Electrotrophic activity and electrosynthetic acetate production by Desulfobacterium autotrophicum HRM2.

Electroautotrophic microorganisms accept electrons from a cathode as source of reducing equivalents to drive CO2 fixation by poorly understood mechanisms. Acetogenic bacteria were the first group found to possess the capability for electroautotrophic metabolism in pure culture with associated electrosynthesis of acetate as primary metabolite. Identification of additional electrotrophic species can contribute to our understanding of this unusual form of metabolism. Here, bioelectrochemical techniques, chemical analysis and microscopy were used to determine electrotrophic metabolism of Desulfobacterium autotrophicum HRM2. Chronoamperometry showed increasing current uptake over 21 days of incubation in duplicate bioelectrochemical system sets. Linear sweep voltammetry indicated peak current uptake at -243 mV. High performance liquid chromatography (HPLC) analysis quantified acetate accumulation in anaerobic minimal media containing inorganic carbon as sole carbon source, consistent with electrosynthesis. Scanning electron microscopy and live/dead staining by epifluorescence microscopy analysis indicated viable 1-2 µm cells after 76 days of cultivation under electroautotrophic conditions. The genome of Db. autotrophicum HRM2 is fully sequenced and, thus, could provide insight into the biochemical and physiological mechanisms by which electrotrophic cells utilize cathode-derived electrons. This research expands the diversity of facultative autotrophs capable of electrotrophic metabolism to include the sulfate-reducing marine bacterium Db. autotrophicum HRM2.

Quantitative characterization of hemozoin in Plasmodium berghei and vivax.

The incidence and global distribution of chloroquine resistant (CR) Plasmodium vivax infection has increased since emerging in 1989. The mechanism of resistance in CR P. vivax has not been defined. The resistance likely relates to the formation and disposition of hemozoin as chloroquine's primary mechanism of action involves disruption of hemozoin formation. CR P. berghei strains, like CR P. vivax strains, are confined to reticulocyte host cells and reportedly they do not accumulate appreciable intraerythrocytic hemozoin. Reports comparing hemozoin production between P. vivax strains and CR to chloroquine sensitive (CS) P. berghei are absent. Here we compare in vivo patterns of hemozoin formation and distribution in blood, spleen and liver tissue of male Swiss mice infected with CS or CR P. berghei not treated with chloroquine and CR P. berghei also treated with chloroquine. Light microscopy, laser desorption mass spectrometry and a colorimetric hemozoin assay detect trace hemozoin in the blood of CR P. berghei infected mice but significant hemozoin accumulation in liver and spleen tissue. Field emission in lens scanning electron microscopy reveals CR P. berghei hemozoin crystals are morphologically smaller but similar to those formed by CS parasites. CR P. berghei produces approximately five-fold less total hemozoin than CS strain. Lipid analysis of CS and CR P. berghei sucrose gradient purified bloodstage hemozoin indicates a similar lipid environment around the isolated hemozoin, predominately monopalmitic glycerol and monostearic glycerol. In contrast to CR and CS P. berghei, colorimetric hemozoin analysis of P. vivax strains indicates similar amounts of hemozoin are produced despite differing chloroquine sensitivities. These results suggest CR P. berghei forms significant hemozoin which accumulates in liver and spleen tissues and that the P. vivax chloroquine resistance mechanism differs from P. berghei.

Enhanced start-up of anaerobic facultatively autotrophic biocathodes in bioelectrochemical systems.

Biocathodes in bioelectrochemical systems (BESs) can be used to convert CO2 into diverse organic compounds through a process called microbial electrosynthesis. Unfortunately, start-up of anaerobic biocathodes in BESs is a difficult and time consuming process. Here, a pre-enrichment method was developed to improve start-up of anaerobic facultatively autotrophic biocathodes capable of using cathodes as the electron donor (electrotrophs) and CO2 as the electron acceptor. Anaerobic enrichment of bacteria from freshwater bog sediment samples was first performed in batch cultures fed with glucose and then used to inoculate BES cathode chambers set at -0.4V (versus a standard hydrogen electrode; SHE). After two weeks of heterotrophic operation of BESs, CO2 was provided as the sole electron acceptor and carbon source. Consumption of electrons from cathodes increased gradually and was sustained for about two months in concert with a significant decrease in cathode chamber headspace CO2. The maximum current density consumed was -34 ± 4 mA/m(2). Biosynthesis resulted in organic compounds that included butanol, ethanol, acetate, propionate, butyrate, and hydrogen gas. Bacterial community analyses based on 16S rRNA gene clone libraries revealed Trichococcus palustris DSM 9172 (99% sequence identity) as the prevailing species in biocathode communities, followed by Oscillibacter sp. and Clostridium sp. Isolates from autotrophic cultivation were most closely related to Clostridium propionicum (99% sequence identity; ZZ16), Clostridium celerecrescens (98-99%; ZZ22, ZZ23), Desulfotomaculum sp. (97%; ZZ21), and Tissierella sp. (98%; ZZ25). This pre-enrichment procedure enables simplified start-up of anaerobic biocathodes for applications such as electrofuel production by facultatively autotrophic electrotrophs.

Treatability studies on different refinery wastewater samples using high-throughput microbial electrolysis cells (MECs).

High-throughput microbial electrolysis cells (MECs) were used to perform treatability studies on many different refinery wastewater samples all having appreciably different characteristics, which resulted in large differences in current generation. A de-oiled refinery wastewater sample from one site (DOW1) produced the best results, with 2.1±0.2 A/m(2) (maximum current density), 79% chemical oxygen demand removal, and 82% headspace biological oxygen demand removal. These results were similar to those obtained using domestic wastewater. Two other de-oiled refinery wastewater samples also showed good performance, with a de-oiled oily sewer sample producing less current. A stabilization lagoon sample and a stripped sour wastewater sample failed to produce appreciable current. Electricity production, organics removal, and startup time were improved when the anode was first acclimated to domestic wastewater. These results show mini-MECs are an effective method for evaluating treatability of different wastewaters.

Enrichment of microbial electrolysis cell biocathodes from sediment microbial fuel cell bioanodes.

Electron-accepting (electrotrophic) biocathodes were produced by first enriching graphite fiber brush electrodes as the anodes in sediment-type microbial fuel cells (sMFCs) using two different marine sediments and then electrically inverting the anodes to function as cathodes in two-chamber bioelectrochemical systems (BESs). Electron consumption occurred at set potentials of -439 mV and -539 mV (versus the potential of a standard hydrogen electrode) but not at -339 mV in minimal media lacking organic sources of energy. Results at these different potentials were consistent with separate linear sweep voltammetry (LSV) scans that indicated enhanced activity (current consumption) below only ca. -400 mV. MFC bioanodes not originally acclimated at a set potential produced electron-accepting (electrotrophic) biocathodes, but bioanodes operated at a set potential (+11 mV) did not. CO(2) was removed from cathode headspace, indicating that the electrotrophic biocathodes were autotrophic. Hydrogen gas generation, followed by loss of hydrogen gas and methane production in one sample, suggested hydrogenotrophic methanogenesis. There was abundant microbial growth in the biocathode chamber, as evidenced by an increase in turbidity and the presence of microorganisms on the cathode surface. Clone library analysis of 16S rRNA genes indicated prominent sequences most similar to those of Eubacterium limosum (Butyribacterium methylotrophicum), Desulfovibrio sp. A2, Rhodococcus opacus, and Gemmata obscuriglobus. Transfer of the suspension to sterile cathodes made of graphite plates, carbon rods, or carbon brushes in new BESs resulted in enhanced current after 4 days, demonstrating growth by these microbial communities on a variety of cathode substrates. This report provides a simple and effective method for enriching autotrophic electrotrophs by the use of sMFCs without the need for set potentials, followed by the use of potentials more negative than -400 mV.

Role of the photosynthetic electron transfer chain in electrogenic activity of cyanobacteria.

Certain anaerobic bacteria, termed electrogens, produce an electric current when electrons from oxidized organic molecules are deposited to extracellular metal oxide acceptors. In these heterotrophic "metal breathers", the respiratory electron transport chain (R-ETC) works in concert with membrane-bound cytochrome oxidases to transfer electrons to the extracellular acceptors. The diversity of bacteria able to generate an electric current appears more widespread than previously thought, and aerobic phototrophs, including cyanobacteria, possess electrogenic activity. However, unlike heterotrophs, cyanobacteria electrogenic activity is light dependent, which suggests that a novel pathway could exist. To elucidate the electrogenic mechanism of cyanobacteria, the current studies used site-specific inhibitors to target components of the photosynthetic electron transport chain (P-ETC) and cytochrome oxidases. Here, we show that (1) P-ETC and, particularly, water photolysed by photosystem II (PSII) is the source of electrons discharged to the environment by illuminated cyanobacteria, and (2) water-derived electrons are transmitted from PSII to extracellular electron acceptors via plastoquinone and cytochrome bd quinol oxidase. Two cyanobacterial genera (Lyngbya and Nostoc) displayed very similar electrogenic responses when treated with P-ETC site-specific inhibitors, suggesting a conserved electrogenic pathway. We propose that in cyanobacteria, electrogenic activity may represent a form of overflow metabolism to protect cells under high-intensity light. This study offers insight into electron transfer between phototrophic microorganisms and the environment and expands our knowledge into biologically based mechanisms for harnessing solar energy.

Light-dependent electrogenic activity of cyanobacteria.

BACKGROUND: Cyanobacteria account for 20-30% of Earth's primary photosynthetic productivity and convert solar energy into biomass-stored chemical energy at the rate of approximately 450 TW [1]. These single-cell microorganisms are resilient predecessors of all higher oxygenic phototrophs and can be found in self-sustaining, nitrogen-fixing communities the world over, from Antarctic glaciers to the Sahara desert [2]. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that diverse genera of cyanobacteria including biofilm-forming and pelagic strains have a conserved light-dependent electrogenic activity, i.e. the ability to transfer electrons to their surroundings in response to illumination. Naturally-growing biofilm-forming photosynthetic consortia also displayed light-dependent electrogenic activity, demonstrating that this phenomenon is not limited to individual cultures. Treatment with site-specific inhibitors revealed the electrons originate at the photosynthetic electron transfer chain (P-ETC). Moreover, electrogenic activity was observed upon illumination only with blue or red but not green light confirming that P-ETC is the source of electrons. The yield of electrons harvested by extracellular electron acceptor to photons available for photosynthesis ranged from 0.05% to 0.3%, although the efficiency of electron harvesting likely varies depending on terminal electron acceptor. CONCLUSIONS/SIGNIFICANCE: The current study illustrates that cyanobacterial electrogenic activity is an important microbiological conduit of solar energy into the biosphere. The mechanism responsible for electrogenic activity in cyanobacteria appears to be fundamentally different from the one exploited in previously discovered electrogenic bacteria, such as Geobacter, where electrons are derived from oxidation of organic compounds and transported via a respiratory electron transfer chain (R-ETC) [3], [4]. The electrogenic pathway of cyanobacteria might be exploited to develop light-sensitive devices or future technologies that convert solar energy into limited amounts of electricity in a self-sustainable, CO(2)-free manner.

Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-MS/MS with multiple reaction monitoring.

An AccQ*Tag ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ*Tag-UPLC-ESI-MS/MS) method for fast, reproducible, and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with a photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ*Tag amino acids with multiple reaction monitoring, varied from 29 to 39 V, whereas the collision energy varied from 15 to 35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentration ranges of 3 x 10(-3)-25 pmol/muL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the range of 0.08-1.08%. The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ*Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/MS for amino acid quantitation using AccQ*Tag derivatization.

Hemozoin: oil versus water.

Because the quinolines inhibit heme crystallization within the malaria parasite much work has focused on mechanism of formation and inhibition of hemozoin. Here we review the recent evidence for heme crystallization within lipids in diverse parasites and the new implications of a lipid site of crystallization for drug targeting. Within leukocytes hemozoin can generate toxic radical lipid metabolites, which may alter immune function or reduce deformability of uninfected erythrocytes.

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization.

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

Hemozoin formation in Echinostoma trivolvis rediae.

Rediae of the trematode Echinostoma trivolvis, from naturally infected Helisoma trivolvis snails, form a black pigment while inside the snail host. Here we examine the black pigment to show that the insolubility characteristics in detergent and weak base solution are identical to Plasmodium falciparum hemozoin. Laser desorption mass spectrometry of the purified pigment demonstrates the presence of heme. Examination of purified pigment under polarized light microscopy illuminates ordered birefringent crystals. Field emission in lens scanning electron microscopy reveals irregular ovoid crystals of 200-300 nm in diameter. The purified pigment crystals seeded extension of monomeric heme onto the crystal which by Fourier Transform Infrared analysis is beta-hematin. Rediae of a second echinostome parasite, Echinostoma caproni, from experimentally infected Biomphalaria glabrata, do not produce measurable or recoverable heme crystals. These observations are consistent with heme crystal formation by a hematophagous parasite within a non-vertebrate intermediate host.

Nosocomial malaria and saline flush.

An investigation of malaria in a US patient without recent travel established Plasmodium falciparum molecular genotype identity in 2 patients who shared a hospital room. P. falciparum can be transmitted in a hospital environment from patient to patient by blood inoculum if standard precautions are breached.

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters.

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.