Production of nanocalcite crystal by a urease producing halophilic strain of <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis> and analysis of its properties by XRD and SEM

Cementation of salt-containing soils can be achieved by salt-tolerant or halophilic calcite precipitation bacteria. Therefore, the isolation of calcite-producing bacteria in the presence of salt is the first step in the microbial cementation of saline soils. Urease producing bacteria can cause calcite nano-crystals to precipitate by producing urease in the presence of urea and calcium. The purpose of this study was to isolate urease producing halophilic bacteria in order to make calcite precipitate in saline soil. The calcite and the properties of the strains were further analyzed by X-ray diffraction (XRD) and scanning electron microscope equipped with an energy dispersive X-ray detector. In this study, a total of 110 halophilic strains were isolated, from which 58 isolates proved to have the ability of urease production. Four strains were identified to produce nano-calcite using urease activity in the precipitation medium. The XRD studies showed that the size of these particles was in the range of 40–60 nm. Strain H3 revealed that calcite is mostly produced in the precipitation medium containing 5% salt in comparison with other strains. This strain also produced calcite precipitates in the precipitation medium containing 15% salt. Phylogenetic analysis indicated that these isolates are about 99–100% similar to Staphylococcus saprophyticus.     

Bacteria-Induced Cementation in Sandy Soils

Bacteria-induced calcite precipitation (BICP) is a promising technique that utilizes bacteria to form calcite precipitates throughout the soil matrix, leading to an increase in soil strength and stiffness. This research investigated BICP in two types of sands under sterile and nonsterile conditions. Bacteria formation and BICP in the sterilized sand specimens are higher than those in the nonsterilized sand specimens. The development of calcite with time is initially greater for the sand specimens containing calcite. Scanning electron microscope imaging allowed the detection of cementation from calcite precipitation on the surface and pores of the sand matrix. The effects of injecting nutrient mediums and bacteria into the specimens, as well as pH of soil samples on BICP were investigated. The bearing capacity of biologically treated vs. untreated sand specimens were determined especially by laboratory foundation loading tests.     

Characterization of two urease-producing and calcifying Bacillus spp. isolated from cement

Two bacterial strains designated as CT2 and CT5 were isolated from highly alkaline cement samples using the enrichment culture technique. On the basis of various physiological tests and 16S rRNA sequence analysis, the bacteria were identified as Bacillus species. The urease production was 575.87 U/ml and 670.71 U/ml for CT2 and CT5 respectively. Calcite constituted 27.6% and 31% of the total weight of sand samples plugged by CT2 and CT5, respectively. Scanning electron micrography (SEM) analysis revealed the direct involvement of these isolates in calcite precipitation. This is the first report of the isolation and identification of Bacillus species from cement. Based on the ability of these bacteria to tolerate extreme environment of cement, they have potential to be used in remediating the cracks and fissures in various building or concrete structures.     

Biogenic Evidences of Moonmilk Deposition in the Mawmluh Cave,Meghalaya, India

Moonmilk, a microcrystalline secondary cave deposit, actively forms on the floor of Krem Mawmluh – a limestone cave in Meghalaya, Northeastern India. Due to the abundance of micrite and calcified microbial filaments, we hypothesize that these deposits form as a result of ongoing microbial interactions. Consistent with this idea, we report electron microscopic and microbiological evidences for the biological origin of moonmilk in Krem Mawmluh. Scanning electron microscopy indicated abundant calcified microbial filaments, needle calcite, fibre calcites (micro-fibre and nano-fibre calcite crystals), biofilm and microbial filaments in the moonmilk. The total viable culturable microbes showed high population densities for microbes in the moonmilk and moonmilk pool waters. In vitro culture experiments, confirmed the capability of many of the isolated strains to precipitate calcite and some of the identified isolates belonged to the Bacillus sp. and Actinomycetes. These results clearly support the biogenic nature of the deposits.     

不同覆盖方式对底泥内源营养盐释放的控制效果

通过底泥内源营养盐释放控制室内模拟试验,考察了塑料包被、斜发沸石、方解石、石英砂和硝酸钙5种覆盖材料对底泥氮磷释放效率的影响,系统分析了各自优劣程度,为实际环境中不同污染背景水体选择适宜的控制技术提供科学依据.结果表明: 不同覆盖材料对底泥总磷释放的控制效果依次为:塑料包被>硝酸钙>斜发沸石>方解石>石英砂;不同覆盖材料对底泥总氮释放的控制效果依次为:斜发沸石>塑料包被>方解石>石英砂>硝酸钙;不同覆盖材料对底泥硝态氮释放的控制效果依次为:塑料包被>斜发沸石>方解石>石英砂>硝酸钙;不同覆盖材料对底泥铵态氮释放的控制效果依次为:硝酸钙>石英砂>斜发沸石>方解石>塑料包被;温度和底泥内源营养盐释放有对应关系,水样中总磷、总氮和硝态氮浓度会随着温度上升而增加,而铵态氮浓度呈下降趋势.     

Improvement of Geomechanical Properties of Bio-remediated Aeolian Sand

Aeolian sand is widely distributed in the desert. It is susceptible to wind erosion because it consists of fine particles that lack cohesion between them. Wind erosion of Aeolian sand, dune migration, and dust storms in deserts affect the environment and infrastructure severely. Conventional chemical methods for lessening the effect of wind erosion are environmentally unfriendly. This paper examines Aeolian sand bio-remediated by microbially induced calcite precipitations (MICPs). The geomechanical properties of the Aeolian sand remediated by MICP were tested to explore its behavior as a desert bio-crust. The results indicated that a bio-crust of Aeolian sand with suitable strength required 7 days for formation. The remediated Aeolian sand had an average unconfined compression strength of 0.66 MPa and an average inner friction angle of 36° at a bacterial concentration in the OD₆₀₀ range of 0.5–0.8. These findings indicate that formation of bio-crust could serve as protection against desert erosion. The particle characteristics and the porosity of Aeolian sand play an important role in the formation of the bio-crust. Crust from bio-remediated well-graded, high-porosity soil is stronger than crust from bio-remediated, poorly graded, low-porosity soil, given that both soils have the same bacteria concentration.     

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.     

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.     

Soil Bioconsolidation Through Microbially Induced Calcite Precipitation by Lysinibacillus sphaericus WJ-8

Biomineralization is a process that leads to the formation of minerals via a biologically or biotechnologically mediated route. This process is a new and innovative research area in geotechnological engineering and structural engineering because it has wide-ranging implications for the strengthening of soil, sand, stone, and cementitious materials. In the present study, we demonstrated the ability of Lysinibacillus sphaericus WJ-8 to precipitate 15.3 mg/mL of calcite and to degrade 415 μmol/mL of urea over a 120-h period. The cell surface hydrophobicity and sand adhesion of spores were higher than those of vegetative cells (77.2% vs. 24.0% and 54.1% vs. 7.8%, respectively). In addition, the bioconsolidated soil block samples had significantly smaller pores than did the control soil block samples. Scanning electron microscopy and energy dispersive spectroscopy analysis revealed that calcite crystals were frequently formed in the bioconsolidated soil block samples, but did not occur in the control soil block samples. In addition, sharp peaks in the X-ray diffraction spectra indicated that calcite (CaCO₃) crystals constituted the predominant mineral in the bioconsolidated samples, whereas quartz (SiO₂) crystals constituted the predominant mineral in the control samples.     

Bioherbicidal activity of allelopathic bacteria against weeds associated with wheat and their effects on growth of wheat under axenic conditions

Allelopathic bacteria found to selectively inhibit weeds but not wheat in our earlier study were selected to evaluate their impact on three weeds and wheat under axenic conditions. Inoculated seeds of each species were sown in sand jars for 25 days. Results indicated that the applied strains variably inhibited germination of wild oat, little seed canary grass and broad leaved dock from 15.2 to 63.3, 18.5 to 58.7 and 18.4 to 60.5% and dry matter from 12.4 to 65, 22.8 to 81.4 and 21.7 to 71.3% than their controls, respectively. These effects were also evident in other growth parameters. Growth of wheat was significantly improved by four strains while others caused non-significant effects. Selectivity of these strains was also reflected in differential root colonization ability. These strains were characterized for various microbial and biochemical parameters. These strains may further be evaluated for their bioherbicidal activity under natural conditions.     

Ultrastructure and mineral distribution in the tergite cuticle of the beach isopod Tylos europaeus Arcangeli, 1938

The crustacean cuticle is a hierarchically organised material composed of an organic matrix and mineral. It is subdivided into skeletal elements whose physical properties are adapted to their function and the eco-physiological strains of the animal. Using a variety of ultrastructural and analytical techniques we studied the organisation of the tergite cuticle of the sand burrowing beach isopod Tylos europaeus. The surface of the tergites bear epicuticular scales, sensilla and micro-tubercles. A distal layer of the exocuticle is characterised by a low density of organic fibres and the presence of magnesium-calcite. Surprisingly, the mineral forms regions containing polyhedral structures alternating with smooth areas. Between sub-domains within the distal exocuticle calcite varies in its crystallographic orientation. Proximal layers of the exocuticle and the endocuticle are devoid of calcite and the mineral occurs in the form of amorphous calcium carbonate (ACC). Using thin sections of mineralised cuticle we describe for the first time that ACC forms tubes around single protein-chitin fibrils.     

Pseudomonas syringae pv. syringae Uses Proteasome Inhibitor Syringolin A to Colonize from Wound Infection Sites

Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.     

Effect of bacterial extracellular polymers on the saturated hydraulic conductivity of sand columns.

Columns were packed with clean quartz sand, sterilized, and inoculated with different strains of bacteria, which multiplied within the sand at the expense of a continuous supply of fresh nutrient medium. The saturated hydraulic conductivity (HCsat) of the sand was monitored over time. Among the four bacterial strains tested, one formed a capsule, one produced slime layers, and two did not produce any detectable exopolymers. The last two strains were nonmucoid variants of the first two. Only one strain, the slime producer, had a large impact on the HCsat. The production of exopolymers had no effect on either cell multiplication within or movement through the sand columns. Therefore, the HCsat reduction observed with the slime producer was tentatively attributed to the obstruction of flow channels with slime. Compared with the results with Arthrobacter sp. strain AK19 used in a previous study, there was a 100-fold increase in detachment from the solid substratum and movement through the sand of the strains used in this study. All strains induced severe clogging when they colonized the inlet chamber of the columns. Under these conditions, the inlet end was covered by a confluent mat with an extremely low HCsat.     

Acid Stream Water Remediation Using Limestone Sand on Bear Run in Southwestern Pennsylvania

Instream limestone sand application is used at many sites in Pennsylvania to neutralize acidic stream water resulting from acid deposition. However, questions remain about the effectiveness of limestone sand in the treatment of acid waters, such as reduced contact time at high flow, remobilization of aluminum, and adverse effects on macroinvertebrates. A 1‐year evaluation of limestone sand application to Bear Run, an acidic stream in southwestern Pennsylvania, was begun in 1999. Another nearby acid stream, Linn Run, served as a control. Water quality during baseflow and episodic acidification events, along with fish and macroinvertebrates, were monitored to evaluate impacts of the sand application. Hydrogen ion (H ⁺ ) concentration and total dissolved aluminum (TDA) were significantly reduced, and acid neutralizing capacity was significantly increased downstream of the limestone sand application compared with the upstream site on Bear Run. These parameters at the downstream sites were also different (∝ 0.10) from the comparable sites on the control stream. TDA and hydrogen ion concentrations were significantly decreased (∝ 0.10) compared with concentrations before the sand application to Bear Run but not on the control stream. No fish were present upstream of the sand application site, and only a few fish were found downstream at the mouth of Bear Run. Standing crop, number of taxa, and Shannon‐Weaver diversity index values indicated that macroinvertebrate populations were negatively impacted 300 m downstream from the sand application and improved at the site 1,600 m downstream. Although water quality was improved on Bear Run, benefits to macroinvertebrates depended on downstream location, and fish populations did not show improvement.     

Effect of bacterial extracellular polymers on the saturated hydraulic conductivity of sand columns.

Columns were packed with clean quartz sand, sterilized, and inoculated with different strains of bacteria, which multiplied within the sand at the expense of a continuous supply of fresh nutrient medium. The saturated hydraulic conductivity (HCsat) of the sand was monitored over time. Among the four bacterial strains tested, one formed a capsule, one produced slime layers, and two did not produce any detectable exopolymers. The last two strains were nonmucoid variants of the first two. Only one strain, the slime producer, had a large impact on the HCsat. The production of exopolymers had no effect on either cell multiplication within or movement through the sand columns. Therefore, the HCsat reduction observed with the slime producer was tentatively attributed to the obstruction of flow channels with slime. Compared with the results with Arthrobacter sp. strain AK19 used in a previous study, there was a 100-fold increase in detachment from the solid substratum and movement through the sand of the strains used in this study. All strains induced severe clogging when they colonized the inlet chamber of the columns. Under these conditions, the inlet end was covered by a confluent mat with an extremely low HCsat.     

Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus species

To obtain a restoring and protective calcite layer on degraded limestone, five different strains of the Bacillus sphaericus group and one strain of Bacillus lentus were tested for their ureolytic driven calcium carbonate precipitation. Although all the Bacillus strains were capable of depositing calcium carbonate, differences occurred in the amount of precipitated calcium carbonate on agar plate colonies. Seven parameters involved in the process were examined: calcite deposition on limestone cubes, pH increase, urea degrading capacity, extracellular polymeric substances (EPS)-production, biofilm formation, ζ-potential and deposition of dense crystal layers. The strain selection for optimal deposition of a dense CaCO₃ layer on limestone, was based on decrease in water absorption rate by treated limestone. Not all of the bacterial strains were effective in the restoration of deteriorated Euville limestone. The best calcite precipitating strains were characterised by high ureolytic efficiency, homogeneous calcite deposition on limestone cubes and a very negative ζ-potential.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.     

Sugarcane Molasses: A Cheap Carbon Source for Calcite Production in Different Class of Soils using Stimulation of Indigenous Urease-producing Bacteria

Abstract

In this research, we investigated the abilities of three different concentration of sugarcane molasses as a carbon source to stimulate indigenous bacterial growth in different classes of soil, namely poorly graded sand (SP), silty sand (SM), and clayey sand (SC) (according to the Unified classification system). A total of 7, 10, and 15 days after the treatment, direct shear tests were performed on the untreated and treated samples. The calcite content on all direct shear samples was determined to further correlate it with the strength gains in the treated samples. The scanning electron microscopy (SEM) images, EDX analysis, and X-ray diffraction (XRD) patterns were taken before and after treatment for all samples to analyze the microbial-induced calcite precipitation (MICP) process. The SP soil samples showed the highest strength gains and also highest calcite content as compared with other two soil type. The peak cohesion intercept for SP-treated samples increased by 2.7–5.5 times as compared to the untreated samples for molasses concentration of 1–3?g/L, respectively. The treated samples became more dilative with the increase in molasses concentration. The sample with highest molasses concentration showed stiffer behavior in shear than the samples with lower concentration.     

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Specificity of transposition of Tn7 in Vibrio parahaemolyticus

Tn7 was found to transpose at a high frequency from the plasmid, RP4::Tn7, to the chromosome of Vibrio parahaemolyticus. Seven isolates carrying Tn7 insertions were derived from three wild-type strains isolated from geographically distinct areas, and HindIII and BstEII DNA digests of these strains were probed with a ColE1::Tn7 biotinylated probe. The results indicated that V. parahaemolyticus is similar to several other species which have been studied in having a highly preferred site of insertion of Tn7 in the chromosome.