William A. Apel

Toxic Effects of Chromium(VI) on Anaerobic and Aerobic Growth of Shewanella oneidensis MR‐1

Cr(VI) was added to early‐ and mid‐log‐phase Shewanella oneidensis ( S. oneidensis) MR‐1 cultures to study the physiological state‐dependent toxicity of Cr(VI). Cr(VI) reduction and culture growth were measured during and after Cr(VI) reduction. Inhibition of growth was observed when Cr(VI) was added to cultures of MR‐1 growing aerobically or anaerobically with fumarate as the terminal electron acceptor. Under anaerobic conditions, there was immediate cessation of growth upon addition of Cr(VI) in early‐ and mid‐log‐phase cultures. However, once Cr(VI) was reduced below detection limits (0.002 mM), the cultures resumed growth with normal cell yield values observed. In contrast to anaerobic MR‐1 cultures, addition of Cr(VI) to aerobically growing cultures resulted in a gradual decrease of the growth rate. In addition, under aerobic conditions, lower cell yields were also observed with Cr(VI)‐treated cultures when compared to cultures that were not exposed to Cr(VI). Differences in response to Cr(VI) between aerobically and anaerobically growing cultures indicate that Cr(VI) toxicity in MR‐1 is dependent on the physiological growth condition of the culture. Cr(VI) reduction has been previously studied in Shewanella spp., and it has been proposed that Shewanella spp.may be used in Cr(VI) bioremediation systems. Studies of Shewanella spp. provide valuable information on the microbial physiology of dissimilatory metal reducing bacteria; however, our study indicates that S. oneidensis MR‐1 is highly susceptible to growth inhibition by Cr(VI) toxicity, even at low concentrations [0.015 mM Cr(VI)].

Multiple Mechanisms of Uranium Immobilization by Cellulomonas sp. strain ES6

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram‐positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non‐growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate‐based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X‐ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non‐uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)‐phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U‐free controls suggesting simultaneous precipitation of U and PO  43− . In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High‐resolution transmission electron microscopy (HR‐TEM) and energy dispersive X‐ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone‐2,6‐disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non‐uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6. Biotechnol. Bioeng. 2011;108: 264–276.

Effect of Carbon and Energy Source on Bacterial Chromate Reduction

Studies were conducted to evaluate carbon and energy sources suitable to support hexavalent chromium (Cr(VI)) reduction by a bacterial consortium enriched from dichromate-contaminated aquifer sediments. The consortium was cultured under denitrifying conditions in a minimal, synthetic groundwater medium that was amended with various individual potential carbon and energy sources. The effects of these individual carbon and energy sources on Cr(VI) reduction and growth were measured. The consortium was found to readily reduce Cr(VI) with sucrose, acetate, L-asparagine, hydrogen plus carbon dioxide, ethanol, glycerol, glycolate, propylene glycol, or D-xylose as a carbon and energy source. Minimal Cr(VI) reduction was observed when the consortium was cultured with citrate, 2-ketoglutarate, L-lactate, pyruvate, succinate, or thiosulfate plus carbon dioxide as a carbon and energy source when compared with abiotic controls. The consortium grew on all of the above carbon and energy sources, with the highest cell densities reached using D-xylose and sucrose, demonstrating that the consortium is metabolically diverse and can reduce Cr(VI) using a variety of different carbon and energy sources. The results suggest that the potential exists for the enrichment of Cr(VI)-reducing microbial populations in situ by the addition of a sucrose-containing feedstock such as molasses, which is an economical and readily available carbon and energy source.

Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth

Cr(VI) reduction was observed during tests with Shewanella oneidensis MR‐1 (previously named S. putrefaciens MR‐1) while being grown with nitrate or fumarate as electron acceptor and lactate as electron donor. From the onset of anoxic growth on fumarate, we measured a gradual and progressive increase in the specific Cr(VI) reduction rate with incubation time until a maximum was reached at late exponential/early stationary phase. Under denitrifying conditions, the specific Cr(VI) reduction rate was inhibited by nitrite, which is produced during nitrate reduction. However, once nitrite was consumed, the specific reduction rate increased until a maximum was reached, again during the late exponential/early stationary phase. Thus, under both fumarate‐ and nitrate‐reducing conditions, an increase in the specific Cr(VI) reduction rate was observed as the microorganisms transition from oxic to anoxic growth conditions, presumably as a result of induction of enzyme systems capable of reducing Cr(VI). Although Cr(VI) reduction has been studied in MR‐1 and in other facultative bacteria under both oxic and anoxic conditions, a transition in specific reduction rates based on physiological conditions during growth is a novel finding. Such physiological responses provide information required for optimizing the operation of in situ systems for remediating groundwater contaminated with heavy metals and radionuclides, especially those that are characterized by temporal variations in oxygen content. Moreover, such information may point the way to a better understanding of the cellular processes used by soil bacteria to accomplish Cr(VI) reduction.

Low temperature reduction of hexavalent chromium by a microbial enrichment consortium and a novel strain of Arthrobacter aurescens

BackgroundChromium is a transition metal most commonly found in the environment in its trivalent [Cr(III)] and hexavalent [Cr(VI)] forms. The EPA maximum total chromium contaminant level for drinking water is 0.1 mg/l (0.1 ppm). Many water sources, especially underground sources, are at low temperatures (less than or equal to 15 Centigrade) year round. It is important to evaluate the possibility of microbial remediation of Cr(VI) contamination using microorganisms adapted to these low temperatures (psychrophiles).ResultsCore samples obtained from a Cr(VI) contaminated aquifer at the Hanford facility in Washington were enriched in Vogel Bonner medium at 10 Centigrade with 0, 25, 50, 100, 200, 400 and 1000 mg/l Cr(VI). The extent of Cr(VI) reduction was evaluated using the diphenyl carbazide assay. Resistance to Cr(VI) up to and including 1000 mg/l Cr(VI) was observed in the consortium experiments. Reduction was slow or not observed at and above 100 mg/l Cr(VI) using the enrichment consortium. Average time to complete reduction of Cr(VI) in the 30 and 60 mg/l Cr(VI) cultures of the consortium was 8 and 17 days, respectively at 10 Centigrade. Lyophilized consortium cells did not demonstrate adsorption of Cr(VI) over a 24 hour period. Successful isolation of a Cr(VI) reducing organism (designated P4) from the consortium was confirmed by 16S rDNA amplification and sequencing. Average time to complete reduction of Cr(VI) at 10 Centigrade in the 25 and 50 mg/l Cr(VI) cultures of the isolate P4 was 3 and 5 days, respectively. The 16S rDNA sequence from isolate P4 identified this organism as a strain of Arthrobacter aurescens, a species that has not previously been shown to be capable of low temperature Cr(VI) reduction.ConclusionA. aurescens, indigenous to the subsurface, has the potential to be a predominant metal reducer in enhanced, in situ subsurface bioremediation efforts involving Cr(VI) and possibly other heavy metals and radionuclides.

Removal of nitrogen oxides from gas streams using biofiltration

Abstract Nitrogen oxides (NOx) are primary air pollutants, and as such, there is considerable interest in the development of efficient, cost effective technologies to remediate NOx containing emissions. Biofiltration involves the venting of contaminated gas streams through biologically active material such as soil or compost. This technology has been used successfully to control odors as well as volatile organic compounds from a variety of industrial and public sources. The purpose of this study was to evaluate the feasibility of using biofiltration as a means to remediate NOx containing gas streams. Biofiltration studies measuring nitric oxide (NO) removal by bacteria indigenous to wood compost were conducted. Vertical biofilters (21 volume) constructed from glass process pipe (3 in i.d. × 12 in) were loaded with 11 of compost bed medium. Compaction of compost in the biofilters was minimized by the addition of wood chips (15% w/w). A nitrogen gas stream, containing various concentrations of NO (100–500 μl/l), was purged (1 l min−1) through the biofilter under single pass, continuous flow conditions. Adsorption studies comparing NO removal in autoclaved and non-autoclaved biofilters indicated that approximately 3% NO removal was attributed to abiotic uptake. Control of pH in the biofilter was a critical variable for maximum nitric oxide removal. Optimum denitrifying activity occurred at pH levels ranging between 6 and 7. Nitric oxide removal rates increased in biofilters treated with an external carbon and energy source. Biofilters treated with phosphate buffer containing either lactate or dextrose were capable of removing more than 90% of the NO from a 500 μl/l NO gas stream flowing at 1 l min−1.

Bacterial reduction of hexavalent chromium by Enterobacter cloacae strain HO1 grown on sucrose

Chromium(VI) was reduced by Enterobacter cloacae strain HO1 grown with sucrose as a carbon source and nitrate as the initial terminal electron acceptor. Under excess substrate conditions, the Cr(VI) concentration, initially at 5 and 10 mg/l, was reduced to less than 100 mg/l.

Purification and characterization of a novel thermo-alkali-stable catalase from Thermus brockianus.

A novel thermo‐alkali‐stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three‐step procedure that resulted in 65‐fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS‐PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 °C and a pH range from 6 to 10, with optimum activity occurring at 90 °C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half‐lives of 330 h at 80 °C and 3 h at 90 °C. The enzyme also demonstrated excellent stability at 70 °C and alkaline pH with measured half‐lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6‐coordinate low spin state rather than the typical 5‐coordinate high spin state. A Km of 35.5 mM and a Vmax of 20.3 mM/min·mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3‐amino‐1,2,4‐triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o‐dianisidine and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase‐peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.

Screening of Cyanobacterial Species for Calcification

Species of cyanobacteria in the genera Synechococcus and Synechocystis are known to be the catalysts of a phenomenon called “whitings”, which is the formation and precipitation of fine‐grained CaCO3 particles. Whitings occur when the cyanobacteria fix atmospheric CO2 through the formation of CaCO3 on their cell surfaces, which leads to precipitation to the ocean floor and subsequent entombment in mud. Whitings represent one potential mechanism for CO2 sequestration. Research was performed to determine the ability of various strains of Synechocystis and Synechococcus to calcify when grown in microcosms amended with 2.5 mM HCO3‐ and 3.4 mM Ca2+. Results indicated that although all strains tested have the ability to calcify, only two Synechococcus species, strains PCC 8806 and PCC 8807, were able to calcify to the extent that a CaCO3 precipitate was formed. Enumeration of the cyanobacterial cultures during testing indicated that cell density did not appear to have a direct effect on calcification. Factors that had the greatest effect on calcification were CO2 removal and subsequent generation of alkaline pH. Whereas cell density was similar for all strains tested, differences in maximum pH were demonstrated. As CO2 was removed, growth medium pH increased and soluble Ca2+ was removed from solution. The largest increases in growth medium pH occurred when CO2 levels dropped below 400 ppmv. Research presented demonstrates that, under the conditions tested, many species of cyanobacteria in the genera Synechocystis and Synechococcus are able to calcify but only two species of Synechococcus were able to calcify to an extent that led to the precipitation of calcium carbonate.

Bioremediation Potential of Cr(VI)-Contaminated Soil Using Indigenous Microorganisms

Amendment of Cr(VI)-contaminated soil (approx. 200 mg/kg) with various treatments resulted in greater CO2 evolution and Cr(VI) reduction with organic amendments relative to controls receiving no organics, indicating bacterial reduction of Cr(VI) under anaerobic conditions. Isolation of Cr(VI)-reducing, indigenous bacteria, representative of the dominant soil population, further indicated Cr(VI) reduction by indigenous bacteria. Although desorption of Cr(VI) was evident with some treatments, its reduction was not affected.