Upscaling Effects of Soil Improvement by Microbially Induced Calcite Precipitation by Surface Percolation

This study has contributed to the technology of soil stabilization via biocementation based on microbially induced calcite precipitation. The newly described method of in situ soil stabilization by surface percolation to dry soil under free draining environment is tested for its up-scaling potential. Then, 2-m columns of one-dimensional trials indicated that repeated treatments of fine sand (<0.3 mm) could lead to clogging closed at the injection end, resulting in limited cementation depth of less than 1 m. This clogging problem was not observed in 2 m coarse (>0.5 mm) sand columns, allowing strength varying between 850 to 2067 kPa along the entire 2 m depth. Three-dimensional fine sand cementation trials indicated that relatively homogenous cementation in the horizontal direction could be achieved with 80% of cemented sand cementing to a strength between 2 to 2.5 MPa and to a depth of 20 cm. A simple mathematical model elucidated that the cementation depth was dependent on the infiltration rate of the cementation solution and the in-situ urease activity. The model also correctly predicted that repeated treatments would enhance clogging close to the injection point. Both experimental and simulated results suggested that the surface percolation technology was more applicable for coarse sand.     

Halotolerant, alkaliphilic urease-producing bacteria from different climate zones and their application for biocementation of sand

Microbially induced calcium carbonate precipitation (MICP) is a phenomenon based on urease activity of halotolerant and alkaliphilic microorganisms that can be used for the soil bioclogging and biocementation in geotechnical engineering. However, enrichment cultures produced from indigenous soil bacteria cannot be used for large-scale MICP because their urease activity decreased with the rate about 5 % per one generation. To ensure stability of urease activity in biocement, halotolerant and alkaliphilic strains of urease-producing bacteria for soil biocementation were isolated from either sandy soil or high salinity water in different climate zones. The strain Bacillus sp. VUK5, isolated from soil in Ukraine (continental climate), was phylogenetically close in identity (99 % of 16S rRNA gene sequence) to the strain of Bacillus sp. VS1 isolated from beach sand in Singapore (tropical rainforest climate), as well as to the strains of Bacillus sp. isolated by other researchers in Ghent, Belgium (maritime temperate climate) and Yogyakarta, Indonesia (tropical rainforest climate). Both strains Bacillus sp. VS1 and VUK5 had maximum specific growth rate of 0.09/h and maximum urease activities of 6.2 and 8.8 mM of hydrolysed urea/min, respectively. The halotolerant and alkaliphilic strain of urease-producing bacteria isolated from water of the saline lake Dead Sea in Jordan was presented by Gram-positive cocci close to the species Staphylococcus succinus. However, the strains of this species could be hemolytic and toxigenic, therefore only representatives of alkaliphilic Bacillus sp. were used for the biocementation studies. Unconfined compressive strengths for dry biocemented sand samples after six batch treatments with strains VS1and VUK5 were 765 and 845 kPa, respectively. The content of precipitated calcium and the strength of dry biocemented sand at permeability equals to 1 % of initial value were 12.4 g Ca/kg of dry sand and 454 kPa, respectively, in case of biocementation by the strain VS1. So, halotolerant, alkaliphilic, urease-producing bacteria isolated from different climate zones have similar properties and can be used for biocementation of soil.     

Selective enrichment and production of highly urease active bacteria by non-sterile (open) chemostat culture

In general, bioprocesses can be subdivided into naturally occurring processes, not requiring sterility (e.g., beer brewing, wine making, lactic acid fermentation, or biogas digestion) and other processes (e.g., the production of enzymes and antibiotics) that typically require a high level of sterility to avoid contaminant microbes overgrowing the production strain. The current paper describes the sustainable, non-sterile production of an industrial enzyme using activated sludge as inoculum. By using selective conditions (high pH, high ammonia concentration, and presence of urea) for the target bacterium, highly active ureolytic bacteria, physiologically resembling Sporosarcina pasteurii were reproducibly enriched and then continuously produced via chemostat operation of the bioreactor. When using a pH of 10 and about 0.2 M urea in a yeast extract-based medium, ureolytic bacteria developed under aerobic chemostat operation at hydraulic retention times of about 10 h with urease levels of about 60 μmol min^?1 ml^?1 culture. For cost minimization at an industrial scale the costly protein-rich yeast extract medium could be replaced by commercial milk powder or by lysed activated sludge. Glutamate, molasses, or glucose-based media did not result in the enrichment of ureolytic bacteria by the chemostat. The concentration of intracellular urease was sufficiently high such that the produced raw effluent from the reactor could be used directly for biocementation in the field.     

Increasing of Soil Urease Activity by Stimulation of Indigenous Bacteria and Investigation of Their Role on Shear Strength

Abstract

Recent studies have shown that the use of biostimulation is an effective technique to eliminate the environmental side effects of traditional soil improvement methods. The use of indigenous bacteria of soil is a new method through which indigenous bacteria produce carbonate calcium by their urease activity. Stimulation of soil indigenous bacteria with the aim of calcite precipitation can considerably increase the soil shear strength. In this study, indigenous ureolytic bacteria are stimulated by adding nutrients to the soil. Subsequently urease activity of these bacteria in the presence of calcium chloride and nickel chloride causes calcium carbonate to precipitate between the sand particles. The analysis showed that the stimulated soil compared to the control soil was significantly different in terms of the soil engineering properties and the amount of precipitated calcite. Further, the treated and untreated samples were examined using direct shear test, scanning electron microscope (SEM), and energy dispersive X-ray (EDX) analysis. The results showed an increase of 30–67% in ultimate shear strength, 4–18.8% in residual shear strength, 190% in the cohesion intercept, and 16.8% in the angle of internal friction. In addition, imaging and analysis of SEM-EDX indicated the production of large amounts of calcite precipitates on surfaces of soil particles and between them.     

Environmental Factors Affecting the Compressive Strength of Microbiologically Induced Calcite Precipitation-Treated Soil

Some microorganisms such as Sporoscarcina pasteurii precipitate calcium carbonate and are suitable for biocementation. This study aimed to investigate the effects of several factors including concentration of bacteria, chemical reactants, temperature, and pH on precipitation of calcium carbonate. The results showed that after 7 and 14 days of curing, the compressive strength of silty clay soil samples increased steadily as pH increased from 5 to 9. It was observed that pH plays an important role in biocementation. The highest compressive strength (i.e. 92 kPa) was observed when the soil was treated with 50 ml of bacterial solution after 14 days of curing. In addition, it was observed that the highest compressive strength of samples was achieved when the temperature was 40°C.     

Effects of environmental factors on microbial induced calcium carbonate precipitation

Aims: To gain an understanding of the environmental factors that affect the growth of the bacterium Sporosarcina pasteurii, the metabolism of the bacterium and the calcium carbonate precipitation induced by this bacterium to optimally implement the biological treatment process, microbial induced calcium carbonate precipitation (MICP), in situ. Methods and Results: Soil column and batch tests were used to assess the effect of likely subsurface environmental factors on the MICP treatment process. Microbial growth and mineral precipitation were evaluated in freshwater and seawater. Environmental conditions that may influence the ureolytic activity of the bacteria, such as ammonium concentration and oxygen availability, as well as the ureolytic activities of viable and lysed cells were assessed. Treatment formulation and injection rate, as well as soil particle characteristics are other factors that were evaluated for impact on uniform induction of cementation within the soils. Conclusions: The results of the study presented herein indicate that the biological treatment process is equally robust over a wide range of soil types, concentrations of ammonium chloride and salinities ranging from distilled water to full seawater; on the time scale of an hour, it is not diminished by the absence of oxygen or lysis of cells containing the urease enzyme. Significance and Impact of Study: This study advances the biological treatment process MICP towards field implementation by addressing key environmental hurdles faced with during the upscaling process.     

Efficacy of Fe3O4/Starch Nanoparticles on Sporosarcina pasteurii Performance in MICP Process

The microbial induced calcite precipitation (MICP) has been explored using well-known urease producer bacterium Sporosarcina pasteurii for many applications including soil stabilization. Urease enzyme hydrolyzes urea and in the presence of calcium chloride causes calcium carbonate precipitation between sand particles increasing sand stiffness and strength. In this study, the liquefied soil samples from Anzali coast were positioned inside injection columns by standard positioning technique. The columns were treated by injecting S. pasteurii suspension and cementation solution (CaCl₂ and urea). The effect of different conditions consisting of number of injections, injection intervals, flow rate, and ratio of injection solution on unconfined compression strength (USC) of sands formed inside the columns were evaluated. The results indicated that soil strength was increased when ratio of reactant solutions and injection time were elevated. Moreover, the maximum Ca-precipitation in MICP reaction in liquid medium was obtained while Fe₃O₄/starch concentration and time of addition of nanoparticle to culture medium were 10.8?mg/L and 1.4?h, respectively. The USC results showed that the columns injected by bacterial suspension treated by Fe₃O₄/starch under optimized conditions improved the soil strength up to 1200?kPa in comparison to the control column as 220?kPa.     

Influence of temperature on the effectiveness of a biogenic carbonate surface treatment for limestone conservation

So far, most studies on microbiologically induced carbonate precipitation for limestone conservation have been performed at temperatures optimal for the activity of the calcinogenic bacteria (i.e., 20–28 °C). Successful application in practice, however, requires adequate performance in a wide range of environmental conditions. Therefore, the aim of this study was to select microorganisms that are most suited for biodeposition at temperatures relevant for practice. In a first step, ureolytic microorganisms were screened for their growth and ureolytic activity at different temperatures (10, 20, 28, and 37 °C). Large differences in calcinogenic activity could be observed between experiments performed on agar plates and those performed in solution and in limestone. In a second step, the influence of temperature on the performance of the biodeposition treatment with different ureolytic microorganisms was evaluated, both on the consolidative and protective effect of the treatment. In contrast with the experiments on agar plates, the Sporosarcina psychrophila strains failed to produce significant amounts of calcium carbonate on limestone in conditions relevant for practice, even at 10 °C. This resulted in a poor performance of the treatment. From experiments performed on limestone prisms, it appeared that the mesophilic Bacillus sphaericus produced the highest amount of carbonate in the shortest amount of time at all temperatures tested. As a result, compared to the untreated specimens, the highest consolidative (64 % lower weight loss upon sonication) and protective effect (46 % decreased sorptivity) were observed for the treatments with this species. From this study, it appears that among all ureolytic strains tested, B. sphaericus is most suited for biodeposition applications in practice.     

Precipitation of Calcite by Indigenous Microorganisms to Strengthen Liquefiable Soils

Enrichments for indigenous microorganisms capable of hydrolyzing urea in the presence of CaCl₂ were performed on potentially liquefiable saturated soils in both the laboratory and in situ. Following enrichment, treatment of soils with nutrients, CaCl₂ and urea resulted in significant in situ precipitation of calcite, even at depth, by indigenous microorganisms. The biomineralized soils showed properties that indicate calcite precipitation increased their resistance to seismic-induced liquefaction.     

Carbonate Mineral Precipitation for Soil Improvement Through Microbial Denitrification

Microbially induced carbonate precipitation (MICP) and associated biogas production may provide sustainable means of mitigating a number of geotechnical challenges associated with granular soils. MICP can induce interparticle soil cementation, mineral precipitation in soil pore space and/or biogas production to address geotechnical problems such as slope instability, soil erosion and scour, seepage of levees and cutoff walls, low bearing capacity of shallow foundations, and earthquake-induced liquefaction and settlement. Microbial denitrification has potential for improving the mechanical and hydraulic properties of soils because it promotes precipitation of calcium carbonate (CaCO₃) and produces nitrogen (N₂) gas without generating toxic by-products. We evaluated the potential for inducing carbonate precipitation in soil via bacterial denitrification using bench-scale experiments with the facultative anaerobe Pseudomonas denitrificans. Bench-scale experiments were conducted (1) without calcium in an N-rich bacterial growth medium in 2.0 L glass batch reactors and (2) with a source of calcium in sand-filled acrylic columns. Changes of pH, alkalinity, NO₃^? and NO₂^? in the batch reactors and columns, quantification of biogas production and observations of calcium-carbonate precipitation in the sand-filled columns indicate that denitrification led to carbonate precipitation and particle cementation in the pore water as well as a substantial amount of biogas production in both systems. These results document that bacterial denitrification has potential as a soil improvement mechanism.     

Evaluation of Shear Strength Parameters of Sandy Soils upon Microbial Treatment

A new method for soil stabilization known as microbial-induced calcite precipitation (MICP) has been the focus of research in this area during the last decade. In this method, the reaction of microorganisms in the presence of urea and calcium chloride is used to produce calcite. Despite the large numbers of bacteria in soil, Sporobacillus pasteurii (previously known as Bacillus pasteurii) has the most capability to create cementation between soil particles in the MICP method due to its high urease activity. In this paper, the effect of MICP treatment on the shear strength characteristics of a sandy soil was studied. The change in the shear strength of sandy soil upon MICP treatment was measured using a strain-controlled direct shear test before and after treatment of the soil samples. The results showed an increase of 44–86% in the shear strength of the sandy soil after 15 days of MICP treatment compared to the untreated soil. The enhanced shear strength was the result of an increase in both the cohesion intercept and angle of internal friction. The increase in the cohesion intercept was more significant than the increase in the angle of internal friction.     

Influence of Celery on the Remediation of PAHs-contaminated Farm Soil

Polycyclic aromatic hydrocarbons (PAHs) contamination has been considered as one of the major environmental concerns for farmland soil all over the world including China. Due to small per capita land area, to find crops or vegetable, which could not only degrade the PAHs contaminants but also would not concentrate PAHs, was particularly important. Celery was selected as the phytoremediator in this experiment, and the soil enzyme activity, PAHs-degrading microorganisms, and the speciation of PAHs in soil were studied. The results showed that celery could significantly enhance the remediation of PAHs compared with the controlled experiment after 90 days (p< 0.01), and the removal efficiency were 31.29%, 30.79%, and 50.21% in the soil, non-rhizosphere soil, and rhizosphere soil, respectively. The soil enzyme activity and PAHs-degrading microorganisms significantly increased in rhizosphere soil compared with non-rhizosphere soil (p< 0.05), and the bioaccessibility of PAHs in soil could have been enhanced in the presence of celery root exudates. Those would help the bioremediation of PAHs by soil microorganisms. Meanwhile, the concentration of PAHs in the edible portion of celery was only 17.13 ± 1.24 μg/kg, and the bioconcentration factors in the aboveground part of celery were only 0.025. This study provides a potential in-site farmland soil phytoremediation technology that could have practical utility.     

Silicon-Mediated Tomato Resistance Against Ralstonia solanacearum is Associated with Modification of Soil Microbial Community Structure and Activity

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease of Solanaceae crops. In this study, the soil microbial effects of silicon-induced tomato resistance against R. solanacearum were investigated through pot experiment. The results showed that exogenous 2.0 mM Si treatment reduced the disease index of bacterial wilt by 19.18 % to 52.7 % compared with non-Si-treated plants. The uptake of Si was significantly increased in the Si-treated tomato plants, where the Si content was higher in the roots than that in the shoots. R. solanacearum inoculation resulted in a significant increase of soil urease activity and reduction of soil sucrase activity, but had no effects on soil acid phosphatase activity. Si supply significantly increased soil urease and soil acid phosphatase activity under pathogen-inoculated conditions. Compared with the non-inoculated treatment, R. solanacearum infection significantly reduced the amount of soil bacteria and actinomycetes by 52.5 % and 16.5 %, respectively, but increased the ratio of soil fungi/soil bacteria by 93.6 %. After R. solanacearum inoculation, Si amendments significantly increased the amount of soil bacteria and actinomycetes and reduced soil fungi/soil bacteria ratio by 53.6 %. The results suggested that Si amendment is an effective approach to control R. solanacearum. Moreover, Si-mediated resistance in tomato against R. solanacearum is associated with the changes of soil microorganism amount and soil enzyme activity.     

Effect of temperate forest tree species on soil dehydrogenase and urease activities in relation to other properties of soil derived from loess and glaciofluvial sand

We investigated the effects of several tree species on dehydrogenase and urease activities in soils derived from two different parent materials (glaciofluvial sand and loess) in forested areas in southern Poland. We hypothesized that coniferous forests (pine, spruce) alter the soil cation exchange capacity (CEC) and decrease soil pH and, therefore, might decrease soil enzyme activities compared with broadleaf species growing on similar soils. Eight paired plots (12 × 12 m) were established on glaciofluvial sand in pine (Pinus sylvestris) + oak (Quercus robur) and spruce (Picea abies) + pine stands, as well as on loess-derived soils: beech (Fagus sylvatica) + pine and hornbeam (Carpinus betulus) + pine stands. Each plot was a 4 × 4 m grid with 16 sampling points. In soil samples pH, soil texture, and organic carbon, nitrogen, base cation contents, dehydrogenase and urease activities were determined. On both parent materials, the soil pH was lower under coniferous species than under broadleaf species. The acidifying effect of tree species on sandy soil was in the order of spruce = pine > oak, while that on loess was pine > beech > hornbeam. Hornbeam and oak increased the soil pH and stimulated enzyme activity in the soil. The content of fine fraction enhanced potential enzyme activities in soils, thus the loess soils had greater dehydrogenase and urease activity. The results suggest that pine stores more soil organic C in association with silt increasing the pool of stabilized soil organic C.     

Biodegradation of trinitrotoluene (TNT) by indigenous microorganisms from TNT-contaminated soil,and their application in TNT bioremediation

Soil and groundwater contaminated by munitions compounds is a crucial issue in environmental protection. Trinitrotoluene (TNT) is highly toxic and carcinogenic; therefore, the control and remediation of TNT contamination is a critical environmental issue. In this study, the authors characterized the indigenous microbial isolates from a TNT-contaminated site and evaluated their activity in TNT biodegradation. The bacteria Achromobacter sp. BC09 and Citrobacter sp. YC4 isolated from TNT-contaminated soil by enrichment culture with TNT as the sole carbon and nitrogen source (strain BC09) and as the sole nitrogen but not carbon source (strain YC4) were studied for their use in TNT bioremediation. The efficacy of degradation of TNT by indigenous microorganisms in contaminated soil without any modification was insufficient in the laboratory-scale pilot experiments. The addition of strains BC09 and YC4 to the contaminated soil did not significantly accelerate the degradation rate. However, the addition of an additional carbon source (e.g., 0.25% sucrose) could significantly increase the bioremediation efficiency (ca. decrease of 200 ppm for 10 days). Overall, the results suggested that biostimulation was more efficient as compared with bioaugmentation. Nevertheless, the combination of biostimulation and bioaugmentation using these indigenous isolates is still a feasible approach for the development of bioremediation of TNT pollution.     

Effects of Silver Nanoparticles on Soil Enzyme Activity of Different Wetland Plant Soil Systems

In the present study, five soil exoenzymes (dehydrogenase, urease, acid phosphatase, neutral phosphatase, and alkaline phosphatase) were investigated in rhizosphere of wetland plants (Iris wilsonii, Arundo donax, and Typha orientalis) treated with silver nanoparticles (0, 0.024, 0.24, 4.80, and 9.60 μg/g dry soil). It was found that Ag NPs were capable of inhibiting all exoenzyme activities tested in this study, with inhibitory effects especially obvious to higher Ag NPs level (4.80 and 9.60 μg/g dry soil). However, for lower Ag NPs concentration (0.024 μg/g dry soil), the adverse effects on exoenzymes was only found in T. orientalis rhizosphere, the exoenzyme activities in rhizosphere of I. wilsonii were less affected. This study suggested that high concentration Ag NPs could negatively affect all soil exoenzyme activities, while the impacts of low Ag NPs level on exoenzyme activities were mainly related to plant species.     

Investigating the Shear Strength of Biologically Improved Soil Using Two Types of Bacteria

Many types of researches have been carried out on sandy soils to improve the fertility through bacteria. In this regard, after measuring the activity of urease enzymes in urea bacterial sediments, calcium carbonate was applied in Sirjan soil (southeast of Iran), and the native bacteria of this soil were isolated. The strains of these microorganisms, because of the Come and aridity in the region and the severity of the environmental conditions in the area, have a greater resistance to chemical and physical factors and are compatible with the environment of this region. In this study, we tried to use two types of soil bacteria: one is Sporosaercina pasturii, many researchers have been working on this bacterium and the effects of soil improvement, and another is the native bacterium found in Sirjan soil (Acinetobacter calcoaceticus strain Nima). Thirty samples were taken in the same conditions and experiments to evaluate the use of native bacteria of Sirjan in soil remediation by direct shear testing, seismic electronic microscopy, and microscopic scanning (SEM) were performed on the samples. The treatment period for this study was 28?days. The results showed that the angle of internal friction increased for the treated A. calcoaceticus Nima (42%) and S. paturii (39%) compared to untreated samples. Also, adhesion between particles increased by 14.5 times for A. calcoaceticus Nima and 13.5 times for S. paturii. Finally, shear strength for soil treatment increased by4.6 times for A. calcoaceticus Nima and 3.9 times for S. pasturii. The use of indigenous strains in the natural environment due to the adaptation of strains to environmental conditions can increase the production of bio-cementation. It is, therefore, possible to use native bacteria for biologically improved soil as an appropriate alternative rather than traditional methods due to environmental problems.     

Decrease in Hydraulic Conductivity of a Paddy Field using Biocalcification in situ

The hydraulic conductivity of a paddy field (Anthraquic Dystrustept), a silty clay soil containing more than 29% (w/w) of gravel, in Nagoya University Farm was reduced by in situ treatment of subsurface soil using bentonite and biocalcification (microbial calcium carbonate precipitation) through the addition of CaCl₂, urea, and corn steep liquor (CSL). The treatment decreased the hydraulic conductivity of the field from an average of 10^?3 cm/s to a range of 10^?5 to 10^?7 cm/s during 69 days, with reducing the proportion of pores of subsurface soil larger than 75 µm in diameter. The biocalcification effect was observed at 10-cm thickness from the treated subsurface. Laboratory soil core experiments demonstrated that the decrease in the hydraulic conductivity was not attributed to the effect of bentonite but mainly to the effect of biocalcification. The addition of CSL enhanced the urease activity of soil required for biocalcification, even at 4°C, as indicated by a decrease in urease activation energy temperature sensitivity. These experimental results agreed with the gradual decrease in hydraulic conductivity observed in the field when the average daily temperature was 7°C (days 24–69). It was suggested that the biocalcification is a potential technique to reduce the hydraulic conductivity of paddy field.     

Tofu Wastewater as Efficient Nutritional Source in Biocementation for Improved Mechanical Strength of Cement Mortars

Biocementation is an eco-friendly process in sustainable construction; however, the associated cost of nutrients is major obstacle in it. Therefore, the main objective of this study was to utilize tofu wastewater (TW) in biocementation to grow Bacillus cereus and desirable characteristics were compared with that in commercial nutrient broth. There was no significant difference in bacterial growth between two media with TW as preferable media for growth and urease activity on which biocementation depends. Further, an improved compressive strength of sandstone and mortars was achieved with TW, showing important insights into the greater feasibility of TW as biocementation source.     

Ureolytic nitrification at low pH by Nitrosospira spec.

An ureolytic ammonium-oxidizing chemolithotroph belonging to the genus Nitrosospira was shown to nitrify at pH 4.5 in a pH-stat with urea as a substrate. With ammonium as the sole substrate nitrification did not occur at pH values below 5.5. Nitrosomonas europaea ATCC 19718 and Nitrosospira briensis ATCC 25971 did not possess urease activity. The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.