Lactose mother liquor as an alternative nutrient source for microbial concrete production by <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis>

Microbiologically induced calcite precipitation by the bacterium Sporosarcina pasteurii (NCIM 2477) using the industrial effluent of the dairy industry, lactose mother liquor (LML) as growth medium was demonstrated for the first time in this study. The urease activity and the calcite precipitation by the bacterium was tested in LML and compared with the standard media like nutrient media and yeast extract media. Calcite constituted 24.0% of the total weight of the sand samples plugged by S. pasteurii and urease production was found to be 353 U/ml in LML medium. The compressive strength of cement mortar was increased by S. pasteurii in all the media used compared to control. No significant difference in the growth, urease production and compressive strength of mortar among the media suggesting LML as an alternative source for standard media. This study demonstrates that microbial calcite acts as a sealing agent for filling the gaps or cracks and fissures in constructed facilities and natural formations alike.     

Improved strength and durability of fly ash-amended concrete by microbial calcite precipitation

Fly ash acts as a partial replacement material for both Portland cement and fine aggregate. An innovative approach of microbial calcite precipitation in fly ash-amended concrete has been investigated. This is the first report to discuss the role of microbial calcite precipitation in enhancing the durability of fly ash-amended concrete. The present study investigated the effects of Bacillus megaterium ATCC 14581 on compressive strength, water absorption and water impermeability of fly ash-amended mortar and concrete. Mortar specimens were used for compressive strength and water absorption tests, while concrete specimens were used for water impermeability tests. At the fly ash concentrations of 10%, 20% and 40% in mortars, bacterial cell enhanced mortar compressive strength by 19%, 14% and 10%, respectively, compared to control specimens. Treated mortar cubes absorbed more than three times less water than control cubes as a result of microbial calcite deposition. Microbial deposition of a layer of calcite on the surface of the concrete specimens resulted in substantial decrease of water uptake and permeability compared to control specimens without bacteria. Microbial cells also prevented ingress of water effectively in different concentrations of fly ash-amended concrete. Scanning Electron Micrography (SEM) analyses evidenced the direct involvement of bacteria in calcite precipitation. The approach of the present study gives us dual environment friendly advantages. First, use of fly ash-a recovered resource reduces depletion of natural resources and also reduces the energy-intensive manufacturing of other concrete ingredients, leading to savings in both energy usage and emissions of greenhouse gases. And second, use of bacterial cells to improve strength and durability of fly ash-amended concrete further provides greener and economic options.     

Biocalcification using B. pasteurii for strengthening brick masonry civil engineering structures

Microbiologically induced calcite precipitation in bricks by bacterium Bacillus pasteurii (NCIM 2477) using a media especially optimized for urease production (OptU) was demonstrated in this study. Effect of biocalcification activity on compressive strength and water absorption capacity of bricks was investigated. Various other parameters such as pH, growth profile, urease activity, urea breakdown and calcite precipitated were monitored during the 28 days curing period. Efficiency of B. pasteurii to form microbial aided calcite precipitate in OptU media resulted into 83.9 % increase in strength of the bricks as compared to only 24.9 % with standard media, nutrient broth (NB). In addition to significant increase in the compressive strength, bricks treated with B. pasteurii grown in OptU media resulted in 48.9 % reduction in water absorption capacity as compared to control bricks immersed in tap water. Thus it was successfully demonstrated that microbial calcification in optimized media by Bacillus pasteurii has good potential for commercial application to improve the life span of structures constructed with bricks, particularly structures of heritage importance.     

Optimization of growth medium for <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis> in bio-based cement pastes to mitigate delay in hydration kinetics

Microbial-induced calcium carbonate precipitation has been identified as a novel method to improve durability and remediate cracks in concrete. One way to introduce microorganisms to concrete is by replacing the mixing water with a bacterial culture in nutrient medium. In the literature, yeast extract often has been used as a carbon source for this application; however, severe retardation of hydration kinetics has been observed when yeast extract is added to cement. This study investigates the suitability of alternative carbon sources to replace yeast extract for microbial-induced calcium carbonate precipitation in cement-based materials. A combination of meat extract and sodium acetate was identified as a suitable replacement in growth medium for Sporosarcina pasteurii; this alternative growth medium reduced retardation by 75 % (as compared to yeast extract) without compromising bacterial growth, urea hydrolysis, cell zeta potential, and ability to promote calcium carbonate formation.     

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.     

Production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. WN9 KCTC 8951P and its usefulness as a cement mortar admixture

The production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. and its suitability as a viscosity modifying admixture for cement mortar mixing were investigated. After 48 h culture in an optimized medium, cell growth and EPS production were 1,2 g/L and 4.0 g/L, respectively. By adding EPS WN9 to mortar, it was possible to prepare a homogeneous mortar without material segregation and excess air entrapment. The optimal amount of EPS addition to mortar was found to be 0.02 to 0.05%(w/w) of the cement used. Increasing the dosage of EPS WN9 from 0 to 0.05%(w/w) resulted in a setting retardation of 0.14 h to 0.8 h and an increase in the compressive strength of mortar of 10 to 20%.     

Accelerated biodegradation of cured cement paste by Thiobacillus species under simulation condition

Biodegradation is one of the most important types of cement deterioration. Complex microbial populations take part in the biodegradation process of cement-based materials. Studies in this field show that the sulfur-oxidizing bacteria, including Acidithiobacillus thiooxidans, due to sulfuric acid formation, play a key role in this process. In this study, with the accelerated leaching process of calcium hydroxide of cement paste, cured under running tap water and exposed to sterile biogenic sulfuric acid for 6 days, the surface pH of the cement was reduced to a more favorable level for bacterial growth. In this case, the growth of Thiobacillus proceeded in the presence of cured cement paste specimens. After 90 days of exposure to a semi-continuous culture of A. thiooxidans with its pH less than 2 and continuous removal of damaged layers the compressive strength, length and mass of the samples dropped by 96%, 11% and 43%, in the order given. The mechanism of degradation and the structure of degraded specimens were analyzed by test laboratory techniques such as, XRD, SEM and EDAX analyses.     

The development of a method to evaluate bioreceptivity of indoor mortar plastering to fungal growth

The aim of this work was to develop and standardise an accelerated laboratory test for detecting bioreceptivity of indoor mortar to fungal growth. To determine which fungal species were predominant under field conditions, isolation was carried out using mortar samples collected from 41 buildings in two cities of São Paulo State in the South East of Brazil. Cladosporium was found to be the genus most frequently recovered from field specimens. Based on the results of laboratory trials strain C. sphaerospermum was chosen as a test microorganism.Four different mortars, two laboratory-manufactured mortars composed of ordinary Portland cement, high calcium hydrated lime and standardised sand, and two different ready-mixed building mortars from the Brazilian market, were investigated for their susceptibility to colonisation by C. sphaerospermum. Several parameters were tested to determine factors influencing fungal bioreceptivity. The type of mortar, degree of carbonation and pH values of mortars, as well as relative humidity of environment effected colonisation of C. sphaerospermum. All except one mortar samples showed significant fungal growth, however, the growth occurred only at 100% relative humidity. Interaction of C. sphaerospermum with mortar specimens was studied using techniques of scanning and environmental scanning electron microscopy combined with energy dispersive X-ray analysis.     

In memoriam

Microcosm experiments were performed to identify the influence of bacterial cell surfaces on the morphology, mineralogy, size and solubility of CaCO₃ precipitated in response to the enzymatic hydrolysis of urea in an artificial groundwater (AGW) by the ureolytic bacteria, Bacillus pasteurii. In each microcosm, B. pasteurii were contained within a cellulose dialysis membrane (10 K Dalton MWCO), resulting in bacteria-inclusive and bacteria-free AGW solution. Urea hydrolysis by B. pasteurii resulted in the production of ammonium and an increase in pH in the whole AGW solution. This initiated predominantly rhombohedral calcite precipitation at the same critical saturation state ( S _critical = 12) in the B. pasteurii-inclusive and bacteria-free zone of the AGW, indicating the mineralogy and morphology of CaCO₃ precipitation is not controlled by B. pasteurii surfaces. However, the temporal evolution of distinctly different lognormal crystal-size-distributions in the B. pasteurii-inclusive and bacteria-free zone of the AGW resulted from identical changes in bulk solution chemistry. Specifically, B. pasteurii increased the size and size variance of crystals, and led to a greater crystal growth rate throughout the experiments, relative to bacteria-free AGW. Calculated crystal solubility (ln K _S0 ) was lower for crystals > 4000 nm in diameter, reflecting smaller molar surface areas. This suggests that the larger crystals generated in the presence of B. pasteurii have a lower affinity for re-dissolution than those generated in the bacteria-free AGW, which may act as a positive feedback to maintain larger crystal sizes in the presence of B. pasteurii. During ureolysis, higher bacterial concentrations may therefore generate larger and less soluble carbonate crystals. This has important implications for the adaptation of bacterial ureolysis as a method for precipitating calcium carbonate and co-precipitating metals and radionuclides in contaminated aquifers.     

Application of alkaliphilic biofilm-forming bacteria to improve compressive strength of cement-sand mortar

The application of microorganisms in the field of construction material is rapidly increasing worldwide; however, almost all studies that were investigated were bacterial sources with mineral-producing activity and not with organic substances. The difference in the efficiency of using bacteria as an organic agent is that it could improve the durability of cement material. This study aimed to assess the use of biofilm-forming microorganisms as binding agents to increase the compressive strength of cement-sand material. We isolated 13 alkaliphilic biofilmforming bacteria (ABB) from a cement tetrapod block in the West Sea, Korea. Using 16S RNA sequence analysis, the ABB were partially identified as Bacillus algicola KNUC501 and Exiguobacterium marinum KNUC513. KNUC513 was selected for further study following analysis of pH and biofilm formation. Cement-sand mortar cubes containing KNUC513 exhibited greater compressive strength than mineral-forming bacteria (Sporosarcina pasteurii and Arthrobacter crystallopoietes KNUC403). To determine the biofilm effect, Dnase I was used to suppress the biofilm formation of KNUC513. Field emission scanning electron microscopy image revealed the direct involvement of organic-inorganic substance in cement-sand mortar.     

Effect of calcifying bacteria on permeation properties of concrete structures

Microbially enhanced calcite precipitation on concrete or mortar has become an important area of research regarding construction materials. This study examined the effect of calcite precipitation induced by Sporosarcina pasteurii (Bp M-3) on parameters affecting the durability of concrete or mortar. An inexpensive industrial waste, corn steep liquor (CSL), from starch industry was used as nutrient source for the growth of bacteria and calcite production, and the results obtained with CSL were compared with those of the standard commercial medium. Bacterial deposition of a layer of calcite on the surface of the specimens resulted in substantial decrease of water uptake, permeability, and chloride penetration compared with control specimens without bacteria. The results obtained with CSL medium were comparable to those obtained with standard medium, indicating the economization of the biocalcification process. The results suggest that calcifying bacteria play an important role in enhancing the durability of concrete structures.     

Assessment of a Biostimulated or Bioaugmented Calcification System with Bacillus pasteurii in a Simulated Soil Environment

To evaluate how native soil microorganism and nutrients interact in a bioaugmented or biostimulated calcification process, batch experiments were conducted in saturated soil extract either amended with Bacillus pasteurii and/or NB-NH₄Cl media (nutrient broth, 3.0 g/L; NH₄Cl, 10.0 g/L; NaHCO₃, 2.12 g/L). The NB-NH₄Cl medium was chosen out of three potential candidates, where B. pasteurii precipitated the maximum amount of calcium (>95 %) as calcite in 12 h of incubation. The addition of B. pasteurii into saturated soil extract marginally improved the calcification to 4.26 % compared to the unamended control (3.2 %). Calcification with B. pasteurii in autoclaved soil extract was even better (18.75 %). However, the addition of the NB-NH₄Cl medium into the soil extract could significantly improve the calcification irrespective of whether B. pasteurii was added (88.46 %) or not (77.04 %). It suggests that soil microbial activity was not limiting even though soil nutrient was inadequate. It also indicates a possible negative interaction between soil microorganisms and B. pasteurii under nutrient-limited conditions.     

Development of bioconcrete material using an enrichment culture of novel thermophilic anaerobic bacteria

In the biosphere, bacteria can function as geo-chemical agents, promoting the dispersion, fractionation and/or concentration of materials. Microbial mineral precipitation is resulted from metabolic activities of microorganisms. Based on this biomineralogy concept, an attempt has been made to develop bioconcrete material incorporating of an enrichment culture of thermophilic and anaerobic bacteria within cement-sand mortar/concrete. The results showed a significant increase in compressive strength of both cement-sand mortar and concrete due to the development of filler material within the pores of cement sand matrix. Maximum strength was observed at concentration 10(5)cell/ml of water used in mortar/concrete. Addition of Escherichia coil or media composition on mortar showed no such improvement in strength.     

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.     

Production and Partial Purification of Antibiotic Materials Formed by Physarum gyrosum

Pure cultures of Physarum gyrosum were grown on agar plates with autoclaved Escherichia coli suspensions as the growth medium. Portions of such agar, after growth of the slime mold, contained diffusible materials that inhibited the growth of Bacillus subtilis, B. cereus, E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Paper chromatography of extracts of such cultures revealed at least three different active fractions. Preliminary fractionations increased the specific activity by one order of magnitude, probably in part by removal of inactive material and in part by separating active components. The fractionations also demonstrated the multiplicity of the antibiotic activity. Fractions variously obtained always retarded the growth of the bacterial species listed above.     

Antibacterial protection in Marthasterias glacialis eggs: characterization of lysozyme-like activity

Eggs from Marthastherias glacialis exert antibacterial action on marine bacterial strains and show a lysozyme-like activity. This one depends on pH and ionic strength of sample and reacting medium. This hydrolase, purified by gel filtration and ion-exchange chromatography, could be responsible for the bacterial growth inhibitory activity observed.     

Development of Bacterial Contamination during Production of Yeast Extracts

Baker’s yeast suspensions having bacterial populations of 10⁶ and 10⁸ CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.     

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.     

Symbiosis of Arbuscular Mycorrhizal Fungi and Robinia pseudoacacia L. Improves Root Tensile Strength and Soil Aggregate Stability

Robinia pseudoacacia L. (black locust) is a widely planted tree species on Loess Plateau for revegetation. Due to its symbiosis forming capability with arbuscular mycorrhizal (AM) fungi, we explored the influence of arbuscular mycorrhizal fungi on plant biomass, root morphology, root tensile strength and soil aggregate stability in a pot experiment. We inoculated R. pseudoacacia with/without AM fungus (Rhizophagus irregularis or Glomus versiforme), and measured root colonization, plant growth, root morphological characters, root tensile force and tensile strength, and parameters for soil aggregate stability at twelve weeks after inoculation. AM fungi colonized more than 70% plant root, significantly improved plant growth. Meanwhile, AM fungi elevated root morphological parameters, root tensile force, root tensile strength, Glomalin-related soil protein (GRSP) content in soil, and parameters for soil aggregate stability such as water stable aggregate (WSA), mean weight diameter (MWD) and geometric mean diameter (GMD). Root length was highly correlated with WSA, MWD and GMD, while hyphae length was highly correlated with GRSP content. The improved R. pseudoacacia growth, root tensile strength and soil aggregate stability indicated that AM fungi could accelerate soil fixation and stabilization with R. pseudoacacia, and its function in revegetation on Loess Plateau deserves more attention.     

Effect of abscisic acid and cytokinins on cultured zygotic embryos of Coffea arabicacv.Cauvery (Catimor)

Immature zygotic embryos of Coffea arabica L.cv.Cauvery (Catimor) were sequentially cultured on different modifications of Murashige and Skoog's (MS) medium to test the effect of abscisic acid and cytokinins. The type of response depended on the medium strength, the growth regulator combinations as well as the period of initial culture in both abscisic acid or cytokinin supplemented media. Increasing concentration of abscisic acid from 0.4 to 18.9 μM enhanced the quiescence of the zygotic embryos. All the cytokinins promoted germination but Kinetin and isopentenyladenine (2-IP) were less effective than benzyl amino purine (BAP). The maximum mature embryos were obtained when immature embryos were cultured initially for 30 days on full strength MS medium with 3.8 μM ABA, followed by 60 days on half strength MS medium with 0.1 μM BAP and finally on half strength MS media with out growth regulator for next 60 days.