Substrate degradation kinetics, microbial diversity, and current efficiency of microbial fuel cells supplied with marine plankton

The decomposition of marine plankton in two-chamber, seawater-filled microbial fuel cells (MFCs) has been investigated and related to resulting chemical changes, electrode potentials, current efficiencies, and microbial diversity. Six experiments were run at various discharge potentials, and a seventh served as an open-circuit control. The plankton consisted of a mixture of freshly captured phytoplankton and zooplankton (0.21 to 1 mm) added at an initial batch concentration of 27.5 mmol liter(-1) particulate organic carbon (OC). After 56.7 days, between 19.6 and 22.2% of the initial OC remained, sulfate reduction coupled to OC oxidation accounted for the majority of the OC that was degraded, and current efficiencies (of the active MFCs) were between 11.3 and 15.5%. In the open-circuit control cell, anaerobic plankton decomposition (as quantified by the decrease in total OC) could be modeled by three terms: two first-order reaction rate expressions (0.79 day(-1) and 0.037 day(-1), at 15 degrees C) and one constant, no-reaction term (representing 10.6% of the initial OC). However, in each active MFC, decomposition rates increased during the third week, lagging just behind periods of peak electricity generation. We interpret these decomposition rate changes to have been due primarily to the metabolic activity of sulfur-reducing microorganisms at the anode, a finding consistent with the electrochemical oxidization of sulfide to elemental sulfur and the elimination of inhibitory effects of dissolved sulfide. Representative phylotypes, found to be associated with anodes, were allied with Delta-, Epsilon-, and Gammaproteobacteria as well as the Flavobacterium-Cytophaga-Bacteroides and Fusobacteria. Based upon these results, we posit that higher current efficiencies can be achieved by optimizing plankton-fed MFCs for direct electron transfer from organic matter to electrodes, including microbial precolonization of high-surface-area electrodes and pulsed flowthrough additions of biomass.     

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H₂S) and methane (CH₄) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H₂S and CH₄ production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H₂S emission was notably high (290.7 ± 72.3 mg S-H₂S liter⁻¹ day⁻¹), whereas emissions of CH₄ remained low (17.9 ± 15.9 mg COD-CH₄ liter⁻¹ day⁻¹). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH₄ emissions increased after 1 year of biofilm growth (327.6 ± 16.6 mg COD-CH₄ liter⁻¹ day⁻¹), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.     

Fungal Growth, Production, and Sporulation during Leaf Decomposition in Two Streams

I examined the activity of fungi associated with yellow poplar (Liriodendron tulipifera) and white oak (Quercus alba) leaves in two streams that differed in pH and alkalinity (a hardwater stream [pH 8.0] and a softwater stream [pH 6.7]) and contained low concentrations of dissolved nitrogen (<35 μg liter⁻¹) and phosphorus (<3 μg liter⁻¹). The leaves of each species decomposed faster in the hardwater stream (decomposition rates, 0.010 and 0.007 day⁻¹ for yellow poplar and oak, respectively) than in the softwater stream (decomposition rates, 0.005 and 0.004 day⁻¹ for yellow poplar and oak, respectively). However, within each stream, the rates of decomposition of the leaves of the two species were not significantly different. During the decomposition of leaves, the fungal biomasses determined from ergosterol concentrations, the production rates determined from rates of incorporation of [¹⁴C]acetate into ergosterol, and the sporulation rates associated with leaves were dynamic, typically increasing to maxima and then declining. The maximum rates of fungal production and sporulation associated with yellow poplar leaves were greater than the corresponding rates associated with white oak leaves in the hardwater stream but not in the softwater stream. The maximum rates of fungal production associated with the leaves of the two species were higher in the hardwater stream (5.8 mg g⁻¹ day⁻¹ on yellow poplar leaves and 3.1 mg g⁻¹ day⁻¹ on oak leaves) than in the softwater stream (1.6 mg g⁻¹ day⁻¹ on yellow poplar leaves and 0.9 mg g⁻¹ day⁻¹ on oak leaves), suggesting that effects of water chemistry other than the N and P concentrations, such as pH or alkalinity, may be important in regulating fungal activity in streams. In contrast, the amount of fungal biomass (as determined from ergosterol concentrations) on yellow poplar leaves was greater in the softwater stream (12.8% of detrital mass) than in the hardwater stream (9.6% of detrital mass). This appeared to be due to the decreased amount of fungal biomass that was converted to conidia and released from the leaf detritus in the softwater stream.     

Generation of Electricity and Analysis of Microbial Communities in Wheat Straw Biomass-Powered Microbial Fuel Cells

Electricity generation from wheat straw hydrolysate and the microbial ecology of electricity-producing microbial communities developed in two-chamber microbial fuel cells (MFCs) were investigated. The power density reached 123 mW/m² with an initial hydrolysate concentration of 1,000 mg chemical oxygen demand (COD)/liter, while coulombic efficiencies ranged from 37.1 to 15.5%, corresponding to the initial hydrolysate concentrations of 250 to 2,000 mg COD/liter. The suspended bacteria found were different from the bacteria immobilized in the biofilm, and they played different roles in electricity generation from the hydrolysate. The bacteria in the biofilm were consortia with sequences similar to those of Bacteroidetes (40% of sequences), Alphaproteobacteria (20%), Bacillus (20%), Deltaproteobacteria (10%), and Gammaproteobacteria (10%), while the suspended consortia were predominately Bacillus (22.2%). The results of this study can contribute to improving understanding of and optimizing electricity generation in microbial fuel cells.Wheat straw is one of the most abundant renewable resources. According to the Food and Agriculture Organization of the United Nations, approximately 1.9 × 10⁹ tons of wheat straw annually are produced worldwide, accompanied by 6.2 × 10⁸ tons of wheat production. Wheat straw is composed of 35 to 45% cellulose and 20 to 30% hemicelluloses with a relatively low lignin content (<20%) (42). The hemicellulose fraction of the straw is easily hydrolyzed to its constituent sugars by a hydrothermal treatment process, forming a carbohydrate-enriched liquid hydrolysate (46). Chemical and biological approaches to sustainable energy production from the liquefied hydrolysates to energy carriers, such as methane, ethanol, and H₂, have been developed. However, many of these approaches encounter technical and economical hurdles (10, 12, 15, 16). An alternative strategy is direct conversion of wheat straw biomass to electrical energy in microbial fuel cells (MFCs).MFCs are bioelectrochemical reactors in which microorganisms mediate the direct conversion of chemical energy stored in organic matter or bulk biomass into electrical energy (12, 15, 16, 40). Various substrates, such as simple carbohydrates, low-molecular-weight organic acids, starch, amino acids, chitin, cellulose, domestic wastewater, food-processing wastewater, recycled paper wastewater, and marine sediment organic matter, have been successfully utilized for power generation in MFCs (16-18, 27, 30, 33). To understand the microbial constraints on various fuel-powered MFCs, microbial communities have been characterized by several groups. Microbial communities from various systems are very different and often diverse, ranging from well-known metal- and anode-reducing bacteria to unknown exoelectrogens (1, 20, 21). It has been found that parameters such as the substrates used as fuels and the inocula used for starting up the MFCs can influence the anode bacterial communities in an MFC, which subsequently influence the efficiency of the MFCs (3, 14, 22, 38, 44). Different pure substrates, such as acetate, glucose, and lactate, were used as fuel to compare the microbial communities that developed in the MFCs. Regardless of the different substrates, all anode communities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone belonging to the family Bacteroidaceae (99% similarity). Firmicutes were only found in glucose-fed MFCs (20). Microbial-community analyses of MFCs powered with complex substrates have also been performed by several researchers, and their results were very diverse. The microbial community in starch wastewater-powered MFC was dominated by unidentified bacteria (35.9%), followed by Betaproteobacteria (25.0%), Alphaproteobacteria (20.1%), and the Cytophaga/Flexibacter/Bacteroides group (19.0%) (21). The anode-attached consortia in a cellulose-powered MFC were related to Clostridium spp., while Comamonas spp. were abundant in the suspended consortia (13). Although many studies have reported the microbial compositions of MFCs, it is still unclear which microbial communities develop as a function of the external parameters.Wheat straw biomass constitutes a large source for bioenergy production and shows promising prospects for electricity generation in MFCs. Therefore, wheat straw biomass was used to study the microbial communities that develop during the operation of an MFC in order to better understand the microbial electrochemical roles and potentially improve MFC performance.The objectives of this study were to (i) test wheat straw hydrolysate as a potential fuel in an MFC for electricity generation and (ii) study the microbial composition and evolution of electricity-producing communities in a two-chamber MFC system. Phylogenetic-diversity analysis of the enriched consortia was conducted to verify the presence of hydrolytic and respiratory anaerobes that could couple hydrolysate oxidation with proton reduction in the anode chamber. This is the first report of exploiting microbial communities for direct conversion of wheat straw hydrolysate to electrical energy in an MFC.     

Comparison of Electrode Reduction Activities of Geobacter sulfurreducens and an Enriched Consortium in an Air-Cathode Microbial Fuel Cell

An electricity-generating bacterium, Geobacter sulfurreducens PCA, was inoculated into a single-chamber, air-cathode microbial fuel cell (MFC) in order to determine the maximum electron transfer rate from bacteria to the anode. To create anodic reaction-limiting conditions, where electron transfer from bacteria to the anode is the rate-limiting step, anodes with electrogenic biofilms were reduced in size and tests were conducted using anodes of six different sizes. The smallest anode (7 cm², or 1.5 times larger than the cathode) achieved an anodic reaction-limiting condition as a result of a limited mass of bacteria on the electrode. Under these conditions, the limiting current density reached a maximum of 1,530 mA/m², and power density reached a maximum of 461 mW/m². Per-biomass efficiency of the electron transfer rate was constant at 32 fmol cell⁻¹ day⁻¹ (178 μmol g of protein⁻¹ min⁻¹), a rate comparable to that with solid iron as the electron acceptor but lower than rates achieved with fumarate or soluble iron. In comparison, an enriched electricity-generating consortium reached 374 μmol g of protein⁻¹ min⁻¹ under the same conditions, suggesting that the consortium had a much greater capacity for electrode reduction. These results demonstrate that per-biomass electrode reduction rates (calculated by current density and biomass density on the anode) can be used to help make better comparisons of electrogenic activity in MFCs.     

Microbial methanol uptake in northeast Atlantic waters

Methanol is the predominant oxygenated volatile organic compound in the troposphere, where it can significantly influence the oxidising capacity of the atmosphere. However, we do not understand which processes control oceanic concentrations, and hence, whether the oceans are a source or a sink to the atmosphere. We report the first methanol loss rates in seawater by demonstrating that ¹⁴C-labelled methanol can be used to determine microbial uptake into particulate biomass, and oxidation to ¹⁴CO₂. We have found that methanol is used predominantly as a microbial energy source, but also demonstrated its use as a carbon source. We report biological methanol oxidation rates between 2.1 and 8.4 nmol l⁻¹ day⁻¹ in surface seawater of the northeast Atlantic. Kinetic experiments predict a V_max of up to 29 nmol l⁻¹ day⁻¹, with a high affinity K_m constant of 9.3 n in more productive coastal waters. We report surface concentrations of methanol in the western English channel of 97±8 n (n=4) between May and June 2010, and for the wider temperate North Atlantic waters of 70±13 n (n=6). The biological turnover time of methanol has been estimated between 7 and 33 days, although kinetic experiments suggest a 7-day turnover in more productive shelf waters. Methanol uptake rates into microbial particles significantly correlated with bacterial and phytoplankton parameters, suggesting that it could be used as a carbon source by some bacteria and possibly some mixotrophic eukaryotes. Our results provide the first methanol loss rates from seawater, which will improve the understanding of the global methanol budget.     

Convergent development of anodic bacterial communities in microbial fuel cells

Microbial fuel cells (MFCs) are often inoculated from a single wastewater source. The extent that the inoculum affects community development or power production is unknown. The stable anodic microbial communities in MFCs were examined using three inocula: a wastewater treatment plant sample known to produce consistent power densities, a second wastewater treatment plant sample, and an anaerobic bog sediment. The bog-inoculated MFCs initially produced higher power densities than the wastewater-inoculated MFCs, but after 20 cycles all MFCs on average converged to similar voltages (470±20 mV) and maximum power densities (590±170 mW m⁻²). The power output from replicate bog-inoculated MFCs was not significantly different, but one wastewater-inoculated MFC (UAJA3 (UAJA, University Area Joint Authority Wastewater Treatment Plant)) produced substantially less power. Denaturing gradient gel electrophoresis profiling showed a stable exoelectrogenic biofilm community in all samples after 11 cycles. After 16 cycles the predominance of Geobacter spp. in anode communities was identified using 16S rRNA gene clone libraries (58±10%), fluorescent in-situ hybridization (FISH) (63±6%) and pyrosequencing (81±4%). While the clone library analysis for the underperforming UAJA3 had a significantly lower percentage of Geobacter spp. sequences (36%), suggesting that a predominance of this microbe was needed for convergent power densities, the lower percentage of this species was not verified by FISH or pyrosequencing analyses. These results show that the predominance of Geobacter spp. in acetate-fed systems was consistent with good MFC performance and independent of the inoculum source.     

Production and Consumption of Dimethylsulfoniopropionate in Marine Microbial Mats

The fate of dimethylsulfoniopropionate (DMSP), a major sulfonium compound in marine ecosystems, was examined in Microcoleus chthonoplastes-dominated microbial mats. Chemical decomposition of DMSP was observed under laboratory conditions at pH values higher than 10.0. pH profiles measured in situ showed that these highly alkaline conditions occurred in microbial mats. Axenic cultures of M. chthonoplastes contained 37.3 μmol of DMSP g of protein⁻¹, which was partially liberated when the cells were subjected to an osmotic shock. DMSP-amended mat slurries showed a rapid turnover of this compound. The addition of glutaraldehyde blocked DMSP decrease, indicating biological consumption. Populations of potential dimethyl sulfide consumers were found in the top 10 mm of the mat.     

Benthic Bacterial and Fungal Productivity and Carbon Turnover in a Freshwater Marsh

Heterotrophic bacteria and fungi are widely recognized as crucial mediators of carbon, nutrient, and energy flow in ecosystems, yet information on their total annual production in benthic habitats is lacking. To assess the significance of annual microbial production in a structurally complex system, we measured production rates of bacteria and fungi over an annual cycle in four aerobic habitats of a littoral freshwater marsh. Production rates of fungi in plant litter were substantial (0.2 to 2.4 mg C g⁻¹ C) but were clearly outweighed by those of bacteria (2.6 to 18.8 mg C g⁻¹ C) throughout the year. This indicates that bacteria represent the most actively growing microorganisms on marsh plant litter in submerged conditions, a finding that contrasts strikingly with results from both standing dead shoots of marsh plants and submerged plant litter decaying in streams. Concomitant measurements of microbial respiration (1.5 to 15.3 mg C-CO₂ g⁻¹ of plant litter C day⁻¹) point to high microbial growth efficiencies on the plant litter, averaging 45.5%. The submerged plant litter layer together with the thin aerobic sediment layer underneath (average depth of 5 mm) contributed the bulk of microbial production per square meter of marsh surface (99%), whereas bacterial production in the marsh water column and epiphytic biofilms was negligible. The magnitude of the combined production in these compartments (~1,490 g C m⁻² year⁻¹) highlights the importance of carbon flows through microbial biomass, to the extent that even massive primary productivity of the marsh plants (603 g C m⁻² year⁻¹) and subsidiary carbon sources (~330 g C m⁻² year⁻¹) were insufficient to meet the microbial carbon demand. These findings suggest that littoral freshwater marshes are genuine hot spots of aerobic microbial carbon transformations, which may act as net organic carbon importers from adjacent systems and, in turn, emit large amounts of CO₂ (here, ~870 g C m⁻² year⁻¹) into the atmosphere.     

Dynamics of Microbial Communities on Marine Snow Aggregates: Colonization, Growth, Detachment, and Grazing Mortality of Attached Bacteria

We studied the dynamics of microbial communities attached to model aggregates (4-mm-diameter agar spheres) and the component processes of colonization, detachment, growth, and grazing mortality. Agar spheres incubated in raw seawater were rapidly colonized by bacteria, followed by flagellates and ciliates. Colonization can be described as a diffusion process, and encounter volume rates were estimated at about 0.01 and 0.1 cm³ h⁻¹ for bacteria and flagellates, respectively. After initial colonization, the abundances of flagellates and ciliates remained approximately constant at 10³ to 10⁴ and ~10² cells sphere⁻¹, respectively, whereas bacterial populations increased at a declining rate to >10⁷ cells sphere⁻¹. Attached microorganisms initially detached at high specific rates of ~10⁻² min⁻¹, but the bacteria gradually became irreversibly attached to the spheres. Bacterial growth (0 to 2 day⁻¹) was density dependent and declined hyperbolically when cell density exceeded a threshold. Bacterivorous flagellates grazed on the sphere surface at an average saturated rate of 15 bacteria flagellate⁻¹ h⁻¹. At low bacterial densities, the flagellate surface clearance rate was ~5 × 10⁻⁷ cm² min⁻¹, but it declined hyperbolically with increasing bacterial density. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduces the main features of the observed microbial population dynamics. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g., antagonistic interactions between bacteria, are of importance in shaping marine snow microbial communities.     

Flowthrough Reactor Flasks for Study of Microbial Metabolism in Sediments

Flowthrough reactor flasks are described that allow continuous low-level nutrient input to mixed anoxic sediments without dilution of the sediment. The flasks were tested by simulating sulfate inputs into sediments collected from a freshwater eutrophic lake. After an initial 2-day adaptation within the reactor system, rates of methane production and sulfate consumption were constant for the duration of a 12-day incubation. A sulfate input rate of 0.15 mmol liter of sediment⁻¹ day⁻¹ resulted in an equivalent rate of sulfate removal, which was unaffected by inputs of acetate (1.0 mmol liter of sediment⁻¹ day⁻¹). The rate of methane production in control reactors, 0.18 mmol liter of sediment⁻¹ day⁻¹, was doubled by the addition of acetate, whereas sulfate consumption was only stimulated by additions of high concentrations of sulfate plus acetate (1.5 and 1.0 mmol liter of sediment⁻¹ day⁻¹, respectively). The reactor system appears to be effective in maintaining the balance between sulfate reduction and methane production in freshwater sediments and is potentially useful for study of the response of sediment populations to varying inputs of naturally occurring substrates, selected inhibitors, or xenobiotic compounds.     

Influence of Sulfide and Temperature on Species Composition and Community Structure of Hot Spring Microbial Mats

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80°C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90°C, and a Chloroflexus mat is formed at 65 to 70°C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one Chloroflexus mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the Chloroflexus mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that Chloroflexus was the dominant mat organism in the low-sulfide spring (1 mg liter⁻¹) below 70°C, whereas Aquificales were dominant in the high-sulfide spring (12 mg liter⁻¹) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of Aquificales on the one hand and temperature and sulfide concentration on the other hand.     

Contributions of Atmospheric CO and Hydrogen Uptake to Microbial Dynamics on Recent Hawaiian Volcanic Deposits

A series of sites were established on Hawaiian volcanic deposits ranging from about 18 to 300 years old. Three sites occurred in areas that supported tropical rain forests; the remaining sites were in areas that supported little or no plant growth. Sites >26 years old consumed atmospheric CO and hydrogen at rates ranging from about 0.2 to 5 mg of CO m⁻² day⁻¹ and 0.1 to 4 mg of H₂ m⁻² day⁻¹, respectively. Respiration, measured as CO₂ production, for a subset of the sites ranged from about 40 to >1,400 mg of CO₂ m⁻² day⁻¹. CO and H₂ accounted for about 13 to 25% of reducing equivalent flow for all but a forested site, where neither substrate appeared significant. Based on responses to chloroform fumigation, hydrogen utilization appeared largely due to microbial uptake. In contrast to results for CO and hydrogen, methane uptake occurred consistently only at the forest site. Increasing deposit age was generally accompanied by increasing concentrations of organic matter and microbial biomass, measured as phospholipid phosphate. Exoenzymatic activities (acid and alkaline phosphatases and α- and β-glucosidases) were positively correlated with deposit age in spite of considerable variability within sites. The diversity of substrates utilized in Biolog Ecoplate assays also increased with deposit age, possibly reflecting changes in microbial community complexity.     

Removing Constraints on the Biomass Production of Freshwater Macroalgae by Manipulating Water Exchange to Manage Nutrient Flux

Freshwater macroalgae represent a largely overlooked group of phototrophic organisms that could play an important role within an industrial ecology context in both utilising waste nutrients and water and supplying biomass for animal feeds and renewable chemicals and fuels. This study used water from the intensive aquaculture of freshwater fish (Barramundi) to examine how the biomass production rate and protein content of the freshwater macroalga Oedogonium responds to increasing the flux of nutrients and carbon, by either increasing water exchange rates or through the addition of supplementary nitrogen and CO₂. Biomass production rates were highest at low flow rates (0.1–1 vol.day⁻¹) using raw pond water. The addition of CO₂ to cultures increased biomass production rates by between 2 and 25% with this effect strongest at low water exchange rates. Paradoxically, the addition of nitrogen to cultures decreased productivity, especially at low water exchange rates. The optimal culture of Oedogonium occurred at flow rates of between 0.5–1 vol.day⁻¹, where uptake rates peaked at 1.09 g.m⁻².day⁻¹ for nitrogen and 0.13 g.m⁻².day⁻¹ for phosphorous. At these flow rates Oedogonium biomass had uptake efficiencies of 75.2% for nitrogen and 22.1% for phosphorous. In this study a nitrogen flux of 1.45 g.m⁻².day⁻¹ and a phosphorous flux of 0.6 g.m⁻².day⁻¹ was the minimum required to maintain the growth of Oedogonium at 16–17 g DW.m⁻².day⁻¹ and a crude protein content of 25%. A simple model of minimum inputs shows that for every gram of dry weight biomass production (g DW.m⁻².day⁻¹), Oedogonium requires 0.09 g.m⁻².day⁻¹ of nitrogen and 0.04 g.m⁻².day⁻¹ of phosphorous to maintain growth without nutrient limitation whilst simultaneously maintaining a high-nutrient uptake rate and efficiency. As such the integrated culture of freshwater macroalgae with aquaculture for the purposes of nutrient recovery is a feasible solution for the bioremediation of wastewater and the supply of a protein resource.     

Low-Molecular-Weight Sulfonates, a Major Substrate for Sulfate Reducers in Marine Microbial Mats

Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 μM HS⁻ h⁻¹ in a stromatolite mat, 38 and 36 μM HS⁻ h⁻¹ in a salt pond mat, and 27 and 18 μM HS⁻ h⁻¹ in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell⁻¹ h⁻¹. Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.     

Biofuel Cells Select for Microbial Consortia That Self-Mediate Electron Transfer

Microbial fuel cells hold great promise as a sustainable biotechnological solution to future energy needs. Current efforts to improve the efficiency of such fuel cells are limited by the lack of knowledge about the microbial ecology of these systems. The purposes of this study were (i) to elucidate whether a bacterial community, either suspended or attached to an electrode, can evolve in a microbial fuel cell to bring about higher power output, and (ii) to identify species responsible for the electricity generation. Enrichment by repeated transfer of a bacterial consortium harvested from the anode compartment of a biofuel cell in which glucose was used increased the output from an initial level of 0.6 W m⁻² of electrode surface to a maximal level of 4.31 W m⁻² (664 mV, 30.9 mA) when plain graphite electrodes were used. This result was obtained with an average loading rate of 1 g of glucose liter⁻¹ day⁻¹ and corresponded to 81% efficiency for electron transfer from glucose to electricity. Cyclic voltammetry indicated that the enhanced microbial consortium had either membrane-bound or excreted redox components that were not initially detected in the community. Dominant species of the enhanced culture were identified by denaturing gradient gel electrophoresis and culturing. The community consisted mainly of facultative anaerobic bacteria, such as Alcaligenes faecalis and Enterococcus gallinarum, which are capable of hydrogen production. Pseudomonas aeruginosa and other Pseudomonas species were also isolated. For several isolates, electrochemical activity was mainly due to excreted redox mediators, and one of these mediators, pyocyanin produced by P. aeruginosa, could be characterized. Overall, the enrichment procedure, irrespective of whether only attached or suspended bacteria were examined, selected for organisms capable of mediating the electron transfer either by direct bacterial transfer or by excretion of redox components.     

Microbial Desulfurization of Alkylated Dibenzothiophenes from a Hydrodesulfurized Middle Distillate by Rhodococcus erythropolis I-19

Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 μmol g dry cell weight⁻¹ min⁻¹, more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (K₀) and half-saturation rate constant (K₁) values were calculated to be 2.8 μmol g dry cell weight⁻¹ min⁻¹ and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent K₁ values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.     

Molecular Phylogenetic and Biogeochemical Studies of Sulfate-Reducing Bacteria in the Rhizosphere of Spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO₄²⁻ reduction rates and geochemical parameters. SO₄²⁻ reduction was rapid in marsh sediments with rates up to 3.5 μmol ml⁻¹ day⁻¹. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO₄²⁻ reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10⁷ ml⁻¹. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.     

Kinetic Studies of Bacterial Sulfate Reduction in Freshwater Sediments by High-Pressure Liquid Chromatography and Microdistillation

Indirect photometric chromatography and microdistillation enabled a simultaneous measurement of sulfate depletion and sulfide production in the top 3 cm of freshwater sediments to be made. The simultaneous measurement of sulfate depletion and sulfide production rates provided added insight into microbial sulfur metabolism. The lower sulfate reduction rates, as derived from the production of acid-volatile ³⁵S²⁻ only, were explained by a conversion of this pool to an undistillable fraction under acidic conditions during incubation. A mathematical model was applied to calculate sulfate reduction from sulfate gradients at the sediment-water interface. To avoid disturbance of these gradients, the sample volume was reduced to 0.2 g (wet weight) of sediment. Sulfate diffusion coefficients in the model were determined (D_s = 0.3 × 10⁻⁵ cm² s⁻¹ at 6°C). The results of the model were compared with those of radioactive sulfate turnover experiments by assessing the actual turnover rate constants (2 to 5 day⁻¹) and pool sizes of sulfate at different sediment depths.