Desulfosporosinus Research Articles

A Thermococci-to-Clostridia Pathway for the Evolution of the Bacteria Domain

With the identification of an archaeal Last Universal Common Ancestor (LUCA) related to the archaeon Methanopyrus, the origin of Bacteria became a choice between autonomic development versus descent from Archaea. The similarity bitscores between paralogous valyl-tRNA synthetase (VARS) and isoleucyl-tRNA synthetase (IARS) suggest that the five oldest bacteria were Mahella australiensis, Thermincola potens, Halobacteroides halobius, Desulfosporosinus orientis and Caldicellulosiruptor lactoaceticus, which were all Clostridia species and hydrogen producers. A search for archaea that could be a candidate Progenitor of Bacteria endowed with an Emden-Myerhof-Parnas type glycolytic pathway and a clostridial-like dark-fermentation mechanism for generating hydrogen pointed to such a role for the anaerobic chemoorganotroph Thermococci, which were known to engage in rapid evolutionary changes at high-biodiversity sites abundant in sugars and other small molecular substrates, and constituted together with Clostridia the two most powerful microbial generators of hydrogen. Moreover, two-domain maximum-likelihood and maximum- parsimony phylogenetic trees for VARS showed that Thermococci and Clostridia formed sister clades on both trees, and close similarity was evident between their VARS sequences, which were consistent with kinship between them. On this basis, it was proposed that the Bacteria domain emerged from a thermocococal or thermococcal-like Progenitor of Bacteria possibly at some high-biodiversity site, through the formation of a Thermincola-proximal Last Bacterial Common Ancestor (LBCA).

Electrophysiology of the Facultative Autotrophic Bacterium Desulfosporosinus orientis.

Electroautotrophy is a novel and fascinating microbial metabolism, with tremendous potential for CO2 storage and valorization into chemicals and materials made thereof. Research attention has been devoted toward the characterization of acetogenic and methanogenic electroautotrophs. In contrast, here we characterize the electrophysiology of a sulfate-reducing bacterium, Desulfosporosinus orientis, harboring the Wood-Ljungdahl pathway and, thus, capable of fixing CO2 into acetyl-CoA. For most electroautotrophs the mode of electron uptake is still not fully clarified. Our electrochemical experiments at different polarization conditions and Fe0 corrosion tests point to a H2- mediated electron uptake ability of this strain. This observation is in line with the lack of outer membrane and periplasmic multi-heme c-type cytochromes in this bacterium. Maximum planktonic biomass production and a maximum sulfate reduction rate of 2 ± 0.4 mM day–1 were obtained with an applied cathode potential of −900 mV vs. Ag/AgCl, resulting in an electron recovery in sulfate reduction of 37 ± 1.4%. Anaerobic sulfate respiration is more thermodynamically favorable than acetogenesis. Nevertheless, D. orientis strains adapted to sulfate-limiting conditions, could be tuned to electrosynthetic production of up to 8 mM of acetate, which compares well with other electroacetogens. The yield per biomass was very similar to H2/CO2 based acetogenesis. Acetate bioelectrosynthesis was confirmed through stable isotope labeling experiments with Na-H13CO3. Our results highlight a great influence of the CO2 feeding strategy and start-up H2 level in the catholyte on planktonic biomass growth and acetate production. In serum bottles experiments, D. orientis also generated butyrate, which makes D. orientis even more attractive for bioelectrosynthesis application. A further optimization of these physiological pathways is needed to obtain electrosynthetic butyrate production in D. orientis biocathodes. This study expands the diversity of facultative autotrophs able to perform H2-mediated extracellular electron uptake in Bioelectrochemical Systems (BES). We characterized a sulfate-reducing and acetogenic bacterium, D. orientis, able to naturally produce acetate and butyrate from CO2 and H2. For any future bioprocess, the exploitation of planktonic growing electroautotrophs with H2-mediated electron uptake would allow for a better use of the entire liquid volume of the cathodic reactor and, thus, higher productivities and product yields from CO2-rich waste gas streams.

Anaerobic Bioreduction of Jarosites and Biofilm Formation by a Natural Microbial Consortium

Jarosite occurs naturally in acid sulphate soils and is a common feature of streams impacted by acid mine drainage (AMD). Biological reduction of iron-sulphate minerals, such as jarosite, has the potential to contribute to the natural attenuation of acid mine drainage sites. The reduction of different jarosites (including minerals containing precious and toxic metals) by a natural bacterial/microbial consortium was examined in this study. Jarosites was used as a sole terminal electron acceptor via the reductive dissolution of Fe(III) minerals. The production of Fe(II) and the presence of sulphate-reducing bacteria in the consortium lead to the precipitation of metal sulphides immobilizing toxic heavy metals. Microbial attachment and biofilm formation of minerals have a great impact on the production and transformation of minerals and can influence the mobility of metals. After the adaptation to different jarosites, a unique specie was found: Desulfosporosinus orientis. Desulfosporosinus species are sulphate-reducing bacteria and can be found in sulphate-rich heavy metal-polluted environments, such as acid mine/rock drainage sites, being responsible for the sulphides formation. D. orientis is an obligate anaerobic microorganism and is able to reduce Fe(III) D. orientis is an obligate anaerobic microorganism and is able to reduce Fe(III). Confocal laser scanning microscopy and fluorescent lectin-binding analyses (FLBA) were used to study the arrangement and composition of the exopolysaccharides/glycoconjugates in biofilms indicating the presence of mannose, glucose, and N-acetylglucosamine residues. This study provides insights to understand the processes leading to the mobility or retention of metals in mine waste and industrial landfill environments.

The effect of gram-positive (Desulfosporosinus orientis) and gram-negative (Desulfovibrio desulfuricans) sulfate-reducing bacteria on iron sulfide mineral precipitation.

Growth of two dissimilatory sulfate-reducing bacteria, Desulfosporosinus orientis (gram-positive) and Desulfovibrio desulfuricans (gram-negative), in a chemically defined culture medium resulted in similar growth rates (doubling times for each culture = 2.8 h) and comparable rates of H2S generation (D. orientis = 0.19 nmol/L S2- per cell per h; D. desulfuricans = 0.12 nmol/L S2- per cell per h). Transmission electron microscopy of whole mounts and thin sections revealed that the iron sulfide mineral precipitates produced by the two cultures were morphologically different. The D. orientis culture flocculated, with the minerals occurring as subhedral plate-like precipitates, which nucleated on the cell wall during exponential growth producing extensive mineral aggregates following cell autolysis and endospore release. In contrast, the D. desulfuricans culture produced fine-grained colloidal or platy iron sulfide precipitates primarily within the bulk solution. Mineral analysis by scanning electron microscopy - energy dispersive spectroscopy indicated that neither culture promoted advanced mineral development beyond a 1:1 Fe:S stoichiometry. This analysis did not detect pyrite (FeS2). The average Fe:S ratios were 1 : 1.09 ± 0.03 at 24 h and 1 : 1.08 ± 0.03 at 72 h for D. orientis and 1 : 1.05 ± 0.02 at 24 h and 1 : 1.09 ± 0.07 at 72 h for D. desulfuricans. The formation of "biogenic" iron sulfides by dissimilatory sulfate-reducing bacteria is influenced by bacterial cell surface structure, chemistry, and growth strategy, i.e., mineral aggregation occurred with cell autolysis of the gram-positive bacterium.

English

Sulfate reducing bacteria (SRB) was successfully isolated from Estuary Dam in Suwung Denpasar, Indonesia. This estuary catches highly polluted water from Badung River which runs across and hence carries pollution due to waste disposal from Denpasar City. SRB was studied in detail for their ability to reduce sulfate to sulfide with organic material as an oxidizing agent. SRB exploration of the estuary ecosystem of the contaminated dam was accomplished through isolation, selection and characterization of the isolates obtained. The result of this study found superior SRB named DPS 1711, DPS 1705 and DPS 1703. The bacteria have the ability to grow at pH 3, room temperature and uses compost as organic substrate. This ability is an important factor for the application of isolates in the treatment of acid mine waste. Isolates have optimum optical density under the pH range of 4 to 7 and the best at pH 5 have a growth rate profile at a temperature range of 25 to 40°C. The isolates observed were Gram-negative stem, motile bacteria which only grow in anaerobic condition. Physiological-biochemical characterization showed the three isolates, namely DPS 1703, DPS 1705 and DPS 1711 were SRB groups identified as Desulfotomaculum orientis. Key words: Sulfate reducing bacteria, polluted waters, estuary dam ecosystem. &nbsp

Marinisporobacter balticus gen. nov., sp. nov., Desulfosporosinus nitroreducens sp. nov. and Desulfosporosinus fructosivorans sp. nov., new spore-forming bacteria isolated from subsurface sediments of the Baltic Sea.

Four novel Gram-stain-positive, endospore-forming bacteria of the order Clostridiales were isolated from subsurface sediments sampled during International Ocean Discovery Program Expedition 347 to the Baltic Sea. One strain (59.4MT) grew as an obligate heterotroph by aerobic respiration and anaerobically by fermentation. Optimum growth was observed with 0.5 % NaCl at 25 °C and pH 7.0-7.3. Analysis of 16S rRNA gene sequences of 59.4MT revealed Alkaliphilus transvaalensis (92.3 % identity), Candidatus Geosporobacter ferrireducens (92.2 %), Geosporobacter subterraneus (91.9 %) and Alkaliphilus peptidifermentans (91.7 %) to be the closest relatives. On the basis of the results of phenotypic and genotypic analyses, we propose that strain 59.4MT represents a novel species within a novel genus, Marinisporobacter balticus gen. nov., sp. nov., with the type strain 59.4MT (=DSM 102940T=JCM 31103T). Three other strains, 59.4F, 59.4BT and 63.6FT, were affiliated with the genus Desulfosporosinus and grew as strictly anaerobic sulfate reducers. These strains additionally used thiosulfate, elemental sulfur, sulfite and DMSO as electron acceptors and hydrogen as an electron donor. Strains 59.4F and 59.4BT had identical 16S rRNA gene sequences, which were most similar to those of Desulfosporosinus lacus (97.8 %), Desulfosporosinus hippei (97.3 %) and Desulfosporosinus orientis (97.3 %). Strain 63.6FT was closely related to D. lacus (97.7 %), Desulfosporosinus meridiei (96.6 %) and D. hippei (96.5 %). The similarity of 16S rRNA gene sequences of strains 59.4BT and 63.6FT was 96.6 %. We propose the new names Desulfosporosinus nitroreducens sp. nov., incorporating strain 59.4F (=DSM 101562=JCM 31104) and the type strain 59.4BT (=DSM 101608T=JCM 31105T), and Desulfosporosinus fructosivorans sp. nov., with the type strain 63.6FT (=DSM 101609T=JCM 31106T).

Microbial Electroreduction: Screening for New Cathodic Biocatalysts

AbstractCathodic microbial electrocatalysis, where microorganisms consume electrons from a cathode to perform biochemical reductions, is currently of high interest. For an effective electrochemical screening of potential new cathode biocatalysts, we developed a time‐saving medium‐throughput potentiostatic BES system to evaluate microbial ability to accept reducing equivalents from an electrode in pure cultures. Of nine tested physiologically and phylogenetically different strains, five promising cathode‐active strains were identified: Acidithiobacillus ferrooxidans (jmax=−50 μA cm−2), Desulfosporosinus orientis (jmax=−4 μA cm−2), Thiobacillus denitrificans and Sulfurimonas denitrificans (jmax=−2 μA cm−2 for both), and Desulfovibrio piger (jmax=−1.5 μA cm−2). From this screening, hypotheses were formulated on possible electron consumption and utilization processes. This work serves as a foundation for deeper investigation and characterization of the identified cathode biocatalysts.

Enrichment and isolation of acidophilic sulfate‐reducing bacteria from Tinto River sediments

Although some acidophilic and alkaliphilic species have been described recently, most of the known sulfate-reducing bacteria (SRB) grow optimally at neutral pH. In this study, sulfate reduction was studied with sediment samples from the extremely acidic Tinto River basin. Stable enrichments of SRB were obtained at pH 4 with glycerol, methanol and hydrogen; at pH 4.5 with lactate and at pH 5.5 with succinate as substrates. Inhibition of sulfate reduction by organic acids below their pKa was observed. Cloning and sequencing of 16S rRNA gene showed that fermentative bacteria (Paludibacter spp., Oscillibacter spp.) and SRB (Thermodesulfobium spp., Desulfosporosinus spp., Desulfitobacterium spp., Desulfotomaculum spp.) were co-enriched. By repeated serial dilutions and streaking on agar plates, four strains of SRB belonging to the Firmicutes phylum were obtained. Two of them show 96% 16S rRNA gene sequence similarity with Desulfosporosinus acidophilus, and a third one with Desulfosporosinus orientis. Another isolate has just 93% rRNA gene sequence similarity with the Desulfosporosinus/Desulfitobacterium cluster and might represent a novel species within a novel genus. One of the Desulfosporosinus strains was further investigated showing maximum growth at pH 5.5, and a pH-dependent inhibitory effect of organic acids and sulfide.

Complete Genome Sequences of Desulfosporosinus orientis DSM765T, Desulfosporosinus youngiae DSM17734T, Desulfosporosinus meridiei DSM13257T, and Desulfosporosinus acidiphilus DSM22704T

Desulfosporosinus species are sulfate-reducing bacteria belonging to the Firmicutes. Their genomes will give insights into the genetic repertoire and evolution of sulfate reducers typically thriving in terrestrial environments and able to degrade toluene (Desulfosporosinus youngiae), to reduce Fe(III) (Desulfosporosinus meridiei, Desulfosporosinus orientis), and to grow under acidic conditions (Desulfosporosinus acidiphilus).

Steel‐Corrosion Inhibitors Derived from Soybean Oil

AbstractSoybean oil derivatives containing a Schiff‐base (SOS‐B) were prepared and evaluated as microbial corrosion inhibitors against sulfate‐reducing bacteria using the gram‐positive Desulfosporosinus orientis bacteria as a representative bacterium. These SOS‐B compounds were also found to be excellent inhibitors against acidic corrosion of carbon steel. These soybean oil derivatives were prepared by ozonation of soybean oil to yield aldehyde functional intermediates which were then reacted with benzylamine to produce a mixture of imine functional triglycerides and linear compounds. The structure of these soy‐based derivatives was confirmed by FTIR and NMR. It was found that the addition of these SOS‐B compounds to D. orientis culture provided a complete inhibition of this bacterium. Furthermore, almost no corrosion of carbon steel panels was observed when the panels were aged in 2N HCl solution containing 10 ppm of these SOS‐B compounds.

BIOTEKNOLOGI LINGKUNGAN UNTUK PENANGGULANGAN LIMBAH MENGANDUNG KROM

The biotechnological approaches for treatment of wastewater are widely studied within the last two decades. We show that this approach can be used for treatment of Cr-contaminated wastewater. The sulfate reducing bacteria Desulfotomaculum orientis ICBB 1204 is used in a laboratory experiment to treat Cr-contained wastewater from metal plating industry and the oil and gas company, PT VICO, East Kalimantan. After adaption of the bacteria for around 10 days in the wastewater containing 100 ppm Cr, the process of Cr reduction took place and the Cr in the medium were reduced 92.7% within 20 days. The similar results were achieved on the treatment of PT VICO’s wastewater. The Cr content in the wastewater decreased from 6.54-6.76 to 0.08-0.09 ppm and the pH increased from 3 to 5 – 6 within 20 days incubation period.Keywords: Cr detoxification, Desulfotomaculum orientis ICBB 1204, sulphate reducing bacteria, wastewater

Desulfosporosinus hippei sp. nov., a mesophilic sulfate-reducing bacterium isolated from permafrost

The sulfate-reducing strain 343T was isolated from ancient permafrost deposits in Siberia, Russia. Based on 16S rRNA gene sequence analyses, this strain was closely related to Desulfosporosinus species, showing 97.9 % 16S rRNA gene sequence similarity to Desulfosporosinus meridiei DSM 13257T, 97.6 % similarity to Desulfosporosinus auripigmenti DSM 13351T, 97.2 % similarity to Desulfosporosinus lacus DSM 15449T and 96.2 % similarity to Desulfosporosinus orientis DSM 765T. The strain was found to contain b-type cytochromes and to reduce only sulfate and thiosulfate using lactate as an electron donor but not sulfite, elemental sulfur, fumarate, nitrate or Fe(III). These data, considered in conjunction with DNA-DNA hybridization data, cell-wall chemotaxonomy and data on physiology, support recognition of strain 343T as representing a distinct and novel species within the genus Desulfosporosinus, namely Desulfosporosinus hippei sp. nov., with the type strain 343T (=DSM 8344T =VKM B-2003T).

A highly sensitive HPLC method for determination of nanomolar concentrations of dipicolinic acid, a characteristic constituent of bacterial endospores

A high-performance liquid chromatographic method with indirect fluorescence detection has been developed for quantification of dipicolinic acid, a major constituent of bacterial endospores. After separation on a reversed-phase column, a post-column reagent of sodium acetate at 1 mol l − 1 with 50 μmol l − 1 terbium chloride was added for complexation of dipicolinic acid. Terbium monodipicolinate complexes formed were quantified by measuring the fluorescence emission maximum at 548 nm after excitation with UV light at 270 nm wavelength. Parameters of post-column complexation were optimized to achieve a detection limit of 0.5 nmol DPA l − 1 , corresponding to about 10 3 Desulfosporosinus orientis endospores per ml. The method was applied to the analysis of spore contamination in tuna and for estimating the endospore numbers in marine sediments.

Sulfidogenesis in Low pH (3.8–4.2) Media by a Mixed Population of Acidophilic Bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8-4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8-4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.

Geochemical, Microbiological, and Geophysical Assessments of Anaerobic Immobilization of Heavy Metals

Bioremediation methods that precipitate contaminants in situ as solid (mineral) phases can provide cost-effective options for removing dissolved metals in contaminated groundwater. The current field-scale experiments demonstrate that indigenous bacteria can be stimulated to remove metals by injection of electron-donating substrates and nutrients into a contaminated aquifer. Groundwater at the investigation site is aerobic and contains high levels of lead, cadmium, zinc, copper, and sulfuric acid (pH = 3.1) derived from a car-battery recycling plant. During the experiments, lead, cadmium, zinc, and copper were almost completely removed by precipitation of solid sulfide phases, as pH increased from 3 to ∼ 5 and Eh dropped from +400 mV to −150 mV. X-ray and transmission electron microscopy (TEM) analyses of filtered material from the treated groundwater indicated the presence of newly formed nanocrystalline metal sulfides. Genetic sequencing indicated that the principal species of sulfate-reducing bacteria involved in the bioremediation process was Desulfosporosinus orientis. Geochemical modeling shows that oxidation of added substrates and subsequent bacterial sulfate reduction produced desired geochemical conditions (i.e., decreasing Eh and increasing pH) for the precipitation and sorption of metal sulfides. Geophysical survey results suggest that bioremediation lowers electrical conductance of groundwater and possibly increases the magnetic susceptibility of porous media. This study demonstrates that integrated geochemical, geophysical, and microbiological analyses, combined with theoretical modeling, can successfully track and predict the progress of subsurface bioremediation.

Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms.

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone reduces corrosion from Desulfotomaculum orientis.

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming and biofilm formation of Gram-positive bacteria (Ren et al., 2002, Lett Appl Microbiol 34: 293-299). In the present study, the Gram-positive sulphate-reducing bacterium (SRB), Desulfotomaculum orientis, was used to study the inhibition of mild steel corrosion due to the addition of furanone. The weight loss from batch coupon experiments incubated with 40 microg x ml(-1) furanone was reduced fivefold compared with samples that lacked furanone. Analysis of the metal surface with environmental scanning electron microscopy further confirmed the protection afforded by the addition of furanone. In agreement with the corrosion inhibition, most probable number (MPN) analysis showed that 20 and 40 microg x ml(-1) furanone inhibited 58% and 96% of the D. orientis growth respectively. Hence, furanone has the potential to inhibit microbial-induced corrosion related to Gram-positive bacteria.

Reclassification of Desulfotomaculum auripigmentum as Desulfosporosinus auripigmenti corrig., comb. nov.

The species Desulfotomaculum auripigmentum is reclassified as Desulfosporosinus auripigmenti corrig., comb. nov. on the basis of morphological and physiological traits, phylogenetic position and chemotaxonomic properties. Characteristics supplementary to those provided in the original description reveal that the type strain, DSM 13351(T) (=ATCC 700205(T)), forms oval, subterminal to terminal spores, possesses LL-diaminopimelic acid and contains MK-7 as the predominant menaquinone, while the whole-cell methanolysate contains even-carbon, straight-chain saturated and mono-unsaturated fatty acids and 1,1-dimethylacetals as major components. DNA-DNA reassociation values below 30 % for Desulfosporosinus orientis DSM 765(T) and Desulfosporosinus meridiei DSM 13257(T) demonstrate that strain DSM 13351(T) shows sufficient genomic differences to maintain its species status. Lack of motility, a smaller cell diameter and the ability to use malate and glycerol as electron donors and fumarate and arsenate as electron acceptors are the main properties that differentiate Desulfosporosinus auripigmenti from the other two species of the genus.

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water.

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50-90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.

Effect of pH on the anaerobic microbial cycling of sulfur in mining-impacted freshwater lake sediments

Lakes caused by coal strip-mining processes are characterized by low pH, low nutrient status, and high concentrations of Fe(II) and sulfate due to the oxidation of pyrite. Most microbiological studies of sulfidic mine tailings concentrate on processes in the oxic zone, and on the ability of acidophilic bacteria to promote the production of toxic leachates. Few studies have studied microbial processes in anoxic sediment zones, where the reduction of Fe(III) often is the dominant electron-accepting process in acidic sediments, and the reduction of sulfate occurs in sediments of elevated pH. In this study, microcosm experiments were performed with sediments of a coal mining-impacted lake to study the microbial turnover of sulfur under anoxic conditions. Microcosms with sediment of the zone of maximum sulfate-reducing activity indicated that sulfidic minerals subsequently formed due to the reduction of sulfate were not stable under anoxic conditions. Supplemental Na 2S stimulated the formation of Fe(II) and sulfate, whereas supplemental S 0 had no effect under pH 5 conditions. Low numbers of S 0-utilizing Fe(III)-reducing bacteria were enumerated in this sediment zone. In contrast, sediment microcosms of the upper acidic sediment zone demonstrated that supplemental elemental S 0 stimulated the formation of Fe(II) and sulfate, whereas supplemental Na 2S had no effect. Most probable number estimates demonstrated that S 0-utilizing Fe(III)-reducing bacteria cultivated at pH 3 approximated to 1% of the total direct counts of bacteria in the upper acidic sediment zone. Sulfate reducers cultured at pH 5.2 were more abundant than sulfate reducers cultured at pH 6.4 in both sediment zones. A sulfate-reducing bacterium, Lau III, was isolated at the in situ pH of 5.2 from the highest growth positive dilution series from the zone of maximum sulfate-reducing activity. Analysis of the 16S rRNA gene sequence (1498 base pairs) of Lau III demonstrated that it was a member of the Clostridium–Bacillus subphylum of gram-positive bacteria related to Desulfosporosinus orientis Y11571 (96.4% 16S rRNA gene sequence similarity). Lau III was a spore-forming, lactate-utilizing sulfate reducer capable of growing at a pH range of 4.9–6.1 and a pH optimum of 5.5. These collective results indicate that, (i) acid-tolerant sulfate reducers are involved in the reduction of sulfate; (ii) sulfide or sulfidic minerals are reoxidized under anoxic conditions; and (iii) the anaerobic turnover of sulfur is affected by the pH gradient in this impacted heterogeneous lake sediment.