Expression and secretion of active Moringa oleifera coagulant protein in Bacillus subtilis.

Cationic polypeptide proteins found in the seeds of the tropical plant Moringa oleifera have coagulation efficiencies similar to aluminum and ferric sulfates without their recalcitrant nature. Although these proteins possess great potential to augment or replace traditional coagulants in water treatment, harvesting active protein from seeds is laborious and not cost-effective. Here, we describe an alternative method to express and secrete active M. oleifera coagulant protein (MO) in Bacillus subtilis. A plasmid library containing the MO gene and 173 different types of secretory signal peptides was created and cloned into B. subtilis strain RIK1285. Fourteen of 440 clones screened were capable of secreting MO with yields ranging from 55 to 122 mg/L of growth medium. The coagulant activity of the highest MO secreting clone was evaluated when grown on Luria broth, and cell-free medium from the culture was shown to reduce turbidity in a buffered kaolin suspension by approximately 90% compared with controls without the MO gene. The clone was also capable of secreting active MO when grown on a defined synthetic wastewater supplemented with 0.5% tryptone. Cell-free medium from the strain harboring the MO gene demonstrated more than a 2-fold reduction in turbidity compared with controls. Additionally, no significant amount of MO was observed without the addition of the synthetic wastewater, suggesting that it served as a source of nutrients for the effective expression and translocation of MO into the medium.

A Versatile Strategy for Characterization and Imaging of Drip Flow Microbial Biofilms.

The inherent architectural and chemical complexities of microbial biofilms mask our understanding of how these communities form, survive, propagate, and influence their surrounding environment. Here we describe a simple and versatile workflow for the cultivation and characterization of model flow-cell-based microbial ecosystems. A customized low-shear drip flow reactor was designed and employed to cultivate single and coculture flow-cell biofilms at the air-liquid interface of several metal surfaces. Pseudomonas putida F1 and Shewanella oneidensis MR-1 were selected as model organisms for this study. The utility and versatility of this platform was demonstrated via the application of several chemical and morphological imaging techniques-including matrix-assisted laser desorption/ionization mass spectrometry imaging, secondary ion mass spectrometry imaging, and scanning electron microscopy-and through the examination of model systems grown on iron substrates of varying compositions. Implementation of these techniques in combination with tandem mass spectrometry and a two-step imaging principal component analysis strategy resulted in the identification and characterization of 23 lipids and 3 oligosaccharides in P. putida F1 biofilms, the discovery of interaction-specific analytes, and the observation of several variations in cell and substrate morphology present during microbially influenced corrosion. The presented workflow is well-suited for examination of both single and multispecies drip flow biofilms and offers a platform for fundamental inquiries into biofilm formation, microbe-microbe interactions, and microbially influenced corrosion.

Perchlorate reduction using free and encapsulated Azospira oryzae enzymes.

Existing methods for perchlorate remediation are hampered by the common co-occurrence of nitrate, which is structurally similar and a preferred electron acceptor. In this work, the potential for perchlorate removal using cell-free bacterial enzymes as biocatalysts was investigated using crude cell lysates and soluble protein fractions of Azospira oryzae PS, as well as soluble protein fractions encapsulated in lipid and polymer vesicles. The crude lysates showed activities between 41 700 to 54 400 U L(-1) (2.49 to 3.06 U mg(-1) total protein). Soluble protein fractions had activities of 15 400 to 29 900 U L(-1) (1.70 to 1.97 U mg(-1)) and still retained an average of 58.2% of their original activity after 23 days of storage at 4 °C under aerobic conditions. Perchlorate was removed by the soluble protein fraction at higher rates than nitrate. Importantly, perchlorate reduction occurred even in the presence of 500-fold excess nitrate. The soluble protein fraction retained its function after encapsulation in lipid or polymer vesicles, with activities of 13.8 to 70.7 U L(-1), in agreement with theoretical calculations accounting for the volume limitation of the vesicles. Further, encapsulation mitigated enzyme inactivation by proteinase K. Enzyme-based technologies could prove effective at perchlorate removal from water cocontaminated with nitrate or sulfate.

Enzyme-mediated assimilation of DNA-functionalized single-walled carbon nanotubes.

When pyrimidine-functionalized carbon nanotubes were incubated with single-stranded DNA ligase, formations of macroscopic aggregates were observed. Wet-cell transmission electron microscopy imaging revealed that the nanotubes were radially bound to form a 3D latticelike structure. These structures were not observed in control reactions lacking ligase or adenosine triphosphate. Raman spectroscopy analysis revealed no spectra indicative of carbon nanotubes in ligase-unamended controls; however, spectra were observed in radial breathing mode and in the G and G' bands in reactions containing ligase. Furthermore, the addition of deoxyribonuclease to the ligated reactions dispersed the aggregates, and a reduction in Raman spectral intensity was observed.

Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a Desulfovibrio species under anaerobic conditions.

Past handling practices associated with the manufacturing and processing of the high explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has resulted in extensive environmental contamination. In-situ biodegradation is a promising technology for remediating RDX contaminated sites but often relies on the addition of a cosubstrate. A sulfate-reducing bacterium isolated from an RDX-degrading enrichment culture was studied for its ability to grow on RDX as a sole source of carbon and nitrogen and for its ability to mineralize RDX in the absence of a cosubstrate. The results showed the isolate degraded 140 muM RDX in 63 days when grown on RDX as a carbon source. Biomass within the carbon limited culture increased 9-fold compared to the RDX unamended controls. When the isolate was incubated with RDX as sole source of nitrogen it degraded 160 muM RDX in 41 days and exhibited a 4-fold increase in biomass compared to RDX unamended controls. Radiolabeled studies under carbon limiting conditions with (14)C-hexahydro-1,3,5-trinitro-1,3,5-triazine confirmed mineralization of the cyclic nitramine. After 60 days incubation 26% of the radiolabel was recovered as (14)CO(2), while in the control bottles less than 1% of the radiolabel was recovered as (14)CO(2). Additionally, approximately 2% of the radiolabeled carbon was found to be associated with the biomass. The 16S rDNA gene was sequenced and identified the isolate as a novel species of Desulfovibrio, having a 95.1% sequence similarity to Desulfovibrio desulfuricans. This is the first known anaerobic bacterium capable of mineralizing RDX when using it as a carbon and energy source for growth.

Analysis of bacterial community diversity in anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) using 16S rRNA gene clone libraries.

Clone libraries were used to evaluate the effects of 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) on bacterial populations within three anaerobic bioreactors. Prior to the addition of DNAN and MNA greater than 69% of the clones in each reactor were identified as a single Desulfuromonales species. However, after 60 days of treatment the Desulfuromonales distribution decreased to less than 13% of the distribution and a clone identified as a Levilinea sp. became the dominant organism at greater than 27% of the clone distribution in each reactor suggesting the species may play an important roll in the reduction of DNAN and MNA.

Anaerobic biotransformation of explosives in aquifer slurries amended with ethanol and propylene glycol.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoluene (TNT) are explosives that are frequently found as environmental contaminants on military installations. Hydrogen has been shown to support the anaerobic transformation of these explosives. We investigated ethanol and propylene glycol as electron donors for providing syntrophically produced H2 for stimulating the anaerobic biodegradation of explosives in contaminated soil. The study was conducted using anoxic microcosms constructed with slurries of the contaminated soil and groundwater. The addition of 5mM ethanol and propylene glycol enhanced the biodegradation of RDX and HMX relative to the control bottles. Ethanol was depleted within about 20 days, resulting in the transient formation of hydrogen, acetate, and methane. The hydrogen headspace concentration increased from 8 ppm to 1838 ppm before decreasing to background concentrations. Propylene glycol was completely degraded after 15 days, forming hydrogen, propionate, and acetate as end-products. The hydrogen headspace concentrations increased from 56 ppm to 628 ppm before decreasing to background concentrations. No methane formation was observed during the incubation period of 48 days. Our findings indicate the addition of ethanol and propylene to the aquifer slurries increased the hydrogen concentrations and enhanced the biotransformation of RDX and HMX in the explosive-contaminated soil.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) serves as a carbon and energy source for a mixed culture under anaerobic conditions.

We studied the anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a mineral medium by a mixed culture. RDX degradation activity was maintained for more than a year with only the addition of RDX. We observed a steady increase in the protein concentration of the culture from 4.8 microg mL(-1) to more than 24.4 microg mL(-1), a >400% increase. There was only a slight increase in protein in the RDX unamended control bottles containing live culture, increasing from 4.8 microg mL(-1) to 7.8 microg mL(-1). Radiolabeled (14)C-RDX confirmed mineralization of the cyclic nitramine to (14)CO(2). After 164 days, 35% of the radiolabel was recovered as 14CO2. This is the first report demonstrating the mineralization of RDX when it serves as a growth substrate for a mixed culture.

Anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Acetobacterium malicum strain HAAP-1 isolated from a methanogenic mixed culture.

In previous work, we studied the anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a methanogenic mixed culture that biodegrades RDX by using H2 as the sole electron donor. Strain HAAP-1 was isolated after enriching for the homoacetogens in a mineral medium containing RDX and an H2-CO2 (80:20) headspace. Strain HAAP-1 degraded 29.0 microM RDX in <14 days and formed 13.0 mM acetate when grown in a mineral medium with an H2-CO2 headspace. Methylenedinitramine was observed as a transient intermediate, indicating ring cleavage had occurred. In live cultures containing an N2-CO2 headspace, RDX was not degraded, and no acetate was formed. The 16S rRNA gene sequence for strain HAAP-1, consisting of 1485 base pairs, had a 99.2% and 99.1% sequence similarity to Acetobacterium malicum and A. wieringae, respectively. This is the first report of RDX degradation by a homoacetogen growing autotrophically and extends the number of genera known to carry out this transformation.

Stimulating the anaerobic biodegradation of explosives by the addition of hydrogen or electron donors that produce hydrogen.

The anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoluene (TNT) by a methanogenic mixed culture was investigated. Microcosms containing a basal medium and the mixed culture were amended with ethanol, propylene glycol (PG), butyrate or hydrogen gas as the electron donor and a mixture of TNT (50 microM), RDX (25 microM), and HMX (8 microM). After 29 days TNT and RDX were completely transformed to unidentified endproducts in the bottles amended with ethanol, hydrogen, or PG, while 53%, 40%, and 22% of the HMX was transformed, respectively. There was no loss of RDX or HMX in the electron donor unamended control bottles. The ethanol and PG were transformed to near stoichiometric amounts of acetate and propionate, suggesting the immediate electron donor supporting the transformation of the explosives was the H2 evolved during the metabolism of the parent substrate. Our findings suggest that the addition of H2 or electron donors that produce H2 may be a useful strategy for enhancing the anaerobic biodegradation of explosives in contaminated groundwater and soils.