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.

Microbial strengthening of loose sand.

AIMS: To test whether the addition of Flavobacterium johnsoniae could increase the strength of saturated Ottawa 30 sand. METHODS AND RESULTS: A box model was built that simulates groundwater-like flow through a main sand compartment. Strength tests were performed at seven locations and at two depths, 10.8 and 20.3 cm below the top of the tank, using a vane shear device before and after the addition of bacteria. After the addition of Fl. johnsoniae, sand samples were obtained from multiple sampling ports on the vertical sides of the box model. The presence of a bacterial biofilm was confirmed by staining these sand samples with SYTO-9 and Alexa Fluor 633 and viewing with a confocal microscope. The average shear strength increases after the addition of Fl. johnsoniae were 15.2-87.5%, depending on the experimental conditions. CONCLUSIONS: Flavobacterium johnsoniae caused a statistically significant increase in the strength of saturated Ottawa 30 sand. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm-forming bacteria can increase the shear strength of saturated sand. The addition of biofilm-forming bacteria to a building site may be an alternate method to mitigate the effects of liquefaction.

Mutations in the alpha and sigma-70 subunits of RNA polymerase affect expression of the mer operon.

The mercury resistance (mer) operon is transcribed from overlapping, divergent promoters: PR for the regulatory gene merR and P(TPCAD) for the structural genes merTPCAD. The dyadic binding site for MerR lies within the 19-bp spacer of the sigma70-dependent P(TPCAD). Unlike typical repressors, MerR does not exclude RNA polymerase from P(TPCAD) but rather forms an inactive complex with RNA polymerase at P(TPCAD) prior to addition of the inducer, the mercuric ion Hg(II). In this "active repression" complex, MerR prevents transcriptional initiation at merTPCAD until Hg(II) is added. When Hg(II) is added, MerR remains bound to the same position and activates transcription of merTPCAD by distorting the DNA of the spacer region. MerR also represses its own transcription from PR regardless of the presence or absence of Hg(II). To explore the role of MerR-RNA polymerase in these processes, we examined mutations in the sigma70 and alpha subunits of RNA polymerase, mutations known to influence other activators but not to impair transcription generally. We assessed the effects of these sigma70 and alpha mutants on unregulated P(TPCAD) and PR transcription (i.e., MerR-independent transcription) and on the two MerR-dependent processes: repression of P(TPCAD) and of PR and Hg(ll)-induced activation of P(TPCAD). Among the MerR-independent effects, we found that mutations in regions 2.1 and 4.2 of rpoD suppress the deleterious effects of nonoptimal promoter spacing. Some C-terminal rpoA mutants also have this property to a considerably lesser degree. Certain "spacer suppressor" variants of rpoA and of rpoD also interfere with the MerR-dependent repression of P(TPCAD) and PR. MerR-Hg(II)-mediated transcriptional activation of P(TPCAD) was also affected in an allele-specific manner by substitutions at position 596 of sigma70 and at positions 311 and 323 of alpha. Thus, certain changes in sigma70 or alpha render them either more or less effective in participating in the topologically novel transcriptional control effected by MerR at the divergent mer operons.

High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector.

Conditions presently have been established for the high-level expression and simplified purification of recombinant human erythropoietin produced in Spodoptera frugiperda cells. Expression, as mediated by infection with a recombinant baculovirus, was accomplished in suspension culture using reduced levels of serum and media supplements experimentally determined to provide optimum levels of factor production (500,000 U/L). Purification of this recombinant human erythropoietin to virtual homogeneity (greater than or equal to 99%) was accomplished via a simple three-step procedure involving isocratic elution from DEAE-Sephacel, reverse-phase high performance liquid chromatography (HPLC) on a C4 medium, and the single-step elution of purified hormone from concanavalin A agarose. Overall, an 890-fold purification was accomplished with a recovery of 80% as assayed in vitro. Biologically, this purified erythropoietin is highly active, possessing a specific activity in vitro of 200,000 U/mg protein. Chemically, this erythropoietin (molecular weight [mol wt] 26,200) appears exceptionally uniform in its oligosaccharide constitution (30%) as contrasted with heterogeneously glycosylated erythropoietins derived from mammalian cells (mol wt 30,000 to 38,000; 40% to 50% complex-type oligosaccharide). Thus, human erythropoietin as presently produced in an insect cell line comprises not only an abundant source of highly active, readily purified hormone for studies of its mechanism of action and cell surface receptor, but also represents a uniquely homogeneous form that should prove advantageous for direct structural analyses.