Azotobacter Vinelandii Strain Research Articles

Competitive fitness and stability of ammonium-excreting Azotobacter vinelandii strains in the soil

Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture.Key points• Genetic engineering for NH4+ excretion imposes a fitness burden on the culture medium• Large phenotypic instability for NH4+-excreting bacteria in culture medium• Lower fitness burden and phenotypic instability for NH4+-excreting bacteria in soil

Effectiveness of a complex microbial preparation for oats and spring barley growing

Today, one of the main challenges for agriculture is to increase grain production and its profitability while reducing the use of chemicals. Addressing this issue is a key to meeting the human need for quality and affordable food. One of the ways to improve the efficiency of grain industry is to use effective microbial-based biological products. They will help to reduce the chemical burden on the environment and produce high quality crops. The aim of this study was to evaluate the effectiveness of treatment and the growing season of spring oats and barley with a complex microbial preparation based on promising strains of Azotobacter vinelandii 7 AI, Azotobacter chroococcum 8 AI and Bacillus megaterium 39 AI isolated by us earlier. Field trials were carried out on the experimental fields of the Skvyra Experimental Station of Organic Production of the Institute of Agroecology and Nature Management of the National Academy of Agrarian Sciences of Ukraine (Ukraine, Kyiv region, Skvyra) in 2022. For the treatment of plants during the growing season, strains of microorganisms and their compositions were used in critical phases of crop development. After 6–7 days, the effect of these compositions on some plant growth parameters was studied. To confirm the hypothesis about the effect of treatment with promising microorganisms on the ability of plants to increase the amount of photosynthetic pigments, a quantitative analysis of plants for their content was performed. The positive effect of plant treatment was found not only on the quantity of the harvest, but also on its quality. The results of the research indicate the effectiveness of the use of a complex microbial preparation based on the proposed strains of microorganisms for such cereals as oats and spring barley. Treatment with a biopreparation based on effective, environmentally friendly and agronomically useful strains of microorganisms improves both growth and photosynthetic processes, which in turn activates their development and enhances the productivity of these crops, while significantly improving the quality of grain.

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

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.

Human osteoblast cells proliferation in biodegradable poly-3-hydroxybutyrate (PHB) scaffolds from a mutant Azotobacter vinelandii strain

Human osteoblast cells proliferation in biodegradable poly-3-hydroxybutyrate (PHB) scaffolds from a mutant Azotobacter vinelandii strain

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster.

Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.

Preparation and application of a functional biological additive for quails based on microbial conversion of plant raw materials

When developing functional feed additives it is of interest to use microorganisms that have the ability to take root in the gastrointestinal tract of poultry and normalize its microflora to have a positive effect on the immune system. One of these bacteria is Propionibacterium shermanii, which has a fortifying, immunostimulating, antimutagenic effect, and can reduce the toxic effect of ultraviolet light and chemical compounds. Azotobacter vinelandii, which has probiotic fortifying, antibiotic properties, is effective as a stimulator of the development of the entire microbiota in a contaminated object, and recycles the formed ammonia in bird droppings, was chosen as another microorganism for creating the additive. The purpose of the research was to develop a functional biological additive based on probiotic microflora, using the by-products of crop processing in the formulations, and to select the norms for its introduction into the diet of quails. As a result of the research the technology of joint cultivation of Propionibacterium shermanii and Azotobacter vinelandii strains was developed, a universal starter culture was created that allows to ferment by-products of processing of plant raw materials, a functional additive with immunostimulating, vitamin and provitamin properties for quails has been obtained. The results of the production test confirmed the effectiveness of the developed additive in the diet of quails. Economic efficiency in the application of the proposed functional biological additive has been shown a reduction in feed consumption per 1 kg of live weight gain by 5,13 %. In addition, the economic effect of using the functional biological additive increases. Thus, the profit in the experimental group was 2738,34 rubles, which was 12,45 % higher than in the control group.

Azotobacter vinelandii helps to combat chromium stress in rice by maintaining antioxidant machinery.

Chromium (Cr) causes toxic effects in plants by generating reactive oxygen species (ROS) which create oxidative environment. Azotobacter vinelandii helps in growth and development of many crops; however, its role in Cr stress tolerance in rice has not been explored. Here, we report the new function of Azotobacter vinelandii strain SRI Az3 (Accession number JQ796077) in providing Cr stress tolerance in Oryza sativa (var. IR64). The efficiency of the strain was checked under different concentrations (50, 100, 150, 200 and 250µM) of Cr stress and it was observed that it provides stress tolerance to rice plant up to 200µM concentration. Different agronomic growth parameters were found to be better in this strain of Azotobacter vinelandii-inoculated rice plants as compared to un-inoculated one. The agronomic growth and photosynthetic characteristics such as net photosynthetic rate (PN), stomatal conductance (gs), intercellular CO2 (Ci) were also found to be significantly increased with increasing concentration of Azotobacter vinelandii inoculation. The activities of antioxidant enzymes were significantly higher (35%) in rice plants inoculated with Azotobacter vinelandii as compared with un-inoculated rice plant. All these positive effects of Azotobacter vinelandii help rice to survive from the toxic effect of Cr.

Deferred control of ammonium cross-feeding in a N2-fixing bacterium-microalga artificial consortium.

There is an increasing interest in the use of N2-fixing bacteria for the sustainable biofertilization of crops. Genetically-optimized bacteria for ammonium release have an improved biofertilization capacity. Some of these strains also cross-feed ammonium into microalgae raising additional concerns on their sustainable use in agriculture due to the potential risk of producing a higher and longer-lasting eutrophication problem than synthetic N-fertilizers. Here we studied the dynamic algal cross-feeding properties of a genetically-modified Azotobacter vinelandii strain which can be tuned to over-accumulate different levels of glutamine synthetase (GS, EC 6.3.1.20) under the control of an exogenous inducer. After switching cells overaccumulating GS into a noninducing medium, they proliferated for several generations at the expense of the previously accumulated GS. Further dilution of GS by cell division slowed-down growth, promoted ammonium-excretion and cross-fed algae. The final bacterial population, and timing and magnitude of algal N-biofertlization was finely tuned in a deferred manner. This tuning was in accordance with the intensity of the previous induction of GS accumulation in the cells. This bacterial population behavior could be maintained up to about 15 bacterial cell generations, until faster-growing and nonammonium excreting cells arose at an apparent high frequency. Further improvements of this genetic engineering strategy might help to align efficiency of N-biofertilizers and safe use in an open environment. KEY POINTS: • Ammonium-excreting bacteria are potential eutrophication agents • GS-dependent deferred control of bacterial growth and ammonium release • Strong but transient ammonium cross-feeding of microalgae.

Efficiency of using biostimulants Agrinos 1 and Agrinos 2 in adaptation of microplants of garden strawberry to ex vitro conditions

This article presents the results of experimental studies of the effect of biostimulants Agrinos 1 and Agrinos 2, containing strains of microorganisms Azotobacter vinelandii and Clostridium pasteurinum, etc., as well as a complex of nutrients (macro-, microelements, protein, amino acids, chitin, chitosan) on the activation of metabolic processes and reduction of stress effects on strawberry plants obtained by in vitro method. The effectiveness of the reaction of biostimulants on the adaptive processes of growth and development of microplants of garden strawberry varieties Alba, Kemiya, Clery has been studied. In the course of the research, a positive effect of biological products on the growth of the vegetative mass of plants was established: an increase in the height of the plants compared to the control by 20-25%, the number of leaves by 25-28% and an increase in the root system of plants by 30-40%. In the Clery variety, the use of the biostimulants promoted the formation of the runners. In addition, the introduction of biostimulants into the soil contributed to the improvement of the quality indicators of the soil. The content of nitrate nitrogen in the soil increased by 18%.

Characterization of a Mo-Nitrogenase Variant Containing a Citrate-Substituted Cofactor.

Nitrogenase converts N2 to NH3 , and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDKCit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDKCit reveals a shift of CO reduction from H2 evolution toward hydrocarbon formation and an opposite shift of N2 reduction from NH3 formation toward H2 evolution. Consistent with a shift in the Mo K-edge energy of NifDKCit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution.

Production of Poly-3-Hydroxybutyrate (P3HB) with Ultra-High Molecular Weight (UHMW) by Mutant Strains of Azotobacter vinelandii Under Microaerophilic Conditions.

Poly-3-hydroxybutyrate (P3HB) is a biopolymer, which presents characteristics similar to those of plastics derived from the petrochemical industry. The thermomechanical properties and biodegradability of P3HB are influenced by its molecular weight (MW). The aim of the present study was to evaluate the changes of the molecular weight of P3HB as a function of oxygen transfer rate (OTR) in the cultures using two strains of Azotobacter vinelandii, a wild-type strain OP, and PhbZ1 mutant with a P3HB depolymerase inactivated. Both strains were grown in a bioreactor under different OTR conditions. An inverse relationship was found between the average molecular weight of P3HB and the OTRmax, obtaining a polymer with a maximal MW (8000-10,000kDa) from the cultures developed at OTRmax of 5mmolL-1h-1 using both strains, with respect to the cultures conducted at 8 and 11mmolL-1h-1, which produced a P3HB between 4000 and 5000kDa. The increase in MW of P3HB was related to the activity of enzymes involved in the synthesis and depolymerization. Overall, our results show that it is possible to modulate the average molecular weight of P3HB by manipulating oxygen transfer conditions with both strains (OP and PhbZ1 mutant) of A. vinelandii.

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

BackgroundThe 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.ResultsIn 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.ConclusionsThese findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.

Molecular weight and viscosifying power of alginates produced by mutant strains of Azotobacter vinelandii under microaerophilic conditions

Alginates are polysaccharides that are of interest in various industrial applications. This is due to the viscosifying properties of alginates, which depends on the weight-average molecular weight. The aim of the present study was to evaluate the changes in alginate quality, in terms of the viscosifying power and weight-average molecular weight of the polymer produced by Azotobacter vinelandii mutant strains in shake flasks under microaerophilic conditions. In cultures developed at oxygen transfer rate (OTR) values close to 5 mmol L−1 h−1, the highest viscosifying power (1.75 L g−1) and weight-average molecular weight (3112 ± 150 kDa) were achieved in cultures performed with the AT9 strain. These values were higher than those obtained for the alginates produced by the parental strain ATCC 9046 grown under similar OTR conditions. In contrast, the alginate produced by the GG9 and OPAlgU + exhibited a very low weight-average molecular weight and therefore a poor viscosifying power. Our results have shown that by the cultivation of AT9 strain under microaerophilic conditions it is possible to obtain a polymer having a high weight-average molecular weight and excellent viscosifying capacity. Therefore, it could be a viable strategy for producing alginates for industrial applications.

A Composite Bioinoculant Based on the Combined Application of Beneficial Bacteria and Fungi

A composite soil bioinoculant containing beneficial bacteria and fungi was developed for biocontrol of plant pathogens, phosphorous mobilization, stem degradation, humification, and nitrogen fixation. A Trichoderma asperellum isolate with outstanding in vitro antagonistic abilities toward a series of plant pathogenic fungi was included as a potential biocontrol component. The selected strain was also shown to promote growth and increase photosynthetic activity of tomato plants. For phosphorous mobilization and stem degradation, a Trichoderma atrobrunneum strain was selected, which produced cellulose-degrading enzymes even in the absence of stem residues, while this ability increased 10–15-fold in the presence of ground maize stem. The strain was also shown to produce large amounts of enzymes liberating organically bound phosphorous, as well as cellulase and xylanase activities in solid-state fermentation on various plant residues. A Streptomyces albus strain with excellent peroxidase-producing abilities was selected as a potential humus-producing component, while an Azotobacter vinelandii strain with the potential to provide excess nitrogen for crops was included for nitrogen fixation. The assembled soil bioinoculant had positive effect on the uptake of certain important macro- and microelements (potassium, sodium, and manganese) from the soil by field-grown tomato plants. The applied screening strategy proved to be applicable for the assembly of a composite soil bioinoculant with notable application potentials.

Effects of coinoculation of Rhizobium with plant growth promoting rhizobacteria on the nitrogen fixation and nutrient uptake of Trifolium repens in low phosphorus soil

Low soil phosphorus (P) availability is a notable limitation for legume nodulation, nitrogen fixation and yield, and it is therefore important to improve legume growth under P-deficient conditions. This study aimed to determine the effects of coinoculation with Rhizobium and plant growth promoting rhizobacter (PGPR) on the nitrogen fixation and nutrient uptake of white clover (Trifolium repens) in a P-deficient soil. White clover plants were inoculated with one of the two Rhizobium strains CHB1120 and CHB1121 alone or coinoculated with either of the Rhizobium strains and one of the two PGPR strains, Bacillus aryabhattai strain Sb and Azotobacter vinelandii strain G31. White clover inoculated with either CHB1120 or CHB1121 alone and coinoculated with one of the two Rhizobium strains and the PGPR significantly enhanced nodule numbers compared with the non-inoculated control. The coinoculation of either CHB1120 or CHB1121 and Sb showed a significantly greater level of nitrogenase than the single inoculation of CHB1120 or CHB1121. In addition, the single inoculation of CHB1120 or CHB1121 and their coinoculation with PGPR significantly increased nitrogen contents in both shoots and roots under P-deficient conditions. The coinoculation of CHB1121 and G31 showed the greatest N content in shoots. Moreover, the single inoculation of the two Rhizobium strains and their coinoculation with G31 and Sb significantly increased the K, Ca, and Mg contents in shoots compared with the non-inoculated control. In summary, the PGPR used in this study can enhance the symbiotic potential of rhizobia under low P conditions.

Preparation of levan and alginate of azotobacter vinelandii strains and use to form adsorbent biocomposites

Preparation of levan and alginate of azotobacter vinelandii strains and use to form adsorbent biocomposites

In vitro investigation to explore the toxicity of different groups of pesticides for an agronomically important rhizosphere isolate Azotobacter vinelandii

In this work, an attempt was made to evaluate the effect of pesticides on growth pattern, surface morphology, cell viability and growth regulators of nitrogen fixing soil bacterium. Pesticide tolerant Azotobacter vinelandii strain AZ6 (Accession no. MG028654) was found to tolerate maximum level of pesticide and displayed multifarious PGP activities. At higher concentrations, pesticides triggered cellular/structural damage and reduced the cell viability as clearly shown under SEM and CLSM. With increase in concentration, pesticides exhibited a significant (p < 0.05) decrease in PGP traits of strain AZ6. Among all three groups of pesticides, herbicides glyphosate and atrazine were most toxic. Kitazin, hexaconazole, metalaxyl, glyphosate, quizalofop, atrazine, fipronil, monocrotophos and imidacloprid at 2400, 1800, 1500, 900, 1200, 900, 1800, 2100 and 2700 μg mL−1, respectively, decreased the production of IAA by 19.5 ± 1.9 (61%), 18.1 ± 1.2 (64%), 36.4 ± 3.4 (28%), 13.1 ± 0.8 (74%), 15.6 ± 1.0 (69%), 7.6 ± 0.5 (83%), 11.9 ± 0.8 (76%), 24.7 ± 1.7 (51%) and 32 ± 2.3 (37%) μg mL−1, respectively, over control (50.7 ± 3.6 μg mL−1). A maximum reduction of 8.4 ± 1.2 (46%), 5.8 ± 0.6 (62%) and 4 ± 0.2 (74%) μg mL−1 in 2, 3-DHBA at 300 (1×), 600 (2×) and 900 (3×) μg mL−1 glyphosate, respectively, While, 32.8 ± 2.7 (19%), 27.2 ± 2 (33%) and 21.5 ± 1.3 (47%) μg mL−1, respectively in the production of SA was observed at 300 (1×), 600 (2×) and 900 (3×) μg mL−1 atrazine, respectively. Likewise, with increase in concentration of pesticides, decrease in P solubilization ability and change in pH of broth was detected. The order of pesticide toxicity to PSE (percent decline over control) at highest concentration was: atrazine (45) > kitazin (44) > metalaxyl (43) > monocrotophos (43) > glyphosate (41) > hexaconazole (39) > quizalofop (33) > imidacloprid (31) > fipronil (25). The present study undoubtedly suggests that even at higher doses of pesticides, A. vinelandii maintained secreting plant growth regulators and this property makes this strain agronomically important microbe for enhancing the growth of plants.

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.

Methods for Generating Microbial Cocultures that Grow in the Absence of Fixed Carbon or Nitrogen.

This work is motivated by both (1) the desire to make interesting products without the reliance on fixed carbon or fixed nitrogen using microbial cocultures, and (2) the desire to develop new methods for growing microbial communities. The bioplastic polyhydroxybutyrate (PHB), for example, is considered prohibitively expensive to make from sugar (compared to polypropylene). Utilizing and building on engineered strains of Synechococcus elongatus and Azotobacter vinelandii, we have combined a nitrogen-fixing organism with a carbon-fixing organism to make PHB from air, water, sunlight, and trace minerals. Our observations of coculture growth in batch culture led us to develop an improved system based on manipulating the osmotic pressure within a hydrogel. The methods we used to develop this coculture are described in detail in the following chapter, including notes detailing some of our additional observations or thoughts on this system.