A polyethylene surrogate for microbial community enrichment and characterization.

Plastic pollution is a vast and increasing problem that has permeated the environment, affecting all aspects of the global food web. Plastics and microplastics have spread to soil, water bodies, and even the atmosphere due to decades of use in a wide range of applications. Plastics include a variety of materials with different properties and chemical characteristics, with polyethylene being a dominant fraction. Polyethylene is also an extremely persistent compound with slow rates of photodegradation or biodegradation. In this study, we developed a method to isolate communities of microbes capable of biodegrading a polyethylene surrogate. This method allows us to study potential polyethylene degradation over much shorter time periods. Using this method, we enriched several communities of microbes that can degrade the polyethylene surrogate within weeks. We also identified specific bacterial strains with a higher propensity to degrade compounds similar to polyethylene. We provide a description of the method, the variability and efficacy of four different communities, and key strains from these communities. This method should serve as a straightforward and adaptable tool for studying polyethylene biodegradation.

Enhanced extracellular ammonium release in the plant endophyte Gluconacetobacter diazotrophicus through genome editing.

IMPORTANCE: Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus. Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe.

Gluconacetobacter diazotrophicus Gene Fitness during Diazotrophic Growth.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.

Rnf1 is the primary electron source to nitrogenase in a high-ammonium-accumulating strain of Azotobacter vinelandii.

The enzyme nitrogenase performs the process of biological nitrogen fixation (BNF), converting atmospheric dinitrogen gas into the biologically accessible ammonia, which is rapidly protonated at physiological pH to yield ammonium. The reduction of dinitrogen requires both ATP and electrons. Azotobacter vinelandii is an aerobic nitrogen-fixing microbe that is a model organism for the study of BNF. Previous reports have described strains of A. vinelandii that are partially deregulated for BNF, resulting in the release of large quantities of ammonium into the growth medium. Determining the source of the electrons required to drive BNF is complicated by the existence of several protein complexes in A. vinelandii that have been linked to BNF in other species. In this work, we used the high-ammonium-accumulating strains of A. vinelandii to probe the source of electrons to nitrogenase by disrupting the Rnf1 and Fix complexes. The results of this work demonstrate the potential of these strains to be used as a tool to investigate the contributions of other enzymes or complexes in the process of BNF. These results provide strong evidence that the Rnf1 complex of A. vinelandii is the primary source of electrons delivered to the nitrogenase enzyme in this partially deregulated strain. The Fix complex under native regulation was unable to provide sufficient electrons to accumulate extracellular ammonium in the absence of the Rnf1 complex. Increased ammonium accumulation could be attained in a strain lacking the Rnf1 complex if the genes of the Fix protein complex were relocated behind the strong promoter of the S-layer protein but still failed to achieve the levels found with just the Rnf1 complex by itself. KEY POINTS: • The Rnf1 complex is integral to ammonium accumulation in A. vinelandii. • The Fix complex can be deleted and still achieve ammonium accumulation in A. vinelandii. • A. vinelandii can be engineered to increase the contribution of the Fix complex to ammonium accumulation.

Canvasing the Substrate-Binding Pockets of the Wax Ester Synthase.

The biosynthesis of wax esters and triglycerides in bacteria is accomplished through the action of the wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase). A hallmark of these enzymes is the broad substrate profile that accepts alcohols, diglycerides, and fatty acyl-CoAs of various carbon chain lengths and degrees of branching. These enzymes have a broad biotechnological potential due to their role in producing high-value lipids or simple fuels similar to biodiesel through biosynthetic routes. Recently, a crystal structure was solved for the wax ester synthase from Marinobacter aquaeolei VT8 (Maqu_0168), providing a much clearer picture of the architecture of this enzyme and enabling a more precise analysis of the important structural features of the protein. In this work, we used the structure to canvas amino acids lining the proposed substrate-binding pockets and tested the effects of exchanging specific residues on the substrate profiles. We also developed an approach to better probe the residues that alter fatty acyl-CoA selectivity, which has proven more difficult to investigate. Our findings provide an improved blueprint for future efforts to understand how these enzymes position substrates for catalysis and to tailor or improve these enzymes in future biosynthetic schemes.

Genomic analysis and characterization of Scenedesmus glucoliberatum PABB004: An unconventional sugar-secreting green alga.

AIMS: In this report, we present Scenedesmus glucoliberatum PABB004, a microalga that was isolated from an association with Paramecium bursaria with the potential for application in fermentative processes and co-culture schemes due to its advantageous high sugar secretion phenotype. METHODS AND RESULTS: We sequenced, assembled and annotated the draft genome and transcriptome for this newly reported strain. The nuclear genome has an exceptionally high GC content of 78%. Our results revealed significant sugar accumulation over a range from 6.2 to 7.8 pH units. The predicted proteome was compared with other green algae that show different sugar secretion phenotypes aiming to help uncover their common features for simple sugar secretion and those unique to S. glucoliberatum PABB004. CONCLUSIONS: The evolutionary history of this organism, inferred from its genomic traits, expands our current understanding of algal mutualistic relationships involving photosynthate exchanges. S. glucoliberatum PABB004 secreted ready-to-use fermentable sugars (glucose and maltose) directly to the extracellular media achieving concentrations greater than 2.7 g/L of free glucose and 1.2 g/L of maltose in batch cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: A draft genome is provided for a new member of an important class of green algae. Scenedesmus glucoliberatum PABB004 secretes high levels of simple sugars over a broad pH range.

Gene Fitness of Azotobacter vinelandii under Diazotrophic Growth.

Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly amenable to genetic manipulation, helping to unravel the intricate importance of different proteins involved in the process of BNF, including the biosynthesis of cofactors that are essential to assembling the complex metal cofactors that catalyze the difficult reaction of nitrogen fixation. Additionally, A. vinelandii accomplishes this feat while growing as an obligate aerobe, differentiating it from many of the nitrogen-fixing bacteria that are associated with plant roots. The ability to function in the presence of oxygen makes A. vinelandii suitable for application in various potential biotechnological schemes. In this study, we employed transposon sequencing (Tn-seq) to measure the fitness defects associated with disruptions of various genes under nitrogen-fixing dependent growth, versus growth with extraneously provided urea as a nitrogen source. The results allowed us to probe the importance of more than 3,800 genes, revealing that many genes previously believed to be important, can be successfully disrupted without impacting cellular fitness. IMPORTANCE These results provide insights into the functional redundancy in A. vinelandii, while also providing a direct measure of fitness for specific genes associated with the process of BNF. These results will serve as a valuable reference tool in future studies to uncover the mechanisms that govern this process.

Evaluation of two thioesterases from Marinobacter aquaeolei VT8: Relationship to wax ester production.

The biosynthesis of lipid-based biofuels is an important aspect of developing sustainable alternatives to conventional oils derived from fossil fuel reserves. Many biosynthetic approaches to biodiesel fuels and oils involve fatty acid derivatives as a precursor, and thioesterases have been employed in various strategies to increase fatty acid pools. Thioesterases liberate fatty acids from fatty acyl-coenzyme A or fatty acyl-acyl carrier protein substrates. The role played by thioesterases has not been extensively studied in model bacteria that accumulate elevated levels of biological oils based on fatty acid precursors. In this report, two primary thioesterases from the wax ester accumulating bacterium Marinobacter aquaeolei VT8 were heterologously expressed, isolated and characterized. These genes were further analyzed at the transcriptional level in the native bacterium during wax ester accumulation, and their genes were disrupted to determine the effect these changes had on wax ester levels. Combined, these results indicate that these two thioesterases do not play an integral role in wax ester accumulation in this natural lipid-accumulating model bacterium.

Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation.

BACKGROUND: The obligate aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. RESULTS: In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. CONCLUSIONS: These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.

Aerobic nitrogen-fixing bacteria for hydrogen and ammonium production: current state and perspectives.

Biological nitrogen fixation (BNF) is accomplished through the action of the oxygen-sensitive enzyme nitrogenase. One unique caveat of this reaction is the inclusion of hydrogen gas (H2) evolution as a requirement of the reaction mechanism. In the absence of nitrogen gas as a substrate, nitrogenase will reduce available protons to become a directional ATP-dependent hydrogenase. Aerobic nitrogen-fixing microbes are of particular interest, because these organisms have evolved to perform these reactions with oxygen-sensitive enzymes in an environment surrounded by oxygen. The ability to maintain a functioning nitrogenase in aerobic conditions facilitates the application of these organisms under conditions where most anaerobic nitrogen fixers are excluded. In recent years, questions related to the potential yields of the nitrogenase-derived products ammonium and H2 have grown more approachable to experimentation based on efforts to construct increasingly more complicated strains of aerobic nitrogen fixers such as the obligate aerobe Azotobacter vinelandii. This mini-review provides perspectives of recent and historical efforts to understand and quantify the yields of ammonium and H2 that can be obtained through the model aerobe A. vinelandii, and outstanding questions that remain to be answered to fully realize the potential of nitrogenase in these applications with model aerobic bacteria.

Efforts toward optimization of aerobic biohydrogen reveal details of secondary regulation of biological nitrogen fixation by nitrogenous compounds in Azotobacter vinelandii.

Biological nitrogen fixation (BNF) through the enzyme nitrogenase is performed by a unique class of organisms known as diazotrophs. One interesting facet of BNF is that it produces molecular hydrogen (H2) as a requisite by-product. In the absence of N2 substrate, or under conditions that limit access of N2 to the enzyme through modifications of amino acids near the active site, nitrogenase activity can be redirected toward a role as a dedicated hydrogenase. In free-living diazotrophs, nitrogenases are tightly regulated to minimize BNF to meet only the growth requirements of the cell, and are often accompanied by uptake hydrogenases that oxidize the H2 by-product to recover the electrons from this product. The wild-type strain of Azotobacter vinelandii performs all of the tasks described above to minimize losses of H2 while also growing as an obligate aerobe. Individual alterations to A. vinelandii have been demonstrated that disrupt key aspects of the N2 reduction cycle, thereby diverting resources and energy toward the production of H2. In this work, we have combined three approaches to override the primary regulation of BNF and redirect metabolism to drive biological H2 production by nitrogenase in A. vinelandii. The resulting H2-producing strain was further utilized as a surrogate to study secondary, post-transcriptional regulation of BNF by several key nitrogen-containing metabolites. The improvement in yields of H2 that were achieved through various combinations of these three approaches was compared and is presented along with the insights into inhibition of BNF by several nitrogen compounds that are common in various waste streams. The findings indicate that both ammonium and nitrite hinder BNF through this secondary inhibition, but urea and nitrate do not. These results provide essential details to inform future biosynthetic approaches to yield nitrogen products that do not inadvertently inhibit BNF.

Phenotypic responses to interspecies competition and commensalism in a naturally-derived microbial co-culture.

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp. HL-48 and Marinobacter sp. HL-58, to ask two fundamental scientific questions: 1) how do the phenotypes of two naturally co-existing species respond to partnership as compared to axenic growth? and 2) how do growth and molecular phenotypes of these species change with respect to competitive and commensal interactions? We hypothesized - and confirmed - that co-cultivation under glucose as the sole carbon source would result in competitive interactions. Similarly, when glucose was swapped with xylose, the interactions became commensal because Marinobacter HL-58 was supported by metabolites derived from Halomonas HL-48. Each species responded to partnership by changing both its growth and molecular phenotype as assayed via batch growth kinetics and global transcriptomics. These phenotypic responses depended on nutrient availability and so the environment ultimately controlled how they responded to each other. This simplified model community revealed that microbial interactions are context-specific and different environmental conditions dictate how interspecies partnerships will unfold.

Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga.

Green algae represent a key segment of the global species capable of photoautotrophic-driven biological carbon fixation. Algae partition fixed-carbon into chemical compounds required for biomass, while diverting excess carbon into internal storage compounds such as starch and lipids or, in certain cases, into targeted extracellular compounds. Two green algae were selected to probe for critical components associated with sugar production and release in a model alga. Chlorella sorokiniana UTEX 1602 - which does not release significant quantities of sugars to the extracellular space - was selected as a control to compare with the maltose-releasing Micractinium conductrix SAG 241.80 - which was originally isolated from an endosymbiotic association with the ciliate Paramecium bursaria. Both strains were subjected to three sequencing approaches to assemble their genomes and annotate their genes. This analysis was further complemented with transcriptional studies during maltose release by M. conductrix SAG 241.80 versus conditions where sugar release is minimal. The annotation revealed that both strains contain homologs for the key components of a putative pathway leading to cytosolic maltose accumulation, while transcriptional studies found few changes in mRNA levels for the genes associated with these established intracellular sugar pathways. A further analysis of potential sugar transporters found multiple homologs for SWEETs and tonoplast sugar transporters. The analysis of transcriptional differences revealed a lesser and more measured global response for M. conductrix SAG 241.80 versus C. sorokiniana UTEX 1602 during conditions resulting in sugar release, providing a catalog of genes that might play a role in extracellular sugar transport.

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation.

Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium.IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket.

Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no. WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD+ cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided.IMPORTANCE This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD+ are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn.

Altering small and medium alcohol selectivity in the wax ester synthase.

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor.

Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.

Patient specific 3D printed phantom for IMRT quality assurance.

The purpose of this study was to test the feasibility of a patient specific phantom for patient specific dosimetric verification.Using the head and neck region of an anthropomorphic phantom as a substitute for an actual patient, a soft-tissue equivalent model was constructed with the use of a 3D printer. Calculated and measured dose in the anthropomorphic phantom and the 3D printed phantom was compared for a parallel-opposed head and neck field geometry to establish tissue equivalence. A nine-field IMRT plan was constructed and dose verification measurements were performed for the 3D printed phantom as well as traditional standard phantoms.The maximum difference in calculated dose was 1.8% for the parallel-opposed configuration. Passing rates of various dosimetric parameters were compared for the IMRT plan measurements; the 3D printed phantom results showed greater disagreement at superficial depths than other methods.A custom phantom was created using a 3D printer. It was determined that the use of patient specific phantoms to perform dosimetric verification and estimate the dose in the patient is feasible. In addition, end-to-end testing on a per-patient basis was possible with the 3D printed phantom. Further refinement of the phantom construction process is needed for routine use.

Draft Genome Sequences of the Alga-Degrading Bacteria Aeromonas hydrophila Strain AD9 and Pseudomonas pseudoalcaligenes Strain AD6.

Aeromonas hydrophila AD9 and Pseudomonas pseudoalcaligenes AD6 have been linked to algal cell degradation. Here we report the draft genomes of A. hydrophila AD9 and P. pseudoalcaligenes AD6 for the investigation of causative agents for algal cell degradation.