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.     

Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement

The mechanical properties of soil (cohesion, friction, stiffness and permeability) are important parameters for engineering constructions and ecosystems in sedimentary environments. BioGrout is an in situ soil strengthening technique involving microbial-induced carbonate precipitation (MICP). This process involves hydrolysis of urea by bacteria containing the enzyme urease in the presence of dissolved calcium ions, resulting in calcium carbonate precipitation. In order to control the BioGrout process for engineering applications, it is necessary to improve understanding of the relevant phenomena and develop efficiencies to enable up-scaling of the technology to suit commercial applications. Control of a homogeneous distribution of bacterial activity in a sand bed is considered crucial in order to prevent clogging during injection and provide homogeneous reinforcement results. This paper describes a methodology to distribute and fix bacteria (with their enzyme activity) relatively homogeneously in a sand bed, before supplying cementation reagents. The methodology is based on a two-phase injection procedure: a bacterial suspension is injected into the sand body, immediately followed by a fixation fluid (i.e. a solution with high salt content). It is proposed that bacteria are retarded by adsorption and filtration processes and are permanently adsorbed to the sand grains when overtaken by the fixation fluid. The presented experimental approach for optimizing bacterial fixation in porous media can be used as a tool to design the treatment protocol for engineering applications in practice.     

Bacterial exopolymer and PHB production in fluctuating ground-water habitats

Abstract The physiological response of a strain of ground-water bacteria to short-term fluctuations in the quality of percolating water was followed in a continuous-flow water-saturated soil column. The cells reduced their production of uronic acids-characterized extracellular polysaccharides by more than 50% when pure ground water was enriched with phosphate and glucose but resumed it gradually as the poor growth conditions returned. Bacteria that were sorbed to the mineral particles in the soil column produced more exopolymers-about six times more in pure ground water-than free-living bacteria, but the composition of the polymer was the same. Glucose, mannose and galacturonic acid accounted for about 90% of the carbohydrate. The cells stored about five times more poly-β-hydroxybutyric acid (PHB) in pure than in enriched ground water, and sorbed cells had five times higher concentrations than free-living cells. The quality variations of the ground water were also reflected in the population size of bacteria, which differed by two to four orders of magnitude between poor and enriched conditions.     

Factors Controlling Microbially Induced Desaturation and Precipitation (MIDP) via Denitrification during Continuous Flow

Three continuous-flow column experiments were conducted to investigate the effects of nitrate loading rate and input concentrations of acetate and calcium on the effectiveness of denitrification to promote microbially induced desaturation and precipitation (MIDP). MIDP differs from microbially induced carbonate precipitation (MICP) in that it relies upon desaturation by biogenic gas production, along with carbonate precipitation, to improve the behavior of saturated granular soil. Denitrification is a stepwise process that is susceptible to inhibition and accumulation of intermediates, like nitrite, due to unfavorable chemical conditions (e.g., low pH). Therefore, nitrate reduction, nitrite accumulation, calcite saturation, and formation of carbonate precipitates were monitored in the columns. Factors investigated in these experiments included the effects of nitrate loading rate and input concentrations of calcium and acetate. Low nitrate-loading rates (i.e., ≤0.7?mol/m²-day) generally led to favorable outcomes (i.e., less accumulation of intermediates and more efficient carbonate precipitation). However, faster precipitation rates associated with low nitrate-loading rates led to smaller carbonate crystals and a less uniform precipitation pattern (i.e., precipitation focused near the nutrient source). Input concentrations of calcium and acetate also affected MIDP, but to a lesser extent than nitrate loading rate. The most important stimulated denitrifying bacteria, identified via 16S rDNA sequencing of suspended and dissolved DNA in the soil columns, were Bacillus species, Pseudomonas species, Brevundimonas species, and members of the Rhizobiaceae family. The results support that lower nitrate loading rates can be beneficial for MIDP although higher nitrate loading rates might be useful for some soil improvement applications.     

Fungi,bacteria and soil pH: the oxalate–carbonate pathway as a model for metabolic interaction

The oxalate–carbonate pathway involves the oxidation of calcium oxalate to low‐magnesium calcite and represents a potential long‐term terrestrial sink for atmospheric CO ₂. In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non‐sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.     

Bacillus megaterium mediated mineralization of calcium carbonate as biogenic surface treatment of green building materials

Microbially induced calcium carbonate precipitation is a biomineralization process that has various applications in remediation and restoration of range of building materials. In the present study, calcifying bacteria, Bacillus megaterium SS3 isolated from calcareous soil was applied as biosealant to enhance the durability of low energy, green building materials (soil–cement blocks). This bacterial isolate produced high amounts of urease, carbonic anhydrase, extra polymeric substances and biofilm. The calcium carbonate polymorphs produced by B. megaterium SS3 were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and Fourier transmission infra red spectroscopy. These results suggested that calcite is the most predominant carbonate formed by this bacteria followed by vaterite. Application of B. megaterium SS3 as biogenic surface treatment led to 40 % decrease in water absorption, 31 % decrease in porosity and 18 % increase in compressive strength of low energy building materials. From the present investigation, it is clear that surface treatment of building materials by B. megaterium SS3 is very effective and eco friendly way of biodeposition of coherent carbonates that enhances the durability of building materials.     

石灰碳铵熏蒸联合生物有机肥对香蕉枯萎病和细菌群落的影响

以连续种植的香蕉枯萎病高发病蕉园为试验点,通过实时定量PCR和高通量测序等方法,研究了田间条件下石灰碳铵熏蒸联合生物有机肥施用对香蕉枯萎病的防治效果,以及对土壤细菌群落结构和组成的影响。结果表明: 与不熏蒸施用有机肥(OF)相比,香蕉枯萎病发病率在施用有机肥前使用石灰碳铵进行熏蒸处理(LAOF)和施用生物有机肥前使用石灰碳铵进行熏蒸处理(LABF)中分别降低了13.3%和21.7%,病原菌的拷贝数分别降低了22.4%和33.0%。与OF处理相比,石灰碳铵熏蒸联合不同肥料施用均显著降低了细菌的丰富度和多样性,形成了明显不同的群落结构,且熏蒸对群落组成的差异产生了决定性的影响。LABF的细菌丰富度和多样性均低于其他处理,群落组成也与其他处理存在明显差异。与OF处理相比,熏蒸处理(LAOF和LABF)显著增加了土壤中水恒杆菌、布鲁式菌和漯河杆菌属的相对丰度,且在LABF中的相对丰度均高于LAOF,水恒杆菌和布鲁式菌的相对丰度差异显著。在田间条件下,施用生物有机肥之前使用石灰碳铵进行熏蒸处理能够显著降低病原菌数量,改变土壤细菌群落结构,激发土壤有益微生物,从而减少香蕉枯萎病的发生。     

Soil carbonate drives local adaptation in Arabidopsis thaliana

High soil carbonate limits crop performance especially in semiarid or arid climates. To understand how plants adapt to such soils, we explored natural variation in tolerance to soil carbonate in small local populations (demes) of Arabidopsis thaliana growing on soils differing in carbonate content. Reciprocal field‐based transplants on soils with elevated carbonate (+C) and without carbonate (?C) over several years revealed that demes native to (+C) soils showed higher fitness than those native to (?C) soils when both were grown together on carbonate‐rich soil. This supports the role of soil carbonate as a driving factor for local adaptation. Analyses of contrasting demes revealed key mechanisms associated with these fitness differences. Under controlled conditions, plants from the tolerant deme A1_(+C) native to (+C) soil were more resistant to both elevated carbonate and iron deficiency than plants from the sensitive T6_(?C) deme native to (?C) soil. Resistance of A1_(+C) to elevated carbonate was associated with higher root extrusion of both protons and coumarin‐type phenolics. Tolerant A1_(+C) also had better Ca‐exclusion than sensitive T6_(?C). We conclude that Arabidopsis demes are locally adapted in their native habitat to soils with moderately elevated carbonate. This adaptation is associated with both enhanced iron acquisition and calcium exclusion.     

The evaluation of plant-available sulphur in soils. II

Summary The availability to oats of adsorbed sulphate in soils and of sulphate impurity in calcium carbonate was studied in pot-culture experiments.When calcium carbonate was added to soils with pH values ranging from 5.7 to 7.4 the uptake of sulphur by oats was increased, due probably to enhanced mineralization of soil organic sulphur. When the calcium carbonate contained sulphate impurity the uptake of sulphur was further increased by an amount comparable with the release of sulphate which could be expected from a reaction of the calcium carbonate with the exchangeable hydrogen of the soil. Sulphate in excess of this amount appeared to be largely unavailable. Uptake of sulphur by oats from calcareous sands containing large amounts of insoluble sulphate associated with calcium carbonate also suggested that soil sulphur in this form had very low availability to plants.Substantial increases in the amounts of sulphur extracted by reagents commonly used for the determination of adsorbed sulphate in soils occurred when soils were airdried at about 20°C. Decreases in adsorbed sulphate in soils following the growth of oats in pot culture confirmed that adsorbed sulphate is readily available to plants.     

Selective medium with nalidixic acid for isolating antibiotic-producing Actinomyces

The majority of actinomycetes belonging to various genera proved to be resistant to nalidixic acid concentrations having an inhibitory effect on bacteria with trailing growth i.e. B. subtilis and B. mycoides. The bacteria prevented isolation of actinomycetes as pure cultures. The use of a selective medium with nalidixic acid for isolation of soil actinomycetes resulted in 20 per cent increase in the number of the actinomycetes isolated as pure cultures. Preliminary treatment of the soil samples with calcium carbonate under moist conditions followed by the inoculation to the medium with nalidixic acid made it possible to increase isolation of actinomycetes at most 100-fold. With this complex method 495 actinomycete cultures were isolated, their antibiotic properties were studied and their taxonomic position at the genus level was determined. The complex method including the preliminary treatment of soil samples with calcium carbonate followed by inoculation to the selective medium with nalidixic acid is efficient and may be recommended for screening organisms producing new antibiotics.     

Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids

The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.     

岩溶洞穴微生物沉积碳酸钙——以贵州石将军洞为例

为探讨洞穴微生物沉积碳酸钙作用对洞穴沉积物的影响,利用传统生物学方法,采集贵州中西部地区石将军洞洞穴沉积物表面的微生物样品,结合洞穴监测数据和理化背景资料,利用B-4培养基和B-4C培养基对洞穴细菌进行筛选和纯化,分离出能沉积碳酸钙的菌种,观察和了解洞穴细菌形成的CaCO3晶体,应用X射线衍射分析仪(XRD)分析细菌形成的CaCO3晶体成分,并利用扫描电子显微镜(SEM)观察晶体结构特征。结果表明:1)在B-4培养基下微生物产生的碳酸钙晶体主要为方解石、球霰石和方解石混合物、球霰石,这种变化与培养基pH值的增幅相关;同时,在添加Mg离子的B-4C培养基下形成的碳酸钙晶体主要为方解石,此外,研究中并未检测到文石晶体。2)通过SEM扫描,发现微生物作用形成的碳酸钙晶体存在不规则六方体、柱状体、四方体层状、半球状等,这些晶体形态在化学作用系统下少见,多见于微生物作用形成的方解石。此外,晶体中微生物作用痕迹明显,微生物作用贯穿于整个沉积过程。     

Nutrient uptake as a contributing explanation for deep rooting in arid and semi-arid ecosystems

Explanations for the occurrence of deep-rooted plants in arid and semi-arid ecosystems have traditionally emphasized the uptake of relatively deep soil water. However, recent hydrologic data from arid systems show that soil water potentials at depth fluctuate little over long time periods, suggesting this water may be rarely utilized or replenished. In this study, we examine the distributions of root biomass, soil moisture and nutrient contents to 10-m depths at five semi-arid and arid sites across southwestern USA. We couple these depth distributions with strontium (Sr) isotope data that show deep (>1 m) nutrient uptake is prevalent at four of the five sites. At all of the sites, the highest abundance of one or more of the measured nutrients occurred deep within the soil profile, particularly for P, Ca²⁺ and Mg²⁺. Phosphate contents were greater at depth than in the top meter of soil at three of five sites. At Jornada, for example, the 2–3 m depth increment had twice the extractable P as the top meter of soil, despite the highest concentrations of P occurring at the surface. The prevalence of such deep resource pools, and our evidence for cation uptake from them, suggest nutrient uptake as a complementary explanation for the occurrence of deep-rooted plants in arid and semi-arid systems. We propose that hydraulic redistribution of shallow surface water to deep soil layers by roots may be the mechanism through which deep soil nutrients are mobilized and taken up by plants.Electronic Supplementary Material Supplementary material is available for this article at     

Improving the Shear Strength of Quartz Sand using the Microbial Method

An increase in urban population and the reduced number of suitable lands for construction projects have necessitated the need for ground improvement methods with no environmentally detrimental effects. Microbial-induced calcite precipitation (MICP) is a relatively environmentally friendly method for soil regeneration. In the present paper, X-ray diffraction (XRD) and scanning electron microscopy (SEM) analyses were performed to investigate the type and method of cement formation in loose quartz sand with different densities stabilized using bacteria. The results showed that the highest content of calcium carbonate cement was produced in the sample with the lowest density. Moreover, after microbial stabilization, the shear strength measured in the direct shear test was increased in the specimen with a lower density. We observed an increase in shear strength from 0.63 kg/cm² before injection for loose sand (γ = 1.5 g/cm³) to 3.92 kg/cm² after injection. In addition, the effect of injection time was investigated and found that shear strength in the two-stage injection per day is greater than that in one- and three-stage injections per day. With prolonging the injection time from one to five days, shear strength was significantly increased from 2.07 to 4.54 kg/cm². Furthermore, prolonging the bacterial treatment period led to a significant increase in the produced carbonate cement and, consequently, enhanced soil shear strength.     

Long-term simulation of in situ biostimulation of polycyclic aromatic hydrocarbon-contaminated soil

A continuous-flow column study was conducted to evaluate the long-term effects of in situ biostimulation on the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soil from a manufactured gas plant site. Simulated groundwater amended with oxygen and inorganic nutrients was introduced into one column, while a second column receiving unamended groundwater served as a control. PAH and dissolved oxygen (DO) concentrations, as well as microbial community profiles, were monitored along the column length immediately before and at selected intervals up to 534?days after biostimulation commenced. Biostimulation resulted in significantly greater PAH removal than in the control condition (73% of total measured PAHs vs. 34%, respectively), with dissolution accounting for a minor amount of the total mass loss (~6%) in both columns. Dissolution was most significant for naphthalene, acenaphthene, and fluorene, accounting for >20% of the total mass removed for each. A known group of PAH-degrading bacteria, 'Pyrene Group 2' (PG2), was identified as a dominant member of the microbial community and responded favorably to biostimulation. Spatial and temporal variations in soil PAH concentration and PG2 abundance were strongly correlated to DO advancement, although there appeared to be transport of PG2 organisms ahead of the oxygen front. At an estimated oxygen demand of 6.2?mg O(2)/g dry soil and a porewater velocity of 0.8?m/day, it took between 374 and 466?days for oxygen breakthrough from the 1-m soil bed in the biostimulated column. This study demonstrated that the presence of oxygen was the limiting factor in PAH removal, as opposed to the abundance and/or activity of PAH-degrading bacteria once oxygen reached a previously anoxic zone.     

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.     

Bacterial Physiological Diversity in the Rhizosphere of Range Plants in Response to Retorted Shale Stress

Bacterial populations were isolated from the soil-root interface and root-free regions of Agropyron smithii Rydb. and Atriplex canescens (Pursh) Nutt. grown in soil, retorted shale, or soil over shale. Bacteria isolated from retorted shale exhibited a wider range of tolerance to alkalinity and salinity and decreased growth on amino acid substrates compared with bacteria from soil and soil-over-shale environments. Exoenzyme production was only slightly affected by growth medium treatment. Viable bacterial populations were higher in the rhizosphere and rhizoplane of plants grown in retorted shale than in plants grown in soil or soil over shale. In addition, a greater number of physiological groups of rhizosphere bacteria was observed in retorted shale compared with soil alone. Two patterns of community similarity were observed in comparisons of bacteria from soil over shale with those from soil and retorted-shale environments. Root-associated populations from soil over shale had a higher proportion of physiological groups in common with those from the soil control than with those from the retorted-shale treatment. However, in non-rhizosphere populations, bacterial groups from soil over shale more closely resembled the physiological groups from retorted shale.     

Comparison of microbially induced calcium carbonate precipitation eligibility using sporosarcina pasteurii and bacillus licheniformis on two different sands

The use of biological means for ground improvement have become popular, which generally works through the process called microbially-induced calcium carbonate precipitation (MICP). Many studies indicate successful application of MICP based improvement with multiple bacteria and on several soils. Given the proven performance of MICP, this study aims to examine the MICP process by comparing the calcium carbonate precipitation ability of widely studied bacteria, i.e., Sporosarcina pasteurii and relatively under-recognized bacteria, i.e., Bacillus licheniformis to outline the formation success. For this purpose, two different sands were tested for observing precipitation behavior using a series of syringe tests. Furthermore, the effect of concentration and inclusion of calcium chloride for nutrition of bacteria, saturation with water, and hybrid use of two bacteria were investigated in some tests for diversification. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive x-ray spectroscopy (EDS) were used for the interpretation of results. Results indicated that Sporosarcina pasteurii had performed superior over Bacillus licheniformis when achieving calcium carbonate precipitation in tests for both sands. In addition, many intriguing SEM images contributed to the literature of MICP monitoring, highlighting the effects of the variables investigated.     

The responses of soil organic carbon mineralization and microbial communities to fresh and aged biochar soil amendments

While biochar soil amendment has been widely proposed as a soil organic carbon (SOC) sequestration strategy to mitigate detrimental climate changes in global agriculture, the SOC sequestration was still not clearly understood for the different effects of fresh and aged biochar on SOC mineralization. In the present study of a two‐factorial experiment, topsoil samples from a rice paddy were laboratory‐incubated with and without fresh or aged biochar pyrolyzed of wheat residue and with and without crop residue‐derived dissolved organic matter (CRM) for monitoring soil organic matter decomposition under controlled conditions. The six treatments included soil with no biochar, with fresh biochar and with aged biochar treated with CRM, respectively. For fresh biochar treatment, the topsoil of a same rice paddy was amended with wheat biochar directly from a pyrolysis wheat straw, the soil with aged biochar was collected from the same soil 6 years following a single amendment of same biochar. Total CO₂ emission from the soil was monitored over a 64 day time span of laboratory incubation, while microbial biomass carbon and phospholipid fatty acid (PLFA) were determined at the end of incubation period. Without CRM, total organic carbon mineralization was significantly decreased by 38.8% with aged biochar but increased by 28.9% with fresh biochar, compared to no biochar. With CRM, however, the significantly highest net carbon mineralization occurred in the soil without biochar compared to the biochar‐amended soil. Compared to aged biochar, fresh biochar addition significantly increased the total PLFA concentration by 20.3%–33.8% and altered the microbial community structure by increasing 17:1ω8c (Gram‐negative bacteria) and i17:0 (Gram‐positive bacteria) mole percentages and by decreasing the ratio of fungi/bacteria. Furthermore, biochar amendment significantly lowered the metabolic quotient of SOC decomposition, thereby becoming greater with aged biochar than with fresh biochar. The finding here suggests that biochar amendment could improve carbon utilization efficiency by soil microbial community and SOC sequestration potential in paddy soil can be enhanced by the presence of biochar in soil over the long run.     

6种土壤微生物提取剂的比较

比较了6种土壤微生物提取剂（6-偏磷酸钠溶液、焦磷酸钠溶液、磷酸盐缓;中液、林格溶液、NaCl溶液和水）对土壤细菌、真菌和放线菌数量的影响。结果表明：0．1％的6-偏磷酸钠（HMP）和焦磷酸钠（PYS）溶液（w／v）提取的细菌、真菌、放线菌数量最多,磷酸缓冲液（pH值7．2）对土壤真菌提取效率与焦磷酸钠和6-偏磷酸钠溶液相似,而对细菌和放线菌的提取效率则低于焦磷酸钠和6-偏磷酸钠溶液,其余3种提取剂的提取效率相对较低。