Microbial communities and their predicted metabolic functions in a desiccating acid salt lake.

The waters of Lake Magic in Western Australia are among the most geochemically extreme on Earth. This ephemeral saline lake is characterized by pH as low as 1.6 salinity as high as 32% total dissolved solids, and unusually complex geochemistry, including extremely high concentrations of aluminum, silica, and iron. We examined the microbial composition and putative function in this extreme acid brine environment by analyzing lake water, groundwater, and sediment samples collected during the austral summer near peak evapoconcentration. Our results reveal that the lake water metagenome, surprisingly, was comprised of mostly eukaryote sequences, particularly fungi and to a lesser extent, green algae. Groundwater and sediment samples were dominated by acidophilic Firmicutes, with eukaryotic community members only detected at low abundances. The lake water bacterial community was less diverse than that in groundwater and sediment, and was overwhelmingly represented by a single OTU affiliated with Salinisphaera. Pathways associated with halotolerance were found in the metagenomes, as were genes associated with biosynthesis of protective carotenoids. During periods of complete desiccation of the lake, we hypothesize that dormancy and entrapment in fluid inclusions in halite crystals may increase long-term survival, leading to the resilience of complex eukaryotes in this extreme environment.

Endotoxin hitchhiking on polymer nanoparticles.

The control of microbial infections is critical for the preparation of biological media including water to prevent lethal septic shock. Sepsis is one of the leading causes of death in the United States. More than half a million patients suffer from sepsis every year. Both gram-positive and gram-negative bacteria are responsible for septic infection by the most common organisms i.e., Escherichia coli and Pseuodomonas aeruginosa. The bacterial cell membrane releases negatively charged endotoxins upon death and enzymatic destruction, which stimulate antigenic response in humans to gram-negative infections. Several methods including distillation, ethylene oxide treatment, filtration and irradiation have been employed to remove endotoxins from contaminated samples, however, the reduction efficiency remains low, and presents a challenge. Polymer nanoparticles can be used to overcome the current inability to effectively sequester endotoxins from water. This process is termed endotoxin hitchhiking. The binding of endotoxin on polymer nanoparticles via electrostatic and hydrophobic interactions offers efficient removal from water. However, the effect of polymer nanoparticles and its surface areas has not been investigated for removal of endotoxins. Poly(ε-caprolactone) (PCL) polymer was tested for its ability to effectively bind and remove endotoxins from water. By employing a simple one-step phase separation technique, we were able to synthesize PCL nanoparticles of 398.3 ± 95.13 nm size and a polydispersity index of 0.2. PCL nanoparticles showed ∼78.8% endotoxin removal efficiency, the equivalent of 3.9 × 10(5) endotoxin units (EU) per ml. This is 8.34-fold more effective than that reported for commercially available membranes. Transmission electron microscopic images confirmed binding of multiple endotoxins to the nanoparticle surface. The concept of using nanoparticles may be applicable not only to eliminate gram-negative bacteria, but also for any gram-positive bacteria, fungi and parasites.

Ability of a haloalkaliphilic bacterium isolated from Soap Lake, Washington to generate electricity at pH 11.0 and 7% salinity.

A variety of anaerobic bacteria have been shown to transfer electrons obtained from organic compound oxidation to the surface of electrodes in microbial fuel cells (MFCs) to produce current. Initial enrichments for iron (III) reducing bacteria were set up with sediments from the haloalkaline environment of Soap Lake, Washington, in batch cultures and subsequent transfers resulted in a culture that grew optimally at 7.0% salinity and pH 11.0. The culture was used to inoculate the anode chamber of a MFC with formate as the electron source. Current densities up to 12.5 mA/m2 were achieved by this bacterium. Cyclic voltammetry experiments demonstrated that an electron mediator, methylene blue, was required to transfer electrons to the anode. Scanning electron microscopic imaging of the electrode surface did not reveal heavy colonization of bacteria, providing evidence that the bacterium may be using an indirect mode of electron transfer to generate current. Molecular characterization of the 16S rRNA gene and restriction fragment length profiles (RFLP) analysis showed that the MFC enriched for a single bacterial species with a 99% similarity to the 16S rRNA gene of Halanaerobium hydrogeniformans. Though modest, electricity production was achieved by a haloalkaliphilic bacterium at pH 11.0 and 7.0% salinity.

Complete genome sequence of the haloalkaliphilic, hydrogen-producing bacterium Halanaerobium hydrogeniformans.

Halanaerobium hydrogenoformans is an alkaliphilic bacterium capable of biohydrogen production at pH 11 and 7% (wt/vol) salt. We present the 2.6-Mb genome sequence to provide insights into its physiology and potential for bioenergy applications.

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

Whole-Genome Identification and Characterization of Bacterial Insertion Sequences Using Bioinformatic Tools.

Insertion sequences are small mobile regions of DNA (transposable elements) found primarily in prokaryotes. The identification of insertion sequences in bacteria is a growing field of study because of their applications in evolution, genetics, and medicine. One of the first steps in characterizing the insertion sequences found in an organism is to perform a genome-wide survey to identify all insertion sequences using in silico methods. This includes a thorough scan of the genome to locate all copies of different families of insertion sequences and the identification of the key characteristics of each element. The results provide an extensive catalog of the insertion sequences which can be used to further other analyses or manipulation of the genome.

Whole-Genome Analysis of Halomonas sp. Soap Lake #7 Reveals It Possesses Putative Mrp Antiporter Operon Groups 1 and 2.

The genus Halomonas possesses bacteria that are halophilic or halotolerant and exhibit a wide range of pH tolerance. The genome of Halomonas sp. Soap Lake #7 was sequenced to provide a better understanding of the mechanisms for salt and pH tolerance in this genus. The bacterium's genome was found to possess two complete multiple resistance and pH antiporter systems, Group 1 and Group 2. This is the first report of both multiple resistance and pH antiporter Groups 1 and 2 in the genome of a haloalkaliphilic bacterium.

Spatial and temporal patterns in the microbial diversity of a meromictic soda lake in Washington State.

The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

Fe(III), Cr(VI), and Fe(III) mediated Cr(VI) reduction in alkaline media using a Halomonas isolate from Soap Lake, Washington.

Hexavalent chromium is one of the most widely distributed environmental contaminants. Given the carcinogenic and mutagenic consequences of Cr(VI) exposure, the release of Cr(VI) into the environment has long been a major concern. While many reports of microbial Cr(VI) reduction are in circulation, very few have demonstrated Cr(VI) reduction under alkaline conditions. Since Cr(VI) exhibits higher mobility in alkaline soils relative to pH neutral soils, and since Cr contamination of alkaline soils is associated with a number of industrial activities, microbial Cr(VI) reduction under alkaline conditions requires attention.Soda lakes are the most stable alkaline environments on earth, and contain a wide diversity of alkaliphilic organisms. In this study, a bacterial isolate belonging to the Halomonas genus was obtained from Soap Lake, a chemically stratified alkaline lake located in central Washington State. The ability of this isolate to reduce Cr(VI) and Fe(III) was assessed under alkaline (pH = 9), anoxic, non-growth conditions with acetate as an electron donor. Metal reduction rates were quantified using Monod kinetics. In addition, Cr(VI) reduction experiments were carried out in the presence of Fe(III) to evaluate the possible enhancement of Cr(VI) reduction rates through electron shuttling mechanisms. While Fe(III) reduction rates were slow compared to previously reported rates, Cr(VI) reduction rates fell within range of previously reported rates.

Complete Genome Sequence of Salinisphaera sp. Strain LB1, a Moderately Halo-Acidophilic Bacterium Isolated from Lake Brown, Western Australia.

Salinisphaera sp. strain LB1 was isolated from Lake Brown, Western Australia, surface water enriched at pH 4.0 and with 5% (wt/vol) NaCl. The complete genome sequence is presented in this report.

NaCl-saturated brines are thermodynamically moderate, rather than extreme, microbial habitats.

NaCl-saturated brines such as saltern crystalliser ponds, inland salt lakes, deep-sea brines and liquids-of-deliquescence on halite are commonly regarded as a paradigm for the limit of life on Earth. There are, however, other habitats that are thermodynamically more extreme. Typically, NaCl-saturated environments contain all domains of life and perform complete biogeochemical cycling. Despite their reduced water activity, ∼0.755 at 5 M NaCl, some halophiles belonging to the Archaea and Bacteria exhibit optimum growth/metabolism in these brines. Furthermore, the recognised water-activity limit for microbial function, ∼0.585 for some strains of fungi, lies far below 0.755. Other biophysical constraints on the microbial biosphere (temperatures of >121°C; pH > 12; and high chaotropicity; e.g. ethanol at >18.9% w/v (24% v/v) and MgCl2 at >3.03 M) can prevent any cellular metabolism or ecosystem function. By contrast, NaCl-saturated environments contain biomass-dense, metabolically diverse, highly active and complex microbial ecosystems; and this underscores their moderate character. Here, we survey the evidence that NaCl-saturated brines are biologically permissive, fertile habitats that are thermodynamically mid-range rather than extreme. Indeed, were NaCl sufficiently soluble, some halophiles might grow at concentrations of up to 8 M. It may be that the finite solubility of NaCl has stabilised the genetic composition of halophile populations and limited the action of natural selection in driving halophile evolution towards greater xerophilicity. Further implications are considered for the origin(s) of life and other aspects of astrobiology.

A case for the protection of saline and hypersaline environments: a microbiological perspective.

Saline and hypersaline environments are known for their unique geochemical properties, microbial populations and aesthetic appeal. Microbial activities and a spectrum of diversity seen in hypersaline environments are distinct with many novel species being identified and reported on a regular basis. Many distinguishing characteristics about the adaptation, morphology, evolutionary history, and potential environmental and biotechnological applications of these organisms are continually investigated. An abundance of interdisciplinary activities and opportunities exist to explore and understand the importance of these environments that potentially hold promising solutions for current and future global issues. Therefore, it is critical to conserve these unique environments and limit the damage inflicted by anthropogenic influences. Increased salinization due to water diversions, undesired freshening, extensive mineral extraction, sewage effluents, pollution due to agricultural runoff and industrial processes, urbanization, and global climate change are factors negatively affecting hypersaline lakes and their surrounding environments. If these harmful effects continue to proceed at the current or even accelerated rates, irrevocable consequences for these environments will occur, resulting in the loss of potential opportunities to gain new knowledge of the biogeochemistry as well as beneficial microbial populations closely associated with these unique and interesting environments.

Treatment of antibiotics and antibiotic resistant bacteria in swine wastewater with free chlorine.

Recent recognition of the occurrence of antibiotics in the environment has highlighted concerns regarding potential threats of antibiotics to humans and wildlife. Antibiotics are commonly applied to animals to prevent diseases and promote growth, making livestock agriculture a major source of antibiotic pollution. The purpose of our study was to examine chlorination technology as a method for preventing the release of antibiotics as well as antibiotic-resistant bacteria into the environment from concentrated animal feeding operations. Wastewaters from various sites of two anaerobic lagoon systems, one aerated and the other not, on a swine facility were investigated. Each system consisted of a primary treatment lagoon and a subsequent polishing lagoon. Free chlorine (or monochloramine for comparison) was applied to oxidize antibiotics and to disinfect lagoon bacteria as well. Results indicate that aeration substantially improves lagoon functionality, thereby adding both organic and ammonia removal. Ammonia present in the wastewaters plays a critical role in antibiotics decomposition and bacterial inactivation due to its rapid competition for free chlorine to form monochloramine. Generally, a chlorine dose close to breakpoint is required to achieve complete removal of antibiotics, leading to high consumption of free chlorine in most of the wastewaters examined. However, because of a low ammonia concentration in the polishing lagoon wastewater of the aerated system, a chlorine dose of 100 mg/L can effectively achieve complete removal of both antibiotics and bacteria. On the basis of our experimental findings, a possible strategy for the treatment of swine wastewater is suggested.

Disinfection of swine wastewater using chlorine, ultraviolet light and ozone.

Veterinary antibiotics are widely used at concentrated animal feeding operations (CAFOs) to prevent disease and promote growth of livestock. However, the majority of antibiotics are excreted from animals in urine, feces, and manure. Consequently, the lagoons used to store these wastes can act as reservoirs of antibiotics and antibiotic-resistant bacteria. There is currently no regulation or control of these systems to prevent the spread of these bacteria and their genes for antibiotic resistance into other environments. This study was conducted to determine the disinfection potential of chlorine, ultraviolet light and ozone against swine lagoon bacteria. Results indicate that a chlorine dose of 30 mg/L could achieve a 2.2-3.4 log bacteria reduction in lagoon samples. However, increasing the dose of chlorine did not significantly enhance the disinfection activity due to the presence of chlorine-resistant bacteria. The chlorine resistant bacteria were identified to be closely related to Bacillus subtilis and Bacillus licheniformis. A significant percentage of lagoon bacteria were not susceptible to the four selected antibiotics: chlortetracycline, lincomycin, sulfamethazine and tetracycline (TET). However, the presence of both chlorine and TET could inactivate all bacteria in one lagoon sample. The disinfection potential of UV irradiation and ozone was also examined. Ultraviolet light was an effective bacterial disinfectant, but was unlikely to be economically viable due to its high energy requirements. At an ozone dose of 100 mg/L, the bacteria inactivation efficiency could reach 3.3-3.9 log.

Mineralogy and Microbial Diversity of the Microbialites in the Hypersaline Storr's Lake, the Bahamas.

Microbialites found in the low-light-intensity, hypersaline waters of Storr's Lake (SL), San Salvador Island, the Bahamas, were investigated with respect to their morphology, mineralogy, and microbial diversity. Previously described microbialite morphologies, as well as a newly identified "multi-cuspate" morphology, were observed at various depths. Electron microscopy analysis revealed the presence of angular, blocky, and needle-shaped crystals with mineralized cyanobacterial filaments and remains of exopolymeric substances. X-ray diffraction studies confirmed the presence of both Mg-calcite and aragonite in the plateau-mushroom and pinnacle mound microbialites, whereas only Mg-calcite was identified in the other microbialite morphotypes. A comprehensive molecular analysis using barcoded pyrosequencing of five different microbial mat communities identified at least 12 dominant bacterial phyla. Cyanobacteria were generally low in abundance and ranged from ∼0.01% in the deeper pinnacle mounds to ∼3.2% in the shallow calcareous knobs. Other photosynthetic members included green nonsulfur bacteria of the phylum Chloroflexi and purple sulfur bacteria of the class Gammaproteobacteria. All mat types contained significant amounts of sulfate-reducing and dehalogenating bacteria. The low light intensity reaching the deeper microbialites, the lack of dominant cyanobacteria, and the abundance of sulfate reducers and Chloroflexi collectively suggest that sulfate reduction and anoxygenic photosynthetic processes influence the carbonate biomineralization process in these systems.

The Use of a Fractional Factorial Design to Determine the Factors That Impact 1,3-Propanediol Production from Glycerol by Halanaerobium hydrogeniformans.

In recent years, biodiesel, a substitute for fossil fuels, has led to the excessive production of crude glycerol. The resulting crude glycerol can possess a high concentration of salts and an alkaline pH. Moreover, current crude glycerol purification methods are expensive, rendering this former commodity a waste product. However, Halanaerobium hydrogeniformans, a haloalkaliphilic bacterium, possesses the metabolic capability to convert glycerol into 1,3-propanediol, a valuable commodity compound, without the need for salt dilution or adjusting pH when grown on this waste. Experiments were performed with different combinations of 24 medium components to determine their impact on the production of 1,3-propanediol by using a fractional factorial design. Tested medium components were selected based on data from the organism's genome. Analysis of HPLC data revealed enhanced production of 1,3-propanediol with additional glycerol, pH, vitamin B12, ammonium ions, sodium sulfide, cysteine, iron, and cobalt. However, other selected components; nitrate ions, phosphate ions, sulfate ions, sodium:potassium ratio, chloride, calcium, magnesium, silicon, manganese, zinc, borate, nickel, molybdenum, tungstate, copper and aluminum, did not enhance 1,3-propanediol production. The use of a fractional factorial design enabled the quick and efficient assessment of the impact of 24 different medium components on 1,3-propanediol production from glycerol from a haloalkaliphilic bacterium.

Inhibition of microbial metabolism in anaerobic lagoons by selected sulfonamides, tetracyclines, lincomycin, and tylosin tartrate.

Antibiotics are used to maintain healthy livestock and to promote weight gain in concentrated animal feed operations. Antibiotics rarely are metabolized completely by livestock and, thus, are often present in livestock waste and in waste-treatment lagoons. The introduction of antibiotics into anaerobic lagoons commonly used for swine waste treatment has the potential for negative impacts on lagoon performance, which relies on a consortium of microbes ranging from fermentative microorganisms to methanogens. To address this concern, the effects of eight common veterinary antibiotics on anaerobic activity were studied. Anaerobic microcosms, prepared from freshly collected lagoon slurries, were amended with individual antibiotics at 10 mg/L for the initial screening study and at 1, 5, and 25 mg/L for the dose-response study. Monitored metabolic indicators included hydrogen, methane, and volatile fatty acid concentrations as well as chemical oxygen demand. The selected antibiotics significantly inhibited methane production relative to unamended controls, thus indicating that antibiotics at concentrations commonly found in swine lagoons can negatively impact anaerobic metabolism. Additionally, historical antibiotic usage seems to be a potential factor in affecting methane production. Specifically, less inhibition of methane production was noted in samples taken from the lagoon with a history of multiple-antibiotic use.

Going from microbial ecology to genome data and back: studies on a haloalkaliphilic bacterium isolated from Soap Lake, Washington State.

Soap Lake is a meromictic, alkaline (∼pH 9.8) and saline (∼14-140 g liter(-1)) lake located in the semiarid area of eastern Washington State. Of note is the length of time it has been meromictic (at least 2000 years) and the extremely high sulfide level (∼140 mM) in its monimolimnion. As expected, the microbial ecology of this lake is greatly influenced by these conditions. A bacterium, Halanaerobium hydrogeniformans, was isolated from the mixolimnion region of this lake. Halanaerobium hydrogeniformans is a haloalkaliphilic bacterium capable of forming hydrogen from 5- and 6-carbon sugars derived from hemicellulose and cellulose. Due to its ability to produce hydrogen under saline and alkaline conditions, in amounts that rival genetically modified organisms, its genome was sequenced. This sequence data provides an opportunity to explore the unique metabolic capabilities of this organism, including the mechanisms for tolerating the extreme conditions of both high salinity and alkalinity of its environment.

A Streamlined Strategy for Biohydrogen Production with Halanaerobium hydrogeniformans, an Alkaliphilic Bacterium.

Biofuels are anticipated to enable a shift from fossil fuels for renewable transportation and manufacturing fuels, with biohydrogen considered attractive since it could offer the largest reduction of global carbon budgets. Currently, lignocellulosic biohydrogen production remains inefficient with pretreatments that are heavily fossil fuel-dependent. However, bacteria using alkali-treated biomass could streamline biofuel production while reducing costs and fossil fuel needs. An alkaliphilic bacterium, Halanaerobiumhydrogeniformans, is described that is capable of biohydrogen production at levels rivaling neutrophilic strains, but at pH 11 and hypersaline conditions. H. hydrogeniformans ferments a variety of 5- and 6-carbon sugars derived from hemicellulose and cellulose including cellobiose, and forms the end products hydrogen, acetate, and formate. Further, it can also produce biohydrogen from switchgrass and straw pretreated at temperatures far lower than any previously reported and in solutions compatible with growth. Hence, this bacterium can potentially increase the efficiency and efficacy of biohydrogen production from renewable biomass resources.