Microbial Transformation of Antibiotics

Microbial transformation of the nucleoside analogue antibiotic showdomycin was performed using some Streptomyces species. Both the growing culture and the resting cells of Streptomyces sp. No. 383 arrested the antibacterial activity of showdomycin. The inactivated showdomycin was isolated from the reaction mixture by carbon chromatography and was identified with an isoshowdomycin sample which has been chemically derived from showdomycin. It is conjectured that the conversion of showdomycin to isoshowdomycin results from isomerization by an enzyme of Streptomyces sp. No. 383.     

Bioautographic Technique for a Rapid Survey of Microbial Populations for the Production of Antibiotics and Other Metabolites

A method is described for the detection of microorganisms capable of producing antibiotics or other metabolites. The microbial population under study was bioautographed against any desired indicator organism. This was easily accomplished by lifting the entire agar layer out of a petri dish on which the microbial population was grown and transferring it to an agar surface seeded with an indicator organism. The use of this technique in the search for metabolites other than antibiotics is pointed out. The advantages of the method, over previously reported methods, are discussed.     

Thermophilic Microbial Metal Reduction

Thermophilic microorganisms can reduce Fe(III), Mn(IV), Cr(VI), U(VI), Tc(VII), Co(III), Mo(VI), Au(I, III), and Hg(II). Ferric iron and Mn(IV) can be used as electron acceptors during growth; the physiological role of the reduction of the other metals is unclear. The process of microbial dissimilatory reduction of Fe(III) is the most thoroughly studied. Iron-reducing prokaryotes have been found in virtually all of the recognized types of terrestrial ecosystems, from hot continental springs to geothermally heated subsurface sediments. Thermophilic iron reducers do not belong to a phylogenetically homogenous group and include representatives of many bacterial and archaeal taxa. Iron reducing thermophiles can couple Fe(III) reduction with oxidation of a wide spectrum of organic and inorganic compounds. In the thermophilic microbial community, they can fulfil both degradative and productive functions. Thermophilic prokaryotes probably carried out global reduction of metals on Earth in ancient times, and, at the same time, they are promising candidates for use in modern biotechnological processes.     

Susceptibility of Coagulase-negative Staphylococci to Lysostaphin and Other Antibiotics

In general, coagulase-negative staphylococci were found to be relatively less susceptible to the lytic action of lysostaphin than coagulase-positive staphylococci. To achieve, arbitrarily, a lysis greater than 75%, it was necessary to use an increased concentration of enzyme or a longer incubation period than that usually required with coagulase-positive strains. For the most part, the cultures studied were sensitive to oxacillin, cloxacillin, dicloxacillin, nafcillin, ancillin, cephalothin, cephaloridine, fusidic acid, lincomycin, novobiocin, and neomycin [median minimal inhibitory concentrations (MIC) of 1.56 mug/ml or less]. Some degree of resistance (median MIC values of 12.5 mug/ml or greater) to benzylpenicillin, ampicillin, methicillin, tetracycline, chloretetracycline, erythromycin, ristocetin, and lysostaphin was found. Ten methicillin-resistant, coagulase-negative staphylococal strains were found to be cross-resistant to all nine of the penicillins tested, but much less resistant to the two cephalosporin analogues. In several instances, some of these strains seemed to be more sensitive to benzylpenicillin and to certain of the semisynthetic penicillins than to methicillin. Of the 18 antibiotics tested with the viable plate count method, the methicillin-resistant strains were found to be the most sensitive to lincomycin and novobiocin.     

Mineral Type Structures Soil Microbial Communities

Soil microorganisms living in close contact with minerals play key roles in the biogeochemical cycling of elements, soil formation, and plant nutrition. Yet, the composition of microbial communities inhabiting the mineralosphere (i.e., the soil surrounding minerals) is poorly understood. Here, we explored the composition of soil microbial communities associated with different types of minerals in various soil horizons. To this effect, a field experiment was set up in which mineral specimens of apatite, biotite, and oligoclase were buried in the organic, eluvial, and upper illuvial horizons of a podzol soil. After an incubation period of two years, the soil attached to the mineral surfaces was collected, and microbial communities were analyzed by means of Illumina MiSeq sequencing of the 16S (prokaryotic) and 18S (eukaryotic) ribosomal RNA genes. We found that both composition and diversity of bacterial, archaeal, and fungal communities varied across the different mineral surfaces, and that mineral type had a greater influence on structuring microbial assemblages than soil horizon. Thus, our findings emphasize the importance of mineral surfaces as ecological niches in soils.     

Effects of Imposed Salinity Gradients on Dissimilatory Arsenate Reduction, Sulfate Reduction, and Other Microbial Processes in Sediments from Two California Soda Lakes

Salinity effects on microbial community structure and on potential rates of arsenate reduction, arsenite oxidation, sulfate reduction, denitrification, and methanogenesis were examined in sediment slurries from two California soda lakes. We conducted experiments with Mono Lake and Searles Lake sediments over a wide range of salt concentrations (25 to 346 g liter⁻¹). With the exception of sulfate reduction, rates of all processes demonstrated an inverse relationship to total salinity. However, each of these processes persisted at low but detectable rates at salt saturation. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes amplified from As(V) reduction slurries revealed that distinct microbial populations grew at low (25 to 50 g liter⁻¹), intermediate (100 to 200 g liter⁻¹), and high (>300 g liter⁻¹) salinity. At intermediate and high salinities, a close relative of a cultivated As-respiring halophile was present. These results suggest that organisms adapted to more dilute conditions can remain viable at high salinity and rapidly repopulate the lake during periods of rising lake level. In contrast to As reduction, sulfate reduction in Mono Lake slurries was undetectable at salt saturation. Furthermore, sulfate reduction was excluded from Searles Lake sediments at any salinity despite the presence of abundant sulfate. Sulfate reduction occurred in Searles Lake sediment slurries only following inoculation with Mono Lake sediment, indicating the absence of sulfate-reducing flora. Experiments with borate-amended Mono Lake slurries suggest that the notably high (0.46 molal) concentration of borate in the Searles Lake brine was responsible for the exclusion of sulfate reducers from that ecosystem.     

Microbial Responses to Microgravity and Other Low-Shear Environments

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

Microbial Mineral Weathering for Nutrient Acquisition Releases Arsenic

Tens of millions of people in Southeast Asia drink groundwater contaminated with naturally occurring arsenic. How arsenic is released from the sediment into the water remains poorly understood. Here, we show in laboratory experiments that phosphate-limited cells of Burkholderia fungorum mobilize ancillary arsenic from apatite. We hypothesize that arsenic mobilization is a by-product of mineral weathering for nutrient acquisition. The released arsenic does not undergo a redox transformation but appears to be solubilized from the apatite mineral lattice during weathering. Analysis of apatite from the source area in the Himalayan basin indicates the presence of elevated levels of arsenic, with an average concentration of 210 mg/kg. The rate of arsenic release is independent of the initial dissolved arsenic concentration and occurs at phosphate levels observed in Bangladesh aquifers. We also demonstrate the presence of the microbial phenotype that releases arsenic from apatite in Bangladesh aquifer sediments and groundwater. These results suggest that microbial mineral weathering for nutrient acquisition could be an important mechanism for arsenic mobilization.Tens of millions of people in Southeast Asia are drinking groundwater containing elevated levels of arsenic that is naturally released from the sediment into the groundwater (22, 49). Multiple mechanisms have been proposed to explain this phenomenon, including arsenic mobilization during reduction of Fe(III) (hydr)oxides (14, 22, 27), arsenate reduction and mobilization (25, 28, 51), and redox cycling and release from arsenic-rich sulfides (30). Each mechanism probably plays a role under certain conditions but cannot fully explain the prevalence of arsenic or all the features of its distribution in groundwater (39, 42).It is well known that bacteria can obtain nutrients from solid-phase minerals (1, 4, 5, 36, 47). Subsurface environments are oligotrophic, and bacteria have been shown to preferentially weather minerals that contain phosphate and ammonia, both required for growth (36, 37). Microbial production of organic acids, ligands, and extracellular polysaccharides could all promote dissolution of minerals (35, 44-46). This is different than microbes using solid-phase minerals in redox processes such as Fe(III) reduction, resulting in the release of toxic metals (2, 24, 28). During redox reactions, microbes use the solid-phase minerals for energy. During nutrient acquisition, microbes access trace nutrients from the minerals for cellular growth (5), and redox alterations may not be occurring. Despite the importance of bacterially promoted mineral weathering for nutrient acquisition, the release of potentially dangerous ancillary elements such as arsenic from minerals to groundwater has not been investigated.We propose a novel mechanism for arsenic mobilization in aquifer systems: microorganisms weather and dissolve minerals to obtain nutrients and, during this process, cause an ancillary release of arsenic into the groundwater. Microcosm experiments were conducted with Burkholderia fungorum, phosphate-free artificial groundwater (AGW), and natural apatite. Apatite from the Himalayas was also analyzed for arsenic. Finally, bacteria extracted from Bangladesh sediments and water samples were incubated on mineral phosphate-solubilizing (MPS) plates to determine if this phenotype is present in arsenic-rich deltaic environments. Results indicate that ancillary arsenic release may mobilize arsenic in aquifer systems.The generalized formula for apatite is Ca₅(PO₄)₃(F,Cl,OH). Purified apatite was obtained from the Himalayas (18) and was digested with hot nitric acid. Digestions were performed to determine the total amount of arsenic associated with the apatite. Approximately 0.5 g of apatite was digested in 5 ml of nitric acid at 90°C for 1 h and then brought to 20 ml with deionized water (8). The total amount of arsenic was measured by inductively coupled plasma mass spectrometry. For microcosm experiments, apatite was purchased from Ward''s Scientific and ground with mortar and pestle to a fine powder. It contained 470 mg/kg arsenic. The percentages of As(V) and As(III) in the solid-phase arsenic from the Ward''s Scientific apatite and one Himalayan sample were determined by X-ray absorption near-edge structure spectroscopic analyses at the Stanford Synchrotron Radiation Laboratory on beam line 11-2 (13, 31, 34).Burkholderia fungorum was utilized because of its ability to weather rocks for nutrient acquisition. Specifically, experiments with basalt and granite have shown that it can weather these rocks and release phosphate into the environment (47, 48). Members of the Burkholderia genus have been isolated from granitic soils (48) and have been shown to be efficient at mineral weathering (40). Unfortunately, little work has been done to characterize microbial populations in the aquifers and soils of Southeast Asia, and isolates were not available (9, 19, 20). The metabolic function of Burkholderia fungorum is indistinguishable from those of other heterotrophic bacteria and therefore was chosen as a model organism (12).Burkholderia fungorum (ATCC BAA-463) was grown overnight at 30°C while being shaken in tryptic soy broth, washed, and resuspended in P-limited AGW. Approximately 5 × 10⁵ cells/ml were added to each microcosm. In abiotic controls, cells were killed by being autoclaved and then added to the microcosm. The P-limited AGW is based on the recipe of Wu et al. (47) and is similar to low-ionic-strength groundwater; per liter of solution, the major components are glucose (0.2 g), NH₄Cl (0.04 g), KCl (5.0 × 10⁻⁴ g), and MgSO₄ (5.0 × 10⁻⁴ g) (added as 1 × 10⁻³ g MgSO₄ · 7H₂O). Glucose was added as the carbon source. Each 500-ml Wheaton bottle contained 200 ml of P-limited AGW and 2 g of apatite. Bottles were shaken at 30°C throughout the course of the experiment. The microcosm experiments were performed under oxic conditions. Experiments included addition of (i) 2 ml of live B. fungorum cells, (ii) 2 ml of killed B. fungorum cells, (iii) a no-cell control, (iv) 2 ml of live B. fungorum cells and an arsenate spike, and (v) 2 ml of live B. fungorum cells with a pH 7 phosphate spike. Samples were spiked with arsenic because recent field studies have shown that arsenic release is independent of the initial arsenic concentration (39). Therefore, a field-relevant method should indicate release in the presence of increasing initial arsenic concentrations. A phosphate spike was included to determine if release occurred under field-relevant phosphate conditions. Abiotic experiments were performed with gluconic acid and the Ward''s Scientific apatite. Eight incubations with 0.2 g of apatite and 20 ml of gluconic acid were performed at concentrations ranging from 314 nM to 3.14 M. Samples were shaken at 200 rpm for 24 h at 37°C.Aliquots were removed from each microcosm at the sampling points for analysis of pH, gluconic acid, glucose, cations, and trace metals. pH was analyzed immediately with an Accumet pH electrode (Fisher Scientific). Glucose and gluconic acid samples were filtered with a Millex-GP 0.22-μm filter (Millipore) and frozen. Glucose was analyzed by the Amplex red glucose kit (Invitrogen) using a Stratagene Mx3005P (La Jolla, CA) system in plate reader mode. Gluconic acid was measured by ion chromatography on a Dionex ICS-2000 (Sunnyvale, CA) with an AS11HC column in gradient mode. Trace metal and cation samples were filtered with a Millex-GP 0.22-μm filter (Millipore) and stored in 1% hydrochloric acid at 4°C. Samples were analyzed by inductively coupled plasma mass spectrometry with a high-resolution Axiom single collector instrument (21). Arsenic species, i.e., As(III) and As(V), were determined by hydride generation atomic fluorescence spectrometry (32).The amount of phosphate remaining in the apatite was not examined because only a small percentage of the apatite was solubilized and it would not be possible to detect these small variations. Previous microcosm experiments with phosphate-limited AGW and basalt or granite have shown that little to no cell growth occurs in the absence of a mineral phosphate source and that apatite was most likely the source of phosphate (47, 48). Plating and flow cytometer analyses during the initial experiments indicated that without phosphate or apatite, significantly less growth occurred. However, the fine-grained apatite in the microcosms made flow cytometer counting and plate counting difficult. Therefore, the mineral solids at the end of one round of incubation were examined by scanning electron microscopy (SEM) to better understand microbe-mineral interactions (7). Samples were prepared inside an anaerobic glove bag. To prepare samples for SEM observations, cell-mineral suspensions were fixed in 2.5% glutaraldehyde in a 0.05 M Na cacodylate buffer solution of pH 7. One droplet of fixed cell-mineral gel was placed on the surface of a glass coverslip that was cleaned with 1 mg/ml polylysine solution prior to use. The sample was allowed to settle down onto the coverslip for 20 min. The sample-coated coverslips were sequentially dehydrated using various proportions of ethanol and distilled water, followed by critical point drying (7). The coverslips were mounted onto SEM stubs and Au coated for observations with a Zeiss Supra 35 VP FEG scanning electron microscope. The only time that the samples were exposed to air was during critical point drying and Au coating. The scanning electron microscope was operated at the accelerating voltage of 15 kV. Short working distance (6 to 10 mm) and low beam current (30 to 40 mA) were used to achieve maximum image resolution.MPS phenotype plates were prepared in two layers (10). The bottom layer consisted of 10 ml of 1% purified agar. The top layer consisted of a nutrient medium with 2% purified agar, 1% glucose, 0.1% MgSO₄ (0.21% MgSO₄·7H₂O), 0.1% NaCl, and 0.5% NH₄Cl. After being autoclaved, 0.5 g sterile hydroxyapatite (Sigma), vitamins, and minerals were added to the top layer of media. The hydroxyapatite was washed 10 times in deionized water to remove sorbed phosphate. The vitamin and mineral mix was altered (23), and no phosphate was present in the MPS phenotype plates except for in the solid-phase hydroxyapatite. Plates were inoculated with cells and incubated aerobically at 30°C. When poured, the plate contents are cloudy because of the presence of solid-phase hydroxyapatite in the agar. The only mechanism causing the plates to become clear is the solubilization of hydroxyapatite. Previous studies have shown that clearings on the plates occur only in the presence of the MPS phenotype (10).Four sediment samples and six water samples from Bangladesh were tested for the presence of the MPS phenotype. All water and sediment samples were from Araihazar Upazila, located 30 km east of Dhaka in a transition zone between the uplifted, low-arsenic Pleistocene Madhupur terrace to the northwest and a high-arsenic region on the opposite bank of the Meghna River to the southeast (50). Groundwater arsenic concentrations in the region range from less than 5 μg/liter to 900 μg/liter (43). Sediments were extracted by combining 10 g of sediment with 20 ml of AGW augmented with 1% Tween 80 and shaken overnight at 30°C (3). The sediment was allowed to settle by gravity, two volumes were plated, 50 μl and 100 μl, and the plates were sealed with Parafilm and incubated at 30°C. Collecting microbially pristine sediment samples from Bangladesh aquifers is difficult and requires novel techniques (33, 41) compared to those traditionally utilized (16, 29). Traditional drilling techniques are not available, and extensive work has been undertaken with the local drillers to obtain pristine samples that are not contaminated by drilling fluid (33, 41). Briefly, during drilling a core or evacuated cylinder is lowered through the drill casing to collect sediment from below the casing. Sediment is therefore collected below the casing from undisturbed sediment. The sediment was stored anaerobically at room temperature until use. These sediments are considered to be pristine with unaltered microbial populations and are our best method for assessing in situ microbial processes (42).Groundwater samples obtained by sterile techniques were also examined. Groundwater from six wells was concentrated by ultrafiltration (15), and the concentrated retentate was utilized to inoculate plates. Briefly, 100 liters of water was concentrated to 250 ml using a Rexeed 25S dialysis filter (Dial Medical Supply, Chester Springs, PA), and 100 μl of this retentate was used to inoculate the plates. The plates were then stored at room temperature in the dark, and growth was observed over the first week. This method was performed with a sterile technique at the wellhead in Bangladesh and greatly decreased the chance of having problems with contamination or long-term storage. Standard MPS plates plus three controls were included in the experiment. The four-plate procedure included (i) standard MPS plates, (ii) MPS plates with no phosphate and no hydroxyapatite, (iii) MPS plates with phosphate but no hydroxyapatite, and (iv) MPS plates with phosphate and hydroxyapatite. The goal of using the additional plates was to better understand under what conditions hydroxyapatite is mobilized and further constrain the phenotype in Bangladesh. Good photographs of the plates could not be obtained at the field clinic, but consistent patterns were observed and reported.Apatite, Ca₅(PO₄)₃(F,Cl,OH), is an abundant source of phosphate and is present in the aquifers and rivers of Bangladesh (6, 22). Dissolution of 13 purified apatite samples from the Himalayas resulted in an average arsenic concentration of 210 mg/kg (all values are 1, 3, 10, 11, 15, 24, 51, 78, 110, 200, 220, 250, and 1,800 mg/kg). These samples are not from Bangladesh but were upgradient within the drainage basin and may represent source material for the delta. These apatite samples were not used in the incubations, as we used the standard mineral, but indicate that apatite is a potential source of phosphate and also arsenic for the shallow aquifers in Bangladesh. Arsenic X-ray absorption near-edge structure analysis on beam line 11-2 at the Stanford Synchrotron Radiation Laboratory of the Ward''s Scientific apatite used in the microcosms and the 51-mg/kg sample from the Himalayas indicated that both samples had approximately 84% ± 6% arsenate and 16% ± 6% arsenite. We assume that the arsenic is replacing the phosphorus in the mineral structure and not just sorbed on the surface, since washes with water do not release the arsenic from the mineral.Aqueous samples were collected from a series of microcosm incubations over time and analyzed. Control incubations with killed cells and without cells did not release phosphorus, calcium, or arsenic, and glucose levels remained unchanged (Fig. ​(Fig.1).1). In live incubations, phosphorus and calcium concentrations increased over time, indicating dissolution of apatite, and glucose levels decreased, indicating microbial respiration (Fig. ​(Fig.1).1). More phosphorus is released than can be consumed by the bacteria, and the concentrations increase over time. More importantly, arsenic levels systematically increased with calcium and phosphorus from 4 to 35 μg/liter arsenic. The increase was consistent in duplicate bottles and experiments (Fig. ​(Fig.1).1). The arsenic was released as oxidized arsenate in all incubations tested, with an average of 87% ± 5% As(V) and 13% ± 5% As(III) in the aqueous phases of the microcosms (Fig. ​(Fig.1E).1E). The percentages of As(V) and As(III) in the aqueous phase were nearly identical to those in the mineral. Therefore, no arsenic redox chemistry was occurring. This indicates that microbes catalyzed the release of arsenic from the apatite. We hypothesize that microbes preferentially weather minerals for nutrient acquisition, and during this process, arsenic contained in the mineral will be released into the water.Open in a separate windowFIG. 1.Change in concentrations over time during microcosm experiments examining arsenic release from apatite. Results are shown for two duplicate microcosms and for the no-cell control. The killed control is shown in Fig. ​Fig.4,4, and the killed control and no-cell control were always nearly identical. (A) Change in calcium concentrations over time. (B) Change in pH over time. (C) Change in glucose concentrations over time. (D) Change in phosphorus concentrations over time. (E) Change in arsenic concentrations over time. The proportion of arsenate As(V) relative to As(V) plus As(III) is indicated at the fourth time point and is presented as a percentage. The arsenic was released as oxidized arsenate in all incubations tested, with an average of 87% ± 5% As(V) and 13% ± 5% As(III) in the aqueous phases of the microcosms. (F) Change in gluconic acid concentrations over time.SEM was performed to better understand mineral dissolution and potential microbe-mineral interactions. In the killed control (Fig. ​(Fig.2A),2A), apatite grains are observed with no cells and no dissolution features. In the live incubations with cells, glucose, and apatite, there is an intimate relationship between the apatite minerals and the cells (Fig. ​(Fig.2B).2B). Cells are observed on the surfaces of the apatite, and dissolution textures are present on the surfaces of the apatite. These observations are consistent with the idea that apatite was weathered by B. fungorum and that during this weathering process, arsenic was released.Open in a separate windowFIG. 2.Secondary electron images from microcosm sediments at the end of 24 h of incubation. (A) Image of apatite grains from the killed control. Microbes were not observed in association with the minerals, and the minerals appear unaltered. (B) Image of apatite grains from a live microcosm amended with B. fungorum and glucose. Rod-shaped cells are observed in close contact with the apatite minerals, and dissolution features are present on the minerals.Further measurements and experiments were conducted to better understand the mechanisms of apatite dissolution and arsenic release (Fig. ​(Fig.3).3). Control incubations with killed cells and without cells did not show a change in pH over the course of the incubations (Fig. ​(Fig.11 and ​and4).4). In live incubations, the pH level systematically decreased (Fig. ​(Fig.1).1). In the no-cell control, gluconic acid levels were constant, whereas in the live incubations, gluconic acid levels increased (Fig. ​(Fig.1E).1E). The production of gluconic acid from glucose during respiration has previously been observed with Burkholderia fungorum (11, 46, 48). Gluconic acid is typically further oxidized by bacteria but can accumulate under stress (12). In order to compare microbial metabolism to abiotic controls, apatite was shaken for 24 h at 30°C in increasing strengths of gluconic acid (Fig. ​(Fig.3).3). The amount of arsenic release was approximately linear on a log-log scale of gluconic acid and arsenic concentrations. In addition, the amount of arsenic released during the live microcosms due to microbial production of gluconic acid was consistent with the abiotic experiments (Fig. ​(Fig.3).3). This indicates that microbial oxidative degradation of glucose to gluconic acid by microbes in intimate contact with apatite is a likely cause of arsenic release from the mineral structure into the water.Open in a separate windowFIG. 3.Gluconic acid concentration versus arsenic concentration from abiotic experiments, along with the average gluconic acid and arsenic concentrations from the live microcosms in Fig. ​Fig.1.1. A log-log relationship was observed between gluconic acid concentrations and the amount of arsenic released from the apatite. In addition, the amount of arsenic released during microcosm experiments was consistent with that of the abiotic experiments.Open in a separate windowFIG. 4.(A) Release of arsenic over time at various initial arsenic concentrations. These experiments were conducted to determine if the release of arsenic occurs at all initial arsenic concentrations, as observed in the field. Microcosms were spiked with arsenate. The killed control is shown for reference, which did not release arsenic. (B) Change in pH over time for the arsenic spike experiments. (C) Change in concentration of arsenic over time at different initial phosphate concentrations. All microcosms contained B. fungorum, and the phosphate was spiked at pH 7. (D) pH changes during the phosphate spike experiments. (E) Release of arsenic as a function of initial phosphate concentrations. The gray bars indicate the distribution of detectable phosphate levels observed in Bangladesh aquifers, as determined by the British Geological Survey (BGS) (22).Additional incubations were conducted to examine the effect of increasing initial arsenic and phosphate concentrations and to better constrain field observations. The amount of arsenic released as a function of initial arsenic concentration was examined because field observations indicate that the release of arsenic occurs at a constant rate and is independent of arsenic concentration (39). Results indicate that during ancillary arsenic release, the amount of arsenic released is independent of the initial arsenic concentration (Fig. ​(Fig.4A).4A). In addition, the pH decreases consistently at all arsenic levels consistent with the mechanism of gluconic acid production (Fig. ​(Fig.4B).4B). We examined the amount of arsenic released as a function of phosphate concentrations to determine if release occurs under field-relevant conditions. The amount of arsenic released decreases as the initial aqueous phosphate concentration increases above 0.1 mM (Fig. 4C and E). The arsenic is released when the pH decreases but remains steady when 10 and 100 mM phosphate are present. The limiting phosphate concentrations are higher than concentrations observed in Bangladesh groundwater (Fig. ​(Fig.4E).4E). Microbially mediated arsenic release from a commonly occurring mineral was documented in the presence of increasing arsenic concentrations and at in situ phosphate levels, indicating that the mechanism is consistent with field observations.In separate experiments, we checked for the presence of the MPS phenotype in bacteria collected from sediments of shallow Bangladesh aquifers. Cells extracted from Bangladesh sediment were incubated on MPS phenotype plates that contained only solid-phase phosphate. Each sample gave rise to colonies surrounded by easily observed clearings (Fig. ​(Fig.5).5). These clearings can be present only if the hydroxyapatite is solubilized. Plates had, on average, about 5 to 10 colonies with clearings, which corresponds to about 100 to 200 colonies per gram. After performing initial sediment incubations, we returned to Bangladesh and incubated MPS plates with groundwater that was concentrated by ultrafiltration using sterile techniques. Four types of plates were incubated as follows: (i) standard MPS plates, (ii) MPS plates with no phosphate and no hydroxyapatite, (iii) MPS plates with phosphate but no hydroxyapatite, and (iv) MPS plates with phosphate and hydroxyapatite. A consistent pattern emerged with the plates (data not shown). The standard MPS plates had colonies and abundant clearings (∼100 colonies). The MPS plates with no phosphate and no hydroxyapatite had colonies but significantly fewer colonies (∼20), indicating that phosphate may have been limited. The MPS plates with phosphate but no hydroxyapatite had significant colonies (∼100). The MPS plates with phosphate and hydroxyapatite had colonies (∼100) but no clearings, indicating that cells were using the soluble phosphate. When phosphate was present in either the solid or aqueous phase, significantly more colonies were observed. This would correspond to about 3 colonies per ml of groundwater. When only solid-phase phosphate as hydroxyapatite was present, clearings were observed. This was corroborating evidence that the MPS phenotype is present in Bangladesh, and under low phosphate conditions, hydroxyapatite is solubilized. This indicates that bacteria capable of solubilizing phosphate from solid-phase hydroxyapatite are present in the shallow aquifers of Bangladesh.Open in a separate windowFIG. 5.MPS phenotype plate containing only solid-phase phosphate as hydroxyapatite was incubated with cells extracted from shallow Bangladesh sediment. The clearings are highlighted by arrows and indicate that phosphate-solubilizing bacteria were extracted from the sediment and are present in the shallow aquifers of Bangladesh. Small clearings are observed around many of the other colonies but do not appear in the photographs.We hypothesize that the required conditions for ancillary arsenic release during microbial mineral weathering appear to include a nutrient limitation, the presence of poorly weathered minerals such as apatite, a supply of organic carbon, and an active microbial population. Each of these conditions is present for Bangladesh aquifers. The MPS phenotype is present in Bangladesh, and the in situ phosphate levels should promote its expression and, hence, mineral weathering with the associated arsenic release. Bangladesh aquifers are organic carbon rich, and organic carbon has been correlated with arsenic (26). Ancillary arsenic release may also occur during weathering of feldspars, biotite, and hornblende. These minerals are common in Bangladesh aquifers (6, 31, 38), and they have been shown to be potential sites of microbial weathering (5, 36, 37, 40).We postulate that nutrient acquisition and ancillary arsenic release are independent of redox conditions and will also occur during fermentation, Fe(III) reduction, and methanogenesis. Importantly, we believe that ancillary arsenic release has been obscured by the overprint of iron cycling. Laboratory and field experiments are showing that Fe and As release are decoupled (17, 33, 42) and that arsenic release occurs at a constant rate across various depositional environments (39). These observations are consistent with our novel mechanism that can occur under all redox conditions. Therefore, we consider ancillary arsenic release to potentially be a more important factor than Fe(III) reduction for controlling arsenic release from the minerals into the groundwater of Bangladesh. This novel mechanism may clarify some of the unexplained geochemical patterns related to the presence of arsenic in groundwater and its worldwide impact on human health.     

Mineral Influence on Microbial Survival During Carbon Sequestration

Geologic carbon sequestration involves the injection of supercritical carbon dioxide into deep saline aquifers. Some of the CO₂ dissolves into the brines, perturbing water chemistry and water-rock interactions, and impacting microbial habitat and survival. In this study 3 model organisms were tested for their ability to survive high pressures of CO₂ exposure in batch cultures: the gram-negative Shewanella oneidensis (SO) strain MR-1, the gram-positive Geobacillus stearothermophilus (GS), and the methanogenic archaeon Methanothermobacter thermoautitrophicus (MT). Results indicate that GS can survive the highest pressures of CO₂ for the longest periods of time while SO is the most sensitive to CO₂ toxicity. Survival was then evaluated for SO with various minerals and rocks representative of deep saline aquifers to determine if minerals enhanced survival. Cultures were exposed to 25 bar of CO₂ for 2 to 8 h and were plated for viable cell counts. Results show that biofilm formation on the mineral surface is important in protecting SO from the harmful effects of CO₂ with quartz sandstones providing the best protection. The release of toxic metals like Al or As from minerals such as clays and feldspars, in contrast, may enhance microbial death under CO₂ stress.     

微生物技术去除抗生素残留污染的研究进展

抗生素进入环境会对生物造成深远的影响,如何去除抗生素的残留引起许多国家的关注。抗生素在环境中主要发生生物降解,而具有抗性的微生物菌株发挥主要的功效,因此近些年利用微生物技术处理抗生素残留污染成为研究热点。本文对具有抗生素降解功能的微生物资源和利用复合菌系处理抗生素残留的生物技术进行概括总结,并对微生物处理技术的不足和发展方向进行展望。     

Environmental Factors That Control Microbial Perchlorate Reduction

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter⁻¹ were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.     

Phenazines and other redox-active antibiotics promote microbial mineral reduction

Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction. Pseudomonas chlororaphis PCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures of P. chlororaphis implies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacterium Shewanella oneidensis MR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.     

Microbial Reduction and Precipitation of Vanadium by Shewanella oneidensis

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V^V). The bacterium reduces V^V (vanadate ion) to V^IV (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V^IV-containing solid.     

Carbonate Mineral Precipitation for Soil Improvement Through Microbial Denitrification

Microbially induced carbonate precipitation (MICP) and associated biogas production may provide sustainable means of mitigating a number of geotechnical challenges associated with granular soils. MICP can induce interparticle soil cementation, mineral precipitation in soil pore space and/or biogas production to address geotechnical problems such as slope instability, soil erosion and scour, seepage of levees and cutoff walls, low bearing capacity of shallow foundations, and earthquake-induced liquefaction and settlement. Microbial denitrification has potential for improving the mechanical and hydraulic properties of soils because it promotes precipitation of calcium carbonate (CaCO₃) and produces nitrogen (N₂) gas without generating toxic by-products. We evaluated the potential for inducing carbonate precipitation in soil via bacterial denitrification using bench-scale experiments with the facultative anaerobe Pseudomonas denitrificans. Bench-scale experiments were conducted (1) without calcium in an N-rich bacterial growth medium in 2.0 L glass batch reactors and (2) with a source of calcium in sand-filled acrylic columns. Changes of pH, alkalinity, NO₃^? and NO₂^? in the batch reactors and columns, quantification of biogas production and observations of calcium-carbonate precipitation in the sand-filled columns indicate that denitrification led to carbonate precipitation and particle cementation in the pore water as well as a substantial amount of biogas production in both systems. These results document that bacterial denitrification has potential as a soil improvement mechanism.     

Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.