Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum.

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation.

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H(2)O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4 -- used as an indicator strain, B. licheniformis T6-5, and B. firmus H(2)O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H(2)O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H(2)O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H(2)O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.

Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates.

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).

Copper-induced inhibition of growth of Desulfovibrio desulfuricans G20: assessment of its toxicity and correlation with those of zinc and lead.

The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using Desulfovibrio desulfuricans G20 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 microM, 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 microM. A Cu(II) concentration of 13.3 microM 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 microM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 microM, 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.

A fluorescence spectroscopic study of phenanthrene sorption on porous silica.

Fluorescence spectroscopic characteristics of sorbed phenanthrene in porous silica provide information about its chemical state such as monomer vs dimer or higher aggregates, as well as a basis for high sensitivity detection. In this study, the chemical state and distribution of phenanthrene sorbed in two types of porous silica particles, mesoporous silica (365 microns particle diameter, 150 A average pore diameter) and microporous silica (custom synthethized, 1 micron particle diameter, 20 A pore diameter), is determined by fluorescence spectroscopy, fluorescence lifetime measurements, and scanning two-photon excitation fluorescence profiling. From the characteristic fluorescence emission spectra, it is found that at loading levels of < or = 4.7 mg/g (phenanthrene/silica) phenanthrene exists as monomers in both meso- and microporous silica particles for phenanthrene loaded from super critical CO2 (SCF). Two-photon excitation fluorescence intensity distribution profiles indicate that for the mesoporous silica particles phenanthrene is adsorbed throughout the entire silica particle. Introduction of water into phenanthrene-loaded mesoporous silica particles causes instantaneous conversion of phenanthrene from monomer to crystalline form at phenantherene loading levels > or = 4.7 micrograms/g due to hydration of the silica surface. In this process, sorption of water molecules expels phenanthrene from the surface sorption sites and causes localized phenanthrene concentration beyond its solubility limit, resulting in crystallization. In comparison this fast conversion is not observed for phenanthrene-loaded microporous silica particles that show extremely slow conversion even for phenanthrene loading levels as high as 4.7 mg/g. This difference is interpreted as reflecting hindered diffusion of phenanthrene in the nearly monodispersed micropores with pore sizes close to the molecular diameter of phenanthrene.

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.

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.

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 biofilms and biofilm reactors.

Scientists and engineers have realized the industrial and environmental significance of biofilm accumulation and activity. The ability to predict and control biofilm formation has led to less fouling and corrosion in industrial systems and a better understanding of biofilm importance in natural aquatic systems. Understanding the fundamental processes contributing to biofilm formation is beneficial to anyone involved with natural or industrial systems where biofilms may play a significant role in determining variables such as bulk water quality, toxic compound biodegradation, or product quality.

A statistical analysis of the effect of substrate utilization and shear stress on the kinetics of biofilm detachment.

One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment. Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed.A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono- and mixed-population biofilms.

Quantitative observations of heterogeneities in Pseudomonas aeruginosa biofilms.

Heterogeneity in a Pseudomonas aeruginosa biofilm was quantified by measuring distributions of thickness in biofilm samples and a distribution of particle sizes in effluent samples. The mean steady-state thickness was approximately 33 microns, but individual measurements ranged from 13.3 to 60.0 microns. Particles exceeding 100 microns3 were observed in the reactor effluent. The results reveal a rough biofilm surface and indicate that most biomass detaches in the form of multicellular particles.