Synergistic effects of citric acid and polyethyleneimine to remove copper from aqueous solutions.

Citric acid and copper are often found in the waste streams from semiconductor manufacturing. They are likely to form complexes, which modify copper speciation. This can lead to changes in sorption mechanisms and the sorption capacity. PEI-agarose adsorbents in a packed bed column are capable of removing these anionic complexes, but the competitive binding between these organic ligands and PEI for copper is not well understood and needs to be explored. The current work focuses on investigating copper sorption by PEI-agarose adsorbent in the presence of citrate ions. Copper binding capacity and copper breakthrough curves are compared and contrasted to results without additional chelator present. The presence of citric acid at the molar ratios of 0.5, 1, and 2 to copper enhances the total copper uptake in a continuous column by 175%, 100% and 75%, respectively. This is a great advantage when wastewater streams contain either low or high amounts of citric acid ligand.

Survival and nutritional requirements of three bacteria isolated from ultrapure water.

Bacteria isolated previously from ultrapure water (UPW) systems were examined for their ability to survive in UPW, with the ultimate goal of elucidating potential carbon and energy sources for the bacteria. Two strains of Ralstonia pickettii isolated from different areas within the UPW system (pretreatment and polishing loop, and referred to as strains 3A1 and MF254A, respectively) and a strain of Bradyrhizobium sp. were compared to increase our understanding of the fundamental behavior of bacteria contaminating UPW. R. pickettii (3A1) grew significantly slower in R2A medium, with a final cell yield much lower than the isolate from the polishing loop. In addition, R. pickettii MF254A showed a broader substrate range than either strain 3A1 or Bradyrhizobium sp. In UPW, there appears to be a threshold cell concentration (approximately 10(6) colony-forming units/ml), whereby the cell numbers remain constant for a prolonged period of 6 months or more. Below this concentration, rapid proliferation is observed until the threshold concentration is attained. Preliminary experiments suggested that nitrogen gas (frequently added to UPW storage tanks) may contribute to growth of Bradyrhizobium sp. Above the threshold concentration, the strain of Ralstonia sp. isolated from the polishing loop was capable of cryptic growth with heat-killed cells in UPW. However, cryptic growth was not observed when the cells supplied as nutrients were killed using UV254 light. Furthermore, cryptic growth did not appear to contribute significantly to proliferation of Bradyrhizobium sp. or Ralstonia sp. 3A1 (isolated from the pretreatment loop). We believe that cryptic growth may aid survival of the bacteria in UPW, but further experiments are warranted to prove this phenomenon conclusively.

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a prospective consortium and its most effective isolate Serratia marcescens.

The biotransformation of hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) has been observed in liquid culture by a consortium of bacteria found in horse manure. Five types of bacteria were found to predominate in the consortium and were isolated. The most effective of these isolates at transforming RDX was Serratia marcescens. The biotransformation of RDX by all of these bacteria was found to occur only in the anoxic stationary phase. The process of bacterial growth and RDX biotransformation was quantified for the purpose of developing a predictive type model. Cell growth was assumed to follow Monod kinetics. All of the aerobic and anoxic growth parameters were determined: micro(max), K(s), and Y(x/s). RDX was found to competitively inhibit cell growth in both atmospheres. Degradation of RDX by Serratia marcescens was found to proceed through the stepwise reduction of the three nitro groups to nitroso groups. Each of these reductions was found to be first order in both component and cell concentrations. The degradation rate constant for the first step in this reduction process by the consortium was 0.022 L/g cells . h compared to 0.033 L/g cells . h for the most efficient isolate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 515-522, 1997.

Biological breakdown of RDX in slurry reactors proceeds with multiple kinetically distinguishable paths.

Biotransformation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) in slurry reactors was studied to determine the importance of supplementation of known biodegraders and the type of nutrient source required. Although addition of bacteria to the system increased the biotransformation rates, the increase may not justify the additional work and cost needed to grow the organisms in a laboratory and mix them into the soil. An inexpensive, rich nutrient source, corn steep liquor, was shown to provide sufficient nutrients to allow for the cometabolic biotransformation of RDX. The rate of RDX transformation was not constant throughout the course of the experiment due to the heterogeneous microbial population. Three kinetically distinct phases were observed. Regardless of the process, RDX biotransformation in slurry reactors was reaction rate limited under the test conditions. Model simulations based on experimental results demonstrate that, at cell densities of 5 g/L, bioremediation of RDX-contaminated soil is an attractive clean-up alternative.

An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics.

Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth-rate differential between plasmid-bearing and plasmid-free cells, and thereby affects the culture dynamics of a plasmid-containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl transferase (CAT). Experimental and theoretical results show that plasmid-bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild-type levels, and thus have more of a metabolic burden than do the plasmid-bearing cells that overproduce CAT. However, when the level of the plasmid-directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid-bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid-containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments.

Genetic control of flocculation in Escherichia coli.

Escherichia coli cells form flocs or aggregates by overproducing type 1 pili. When the pil operon is placed under the control of a tac or lac promoter-operator sequence, the bacterial cells can be induced to form flocs by adding isopropyl-beta-D-thiogalactopyranoside to the culture medium. This phenomenon of genetically induced flocculation can aid in the downstream of biological products. This paper describes the construction of two artificially controlled plasmids which cause cell flocculation. Cell aggregates 50 microns in mean diameter were obtained 1 h after the cells were induced.

Plasmid maintenance and protein overproduction in selective recycle bioreactors.

A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein beta-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions.