Brent M. Peyton

Copper-Induced Inhibition of Growth of Desulfovibrio desulfuricans G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead

ABSTRACT The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using Desulfovibrio desulfuricansG20 in a medium (MTM) developed specifically to test metal toxicity to SRB (R. K. Sani, G. Geesey, and B. M. Peyton, Adv. Environ. Res. 5:269–276, 2001). The effects of Cu(II) toxicity were observed in terms of inhibition in total cell protein, longer lag times, lower specific growth rates, and in some cases no measurable growth. At only 6 μM, Cu(II) reduced the maximum specific growth rate by 25% and the final cell protein concentration by 18% compared to the copper-free control. Inhibition by Cu(II) of cell yield and maximum specific growth rate increased with increasing concentrations. The Cu(II) concentration causing 50% inhibition in final cell protein was evaluated to be 16 μM. A Cu(II) concentration of 13.3 μM showed 50% inhibition in maximum specific growth rate. These results clearly show significant Cu(II) toxicity to SRB at concentrations that are 100 times lower than previously reported. No measurable growth was observed at 30 μM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 μM, increased lag times by 48 and 72 h, respectively, but yielded final cell protein concentrations equivalent to those of the zinc- and lead-free controls. Live/dead staining, based on membrane integrity, indicated that while Cu(II), Zn(II), and Pb(II) inhibited growth, these metals did not cause a loss of D. desulfuricans membrane integrity. The results show that D. desulfuricans in the presence of Cu(II) follows a growth pattern clearly different from the pattern followed in the presence of Zn(II) or Pb(II). It is therefore likely that Cu(II) toxicity proceeds by a mechanism different from that of Zn(II) or Pb(II) toxicity.

Halomonas campisalis sp.nov., a Denitrifying, Moderately Haloalkaliphilic Bacterium

The isolation and characterization of a denitrifying bacterium that is both moderately halophilic and alkaliphilic is described. The organism was isolated for use in the development of a bioprocess that could potentially reduce the costs of ion exchange resin regenerant disposal. The process of ion exchange, after resin regeneration, produces a briny, alkaline waste that is difficult and expensive to dispose. The biological removal of nitrate and subsequent reuse of these brines can potentially provide a cost-saving alternative to disposing of this waste product. To achieve our objective, a moderately halophilic, alkaliphilic bacterium was isolated from sediment samples taken from the salt plain of Alkali Lake in Washington State (USA). The haloalkaliphilic bacterium, designated strain 4A, is motile with rod-shaped cells that are 3 to 5 microm long and 1 microm wide. Electron acceptors used include oxygen, nitrate, and nitrite. In addition, it has similar specific nitrate reduction rates and biomass yields as non-halophilic denitrifying bacteria. It is capable of using a variety of electron donors. This organism can grow at NaCl concentrations ranging from 0.2 to 4.5 M with optimum growth occurring at 1.5 M and pH values ranging from 6 to 12 with 9.5 being the optimum pH. The temperature range for growth of strain 4A is 4-50 degrees C with optimal growth occurring at 30 degrees C. The G + C content is 66 mol%. Phylogenetic analyses based upon 16S rDNA gene sequence placed isolate 4A in the genus Halomonas. In addition, DNA-DNA hybridization experiments clearly indicate that it is a unique species. Phenotypic and phylogenetic studies indicate that isolate 4A represents a new species. We propose the name Halomonas campisalis for this species and strain 4A (ATCC 700597) as the type strain. Due to its denitrification ability, broad carbon utilization range and its high salinity and pH tolerance this organism, and similar ones, hold promise for the treatment of saline, alkaline waste.

Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum.

BackgroundPhaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination.ResultsThe specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc 7 and cyc 10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases).ConclusionsThe results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

Kinetics of phenol biodegradation in high salt solutions

Biological treatment of high-salinity industrial wastewaters using halophilic bacteria can be used to remove organic compounds without first decreasing the salt concentration. While halophilic degradation of phenol and other organics has been investigated, there exists a general absence of kinetic data in current literature to allow evaluation of this treatment alternative. Liquid, soil and sediment samples were collected from three distinct saline environments in the western United States. Samples were enriched in media containing 10% (w/v) NaCl at pH 7.0, with phenol as a substrate. Mixed culture batch experiments were conducted at 30 degrees C with initial phenol concentrations of 50 mg/L. Evaluation of phenol degradation and corresponding cell growth data with Monod and Andrews models indicated that the kinetics were zero-order with respect to phenol. Zero-order specific growth rates ranged from 0.22 to 0.32 h(-1), while observed cell yields were 0.18-0.28 mg cell protein/mg phenol for the five cultures. For one of the cultures, phenol degradation rates were also quantified at concentrations of up to 320 mg/L. Under these conditions, specific growth rates ranged from 0.09 to 0.22 h(-1), decreasing with increasing initial phenol concentrations. Cell yields at these higher initial phenol concentrations ranged from 0.20 to 0.29 mg cell protein/mg phenol.

Composition and Diversity of Microbial Communities Recovered from Surrogate Minerals Incubated in an Acidic Uranium-Contaminated Aquifer

ABSTRACT Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the β-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by β-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.

Kinetics of U(VI) reduction by a dissimilatory Fe(III)‐reducing bacterium under non‐growth conditions

Dissimilatory metal-reducing microorganisms may be useful in processes designed for selective removal of uranium from aqueous streams. These bacteria can use U(VI) as an electron acceptor and thereby reduce soluble U(VI) to insoluble U(IV). While significant research has been devoted to demonstrating and describing the mechanism of dissimilatory metal reduction, the reaction kinetics necessary to apply this for remediation processes have not been adequately defined. In this study, pure culture Shewanella alga strain BrY reduced U(VI) under non-growth conditions in the presence of excess lactate as the electron donor. Initial U(VI) concentrations ranged from 13 to 1680 microM. A maximum specific U(VI) reduction rate of 2.37 micromole-U(VI)/(mg-biomass h) and Monod half-saturation coefficient of 132 microM-U(VI) were calculated from measured U(VI) reduction rates. U(VI) reduction activity was sustained at 60% of this rate for at least 80 h. The initial presence of oxygen at a concentration equal to atmospheric saturation at 22 degrees C delays but does not prevent U(VI) reduction. The rate of U(VI) reduction by BrY is comparable or better than rates reported for other metal reducing species. BrY reduces U(VI) at a rate that is 30% of its Fe(III) reduction rate.

Microbial growth and transport in porous media under denitrification conditions : experiments and simulations

Abstract Soil column experiments were conducted to study bacterial growth and transport in porous media under denitrifying conditions. The study used a denitrifying microbial consortium isolated from aquifer sediments sampled at the U.S. Department of Energys Hanford site. One-dimensional, packed-column transport studies were conducted under two substrate loading conditions. A detailed numerical model was developed to predict the measured effluent cell and substrate concentration profiles. First-order attachment and detachment models described the interphase exchange processes between suspended and attached biomass. Insignificantly different detachment coefficient values of 0.32 and 0.43 day−1, respectively, were estimated for the high and low nitrate loading conditions (48 and 5 mg l−1 NO3, respectively). Comparison of these values with those calculated from published data for aerobically growing organisms shows that the denitrifying consortium had lower detachment rate coefficients. This suggests that, similar to detachment rates in reactor-grown biofilms, detachment in porous media may increase with microbial growth rate. However, available literature data are not sufficient to confirm a specific analytical model for predicting this growth dependence.

Nitrate Reduction with Halomonas campisalis: Kinetics of Denitrification at pH 9 and 12.5% NaCl

Regeneration of ion exchange resins with NaCl produces brine containing high concentrations of nitrate that can be difficult to remove using standard biological, physical, or chemical technologies. In this study. Halomonas campisalis (ATCC #700597) (Mormile et al., 1999) was shown to completely reduce nitrate at 125 g/L NaCl and pH 9. This organism was also used in experiments to determine nitrate-reduction rates and biomass yields. Kinetic parameters were measured separately with glycerol, lactate. acetate, ethanol, and methanol. The specific nitrate-reduction rate coefficient was highest in cultures amended with acetate, while lactate and glycerol (a natural osmoticum in hypersaline environments) had lower reduction rates. No evidence of nitrate reduction was observed when ethanol or methanol was provided as an electron donor. Kinetic modeling provided values for nitrate and nitrite-reduction rate coefficients and for biomass yields. Measured rates and yields were similar to reported parameters obtained from non-halophilic nitrate-reducing cultures under low salt concentrations. Therefore, for highly saline solutions, the use of halophiles to selectively remove nitrate from these brines may represent a viable treatment option.

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)].

Comparison of CO2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii.

Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated since these compounds can be used as precursors for food and fuel. Here, we detail the results of a comprehensive analysis of Chlamydomonas reinhardtii using high or low inorganic carbon concentrations and speciation between carbon dioxide and bicarbonate, and the effects these have on inducing lipid and starch accumulation during nitrogen depletion. High concentrations of CO2 (5%; v/v) produced the highest amount of biofuel precursors, transesterified to fatty acid methyl esters, but exhibited rapid accumulation and degradation characteristics. Low CO2 (0.04%; v/v) caused carbon limitation and minimized triacylglycerol (TAG) and starch accumulation. High bicarbonate caused a cessation of cell cycling and accumulation of both TAG and starch that was more stable than the other experimental conditions. Starch accumulated prior to TAG and then degraded as maximum TAG was reached. This suggests carbon reallocation from starch‐based to TAG‐based carbon storage. Biotechnol. Bioeng. 2013; 110: 87–96.