Kimberly L. Ogden

Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems

ABSTRACT Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4′,6′-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (≥20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.

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.

Efficient extraction method to collect sugar from sweet sorghum

BackgroundSweet sorghum is a domesticated grass containing a sugar-rich juice that can be readily utilized for ethanol production. Most of the sugar is stored inside the cells of the stalk tissue and can be difficult to release, a necessary step before conventional fermentation. While this crop holds much promise as an arid land sugar source for biofuel production, a number of challenges must be overcome. One lies in the inherent labile nature of the sugars in the stalks leading to a short usable storage time. Also, collection of sugars from the sweet sorghum stalks is usually accomplished by mechanical squeezing, but generally does not collect all of the available sugars.ResultsIn this paper, we present two methods that address these challenges for utilization of sweet sorghum for biofuel production. The first method demonstrates a means to store sweet sorghum stalks in the field under semi-arid conditions. The second provides an efficient water extraction method that can collect as much of the available sugar as feasible. Operating parameters investigated include temperature, stalk size, and solid–liquid ratio that impact both the rate of sugar release and the maximal amount recovered with a goal of low water use. The most desirable conditions include 30°C, 0.6 ratio of solid to liquid (w/w), which collects 90 % of the available sugar. Variations in extraction methods did not alter the efficiency of the eventual ethanol fermentation.ConclusionsThe water extraction method has the potential to be used for sugar extraction from both fresh sweet sorghum stalks and dried ones. When combined with current sugar extraction methods, the overall ethanol production efficiency would increase compared to current field practices.

A Comparison of Nannochloropsis salina Growth Performance in Two Outdoor Pond Designs: Conventional Raceways versus the ARID Pond with Superior Temperature Management

The present study examines how climatic conditions and pond design affect the growth performance of microalgae. From January to April of 2011, outdoor batch cultures of Nannochloropsis salina were grown in three replicate 780 L conventional raceways, as well as in an experimental 7500 L algae raceway integrated design (ARID) pond. The ARID culture system utilizes a series of 8–20 cm deep basins and a 1.5 m deep canal to enhance light exposure and mitigate temperature variations and extremes. The ARID culture reached the stationary phase 27 days earlier than the conventional raceways, which can be attributed to its superior temperature management and shallower basins. On a night when the air temperature dropped to −9°C, the water temperature was 18°C higher in the ARID pond than in the conventional raceways. Lipid and fatty acid content ranged from 16 to 25% and from 5 to15%, respectively, as a percentage of AFDW. Palmitic, palmitoleic, and eicosapentaenoic acids comprised the majority of fatty acids. While the ARID culture system achieved nearly double the volumetric productivity relative to the conventional raceways (0.023 versus 0.013 g L−1day−1), areal biomass productivities were of similar magnitude in both pond systems (3.47 versus 3.34 g m−2day−1), suggesting that the ARID pond design has to be further optimized, most likely by increasing the culture depth or operating at higher cell densities while maintaining adequate mixing.

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 106 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. Journal of Industrial Microbiology & Biotechnology (2002) 29, 75–82 doi:10.1038/sj.jim.7000273

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.

Statistical optimization of culture media for growth and lipid production of Chlorella protothecoides UTEX 250

The concentration of NaNO(3), MgSO(4) · 7H(2)O and proteose, in Chlorella protothecoides medium were optimized for algal biomass and lipid production by using response surface methodology with Box-Behnken design. The optimal concentrations were 0.45 g/L of NaNO(3), 6 mg/L of MgSO(4) · 7H(2)O, and 0.25 g/L of proteose for maximum biomass production and 2 mg/L of MgSO(4) · 7H(2)O and no addition of NaNO(3) and proteose for lipid accumulation. In optimized biomass production medium, a final biomass concentration of 1.19 g/L was obtained, which was 1.8 times higher than that in the original medium. For lipid accumulation, a 12.9% lipid content was obtained from the biomass in the lipid production medium, which was three times higher than that from the original medium. The fatty acid profile of algae grown in the optimized medium demonstrated a higher unsaturated fatty acid content (i.e. methyl linoleate (C18:2) and methyl linolenate (C18:3)) than that of the algae grown in the original medium. The results provide a strategy for limiting the amount of nutrients required in large scale outdoor cultivation systems of C. protothecoides to make the production of algal biomass more economically attractive.

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.

Cultivation of Nannochloropsis salina in municipal wastewater or digester centrate

Meaningful use of biofuels for transportation depends on utilization of water from non-traditional, non-potable resources. Here it is hypothesized that (i) reclaimed wastewater or nutrient-rich side streams derived from municipal wastewater treatment are suitable for that purpose and (ii) use of those waters for algal growth can promote water quality through nutrient management. Experiments showed that metals levels in municipal wastewaters are unlikely to inhibit algal growth and lipid production, at least by metals tolerant microalgae like Nannochloropsis salina. Cells grew without inhibition in treated municipal wastewater or centrate derived from wastewater treatment at additions up to 75 percent v/v in their normal growth medium minus nitrogen and phosphorus. Although wastewater provides a suitable nutrient source for algal growth, not enough municipal wastewater is available to support a meaningful biofuels industry without efficient water recycling and nutrient recovery/reuse from spent algae.

Effect of culture conditions on the growth rate and lipid production of microalgae Nannochloropsis gaditana

Microalgae Nannochloropsis gaditana (CCMP527) is an ideal candidate as a renewable and sustainable alternative oil source to traditional fossil fuel. It has advantages including high oil content, short growth cycle, and high biomass yield. Experimental data showed that with 5% CO2 enriched air and tris-HCl buffered f/2-Si medium, a maximum growth rate of 0.48 day−1 and a lipid yield of 0.32 g/l were achieved. Various carbon and nitrogen sources were investigated to determine the range of substrates that may be feasible for cultivation with minimal impact on productivity. The sources evaluated cover some of the compounds found in fertilizers and groundwater. When glucose was added into the medium, microalgae grew faster mixotrophically but the lipid yield was reduced by 86%. Adding high concentrations of nitrogen, 10 mM N in the form of nitrate, ammonium sulfate, glycine, and urea, resulted in variations in lipid yield from 0.02 g/l to 0.27 g/l. However, the growth rate increased slightly to 0.52 day−1 at ...