urease production on urea agar plates 53 . Six highly ureolytic cultures were screened. This was followed by the identi cation of these cultures via 16S rRNA genomic sequencing. The 16S rRNA sequencing was conducted at NCMR (National Centre for Microbial Research, India). The sequences of the isolated strains were analyzed and compared with the highly similar sequences available on the NCBI database utilizing nucleotide BLAST.
The DNA sequences of the isolated strains were aligned with the reference sequences of the obtained highly similar strains by the MUSCLE algorithm and the phylogenetic tree was constructed by the neighbor-joining method using Mega-X software 54 .
The screened isolates were further characterized based on their biochemical properties, growth characteristics, urease activity, and calcium utilization potential. The biochemical properties of the isolates were evaluated using the Microbact 12 A, 12B, and 24 E kit. The isolates were grown in the NBU media for four days at 37°C and 180 rpm in a shaking incubator to evaluate growth characteristics and urease activity. The performance of isolates was compared with the comprehensively used SP (Sporosarcina pasteurii ATCC 11859) and the arti cial consortia. The arti cial consortia were created by mixing an equal proportion of the isolated strains from site 1.
The urease activity (mM urea hydrolyzed per hour) of the bacterial isolates was evaluated following the electrical conductivity method 55 . For assay of urease activity, 1 ml of bacteria is mixed with 9 ml of 1.11 M urea, and the change in electrical conductivity was measured for 5 minutes at room temperature (25±3°C ). The variation in electrical conductivity rate (mS per cm per minute) is measured and the urease activity is calculated taking the dilution factor into account 56 . The ratio of the urease activity and the bacterial optical density (OD 600 ) is de ned as the speci c urease activity (mM urea hydrolyzed h -1 OD 600 -1 ).

Analytical methods for evaluation of biocementation potential of the isolates
The biocementation potential of the e cient bacterial isolate was evaluated following a modi ed method from Dhami et al. 58 1% overnight grown bacterial culture (OD 600 =1) was added in the precipitation media (PM) consisting of 1g/l Nutrient Broth, 2% urea, and 50 mM of CaCl 2 in a shaking incubator at 37°C and 120 rpm. A negative control set consisting of only ask cementation media was also observed for the test duration to check the possibility of abiotic precipitation.
The soluble calcium depletion was evaluated by measuring soluble Ca 2+ for 48 hours by the EDTA titration method 19 . For evaluation of soluble Ca 2+ , 10 ml of the inoculated cementation media was taken out at different time intervals and centrifuged at 4°C and 8000 rpm for 5 minutes. The supernatant was supplemented with 0.5 ml of 5N sodium hydroxide and few drops of hydroxy-naphthol blue indicator and the mixture was titrated against the 250 mM EDTA. The required EDTA to change color from pink to blue was noted and compared with a standard calibration plot of CaCl 2 solutions from a range (5-100 mM).
The performance of isolates was compared with comprehensively used SP (Sporosarcina pasteurii ATCC 11859) and the consortium. A negative control set consisting of only precipitation media was also observed for the test duration to check the possibility of abiotic precipitation.
At the end of four days, the precipitates were collected on Whatman no. 1 lter paper and washed with sterile water as per the protocol of Dhami et al. 47 . The precipitates were air-dried after ltration at room temperature for 48 hours, weighed, and analyzed for gravimetric, microstructural, and mineralogical analysis.

Soil sample preparation
Fine sand equivalent to Brahmaputra riverbank sand was procured from Cook Industrial Minerals, Western Australia. The sand was lled at a 40% relative density of 1.6 g/cc in the containers. For the needle penetration test and calcite content measurements, the soil samples were prepared in Petri dishes of diameter 5 cm and depth 1 cm. After the preliminary investigation on the penetration resistance improvement, samples for the hydraulic ume erosion tests were prepared in a container of length 9.25 cm, width 5.8 cm, and depth 4 cm. Separate samples were made for ume erosion test and needle penetration test as the Needle penetration prior to hydraulic erosion can damage the bio cemented crust formed on the top surface. The soil specimens were treated with 0.5 M of equimolar cementation media, as suggested by Jiang and Soga 32 and Porter et al. 51 . The spraying strategy for the treatment was considered as it is a convenient strategy for the eld application. As per the recommendation of Wang et al. 33 , non-uniform calcite precipitation was observed with sur cial percolation strategy and spraying strategies were suggested as a viable alternative. For uniform precipitation, the biocementation solutions were supplied in three steps, which include the application of xation solution, ureolytic bacteria solution, and cementation solution, as suggested by Harkes et al. 28 . In the current study, one biocementation cycle (BC1) is completed by spraying one pore volume of xation solution, one pore volume of bacterial solution (at optical density ≥ 1), and one pore volume of the cementation solution (two times) consecutively after the retention period of 24 hours for each step. Similarly, samples with two and three biocementation cycles (BC2 and BC3) treatments were prepared to investigate improved soil properties. Details of all the sample prepared for evaluation has been summarized in table 3.   50 and mapped for 25 points. The Chenile needle #22 with a diameter of 0.86 mm, was mounted on a universal testing machine (Shimadzu AGS-X). First, the average needle penetration indices of samples SP-BC1, BS3-BC1, and Consortia-BC1 were compared to investigate the in uence of different strains and the same level of biocementation on soil strength improvement. Later, to quantify the improvement in soil strength, needle penetration resistance was investigated for samples treated with native bacteria-based two and three biocementation cycles (BS3-BC2 and BS3-BC3).

Erodibility test for a bed slope in Hydraulic ume
The sample treated with the most suitable native strain at a different level of biocementation (BS3-BC1, BS3BC2, and BS3-BC3) were further tested and compared with the untreated sand on the hydraulic umebased erosion test. The soil erosion tests were performed in a 12 m long, Arm eld Engineering ltd. S5 tilting ume with the glass walls 320 mm high and 300 mm wide (internal breadth). The treated soil containers were plugged into an acrylic bed slope in the ume (Supplementary data, gure 2). The samples were saturated and then the ow velocity was increased stepwise at intervals of a 5-minute time interval (Supplementary data, gure 3). This stepwise incremental ow methodology was considered based on the study by Clarà Saracho et al. 35 , as the critical ow velocity is expected to increase with the biocementation treatment levels. The ow was operated using the control panel and velocity was measured on three different points near the sample using a pulse velocity meter. The critical ow velocity for the riverbank soil calculated from Briaud' (2008) suggested equation 59 was found to be 0.15 m/s.
Here, V c is the critical ow velocity required to initiate the erosion and D 50 is the mean diameter of the cohesionless soil. The samples were installed 6 meters downstream in the hydraulic ume.

Microstructural analysis and Calcite content of the treated soil
The ZEISS Neon 40 EsB dual FESEM/FIBSEM instrument equipped with Field Emission Scanning Electron Microscopy (FESEM) and Energy-dispersive X-ray spectroscopy (EDX) was used for the microstructural investigation. The air-dried treated soil specimens were mounted on the aluminum stub for its microstructural and elemental composition analysis. The samples were coated with a 10 nm platinum coating. For mineralogical analysis of the precipitates, all the samples were micro-ionized (particle size <10 microns) and analyzed by X-ray diffraction (XRD) on Bruker D8 advanced diffractometer with Nickel ltered Cu-Kα radiation varying 2θ from 5° -100° with a step size of 0.013°.
The phase identi cation was made utilizing COD and ICDD databases with Bruker EVA.
The calcite content of the treated soil was measured by the acid washing method. 5-gram treated soil was collected in triplicates from the crust after the ume erosion test and washed with 20 ml of 1 M HCl.
The calcite content was determined based on the gravimetric difference between the collected soil sample (5g) and retained weight of the acid-washed soil on Whatman lter grade 1 60 .

Statistical Analysis
All samples were analyzed in triplicates, and the average was compared except for the needle penetration test and ume erosion test. The data were analyzed by one-way analysis of variance (ANOVA). For the needle penetration test, the tests were performed at 25 points on the crust and the average value with the standard deviation is reported.