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.

Bioprospecting for fungal enzymes for applications in microalgal biomass biorefineries.

Microalgal biomass is a promising feedstock for biofuels, feed/food, and biomaterials. However, while production and commercialization of single-product commodities are still not economically viable, obtaining multiple products in a biomass biorefinery faces several techno-economic challenges. The aim of this study was to identify a suitable source of hydrolytic enzymes for algal biomass saccharification. Screening of twenty-six fungal isolates for secreted enzymes activity on Chlamydomonas reinhardtii biomass resulted in the identification of Aspergillus niger IB-34 as a candidate strain. Solid-state fermentation on wheat bran produced the most active enzyme preparations. From sixty-five proteins identified by liquid chromatography coupled to mass spectrometry (LC-MS) (ProteomeXchange, identifier PXD034998) from A. niger IB-34, the majority corresponded to predicted secreted proteins belonging to the Gene Ontology categories of catalytic activity/hydrolase activity on glycosyl and O-glycosyl compounds. Skimmed biomass of biotechnologically relevant strains towards the production of commodities, Chlorella sorokiniana and Scenedesmus obliquus, was fully saccharified after a mild pretreatment at 80 °C for 10 min, at a high biomass load of 10% (w/v). The soluble liquid stream, after skimming and saccharification of biomass of both strains, was further converted into ethanol by fermentation with Saccharomyces cerevisiae at a theoretical maximum efficiency, in a separated saccharification and fermentation assays. The resulting insoluble protein, after biomass skimming with an organic solvent and enzymatic saccharification, was highly digestible in an in vitro digestion assay. Proof of concept is presented for an enzyme-assisted biomass biorefinery recovering 81% of the main biomass fractions in a likely suitable form for the conversion of lipids and carbohydrates into biofuels and proteins into feed/food. KEY POINTS: • Twenty-six fungal extracts were analyzed for saccharification of microalgal biomass. • Skimmed biomass was fully enzymatically saccharified and fermented into ethanol. • Up to 81% recovery of biomass fractions suitable for biofuels and feed/food.

Climate-Simulated culturing suggests high microalgal biomass and oil productivities in most of the South American continent.

BACKGROUND: Current production costs of microalgal biomass indicate that only highly-productive cultivation facilities will approach commercial feasibility. Geographical site selection for siting those facilities is critical for achieving target productivities. The aim of this study was to provide a semi-empirical estimation of microalgal biomass and lipids productivity in South America. METHODS AND RESULTS: Simulated-climate was programed in environmental photobioreactors (Phenometrics) for a simulation of cultivation in open raceway ponds at different geographical sites. The mean annual South American biomass productivity of 20-cm deep ponds was 12 ± 4 g · m- 2 · d-1 . The most productive regions were clustered in the subtropical and tropical regions of the continent. Fortaleza (Brazil) showed a low seasonality and a high annual mean productivity of 23 g · m-2 · d-1 in 5-cm deep ponds, closely approaching the productivity target. Lipids accumulation and productivity in Fortaleza showed a high microalgal oil accumulation up to 46% (w/w) and a maximum oil productivity of 5 g · m-2 · d-1 for biomass containing around 20% lipids (w/w). CONCLUSION: This study provides the first semi-empirical estimation of microalgal productivity in South America and supports a high potential of a vast region of the continent.

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.

Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae.

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N2 and CO2 from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N2 from the air into crops.

Plant-adapted Escherichia coli show increased lettuce colonizing ability, resistance to oxidative stress and chemotactic response.

BACKGROUND: Escherichia coli is a widespread gut commensal and often a versatile pathogen of public health concern. E. coli are also frequently found in different environments and/or alternative secondary hosts, such as plant tissues. The lifestyle of E. coli in plants is poorly understood and has potential implications for food safety. METHODS/PRINCIPAL FINDINGS: This work shows that a human commensal strain of E. coli K12 readily colonizes lettuce seedlings and produces large microcolony-like cell aggregates in leaves, especially in young leaves, in proximity to the vascular tissue. Our observations strongly suggest that those cell aggregates arise from multiplication of single bacterial cells that reach those spots. We showed that E. coli isolated from colonized leaves progressively colonize lettuce seedlings to higher titers, suggesting a fast adaptation process. E. coli cells isolated from leaves presented a dramatic rise in tolerance to oxidative stress and became more chemotactic responsive towards lettuce leaf extracts. Mutant strains impaired in their chemotactic response were less efficient lettuce colonizers than the chemotactic isogenic strain. However, acclimation to oxidative stress and/or minimal medium alone failed to prime E. coli cells for enhanced lettuce colonization efficiency. CONCLUSION/SIGNIFICANCE: These findings help to understand the physiological adaptation during the alternative lifestyle of E. coli in/on plant tissues.

Challenges to develop nitrogen-fixing cereals by direct nif-gene transfer.

Some regions of the developing world suffer low cereal production yields due to low fertilizer inputs, among other factors. Biological N2 fixation, catalyzed by the prokaryotic enzyme nitrogenase, is an alternative to the use of synthetic N fertilizers. The molybdenum nitrogenase is an O2-labile metalloenzyme composed of the NifDK and NifH proteins, which biosyntheses require a number of nif gene products. A challenging strategy to increase cereal crop productivity in a scenario of low N fertilization is the direct transfer of nif genes into cereals. The sensitivity of nitrogenase to O2 and the apparent complexity of nitrogenase biosynthesis are the main barriers identified so far. Expression of active NifH requires the products of nifM, nifH, and possibly nifU and nifS, whereas active NifDK requires the products of nifH, nifD, nifK, nifB, nifE, nifN, and possibly nifU, nifS, nifQ, nifV, nafY, nifW and nifZ. Plastids and mitochondria are potential subcellular locations for nitrogenase. Both could provide the ATP and electrons required for nitrogenase to function but they differ in their internal O2 levels and their ability to incorporate ammonium into amino acids.

Metabolic engineering of ammonium release for nitrogen-fixing multispecies microbial cell-factories.

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. In this study we compared the effect of controlling the maximum activation state of the Azotobacter vinelandii glutamine synthase by a point mutation at the active site (D49S mutation) and impairing the ammonium-dependent homeostatic control of nitrogen-fixation genes expression by the ΔnifL mutation on ammonium release by the cells. Strains bearing the single D49S mutation were more efficient ammonium producers under carbon/energy limiting conditions and sustained microalgae growth at the expense of atmospheric N2 in synthetic microalgae-bacteria consortia. Ammonium delivery by the different strains had implications for the microalga׳s cell-size distribution. It was uncovered an extensive cross regulation between nitrogen fixation and assimilation that extends current knowledge on this key metabolic pathway and might represent valuable hints for further improvements of versatile N2-fixing microbial-cell factories.

High lipid productivity of an Ankistrodesmus-Rhizobium artificial consortium.

Microalgae have great potential as alternative productive platforms for sustainable production of bioenergy, food, feed and other commodities. Process optimization to realize the claimed potential often comprises strains selection and improvement and also developing of more efficient cultivation, harvesting and downstream processing technology. In this work we show that inoculation with the bacterium Rhizobium strain 10II resulted in increments of up to 30% in chlorophyll, biomass and lipids accumulation of the oleaginous microalgae Ankistrodesmus sp. strain SP2-15. Inoculated cultures have reached a high lipid productivity of up to 112 mg L(-1) d(-1) after optimization. The resulting biomass presented significant levels of Ω3 fatty acids including stearidonic acid, suggesting potential as an alternative land-based source of essential fatty acids.

Genetic engineering of multispecies microbial cell factories as an alternative for bioenergy production.

There is currently much interest in developing technology to use microlgae or cyanobacteria for the production of bioenergy and biomaterials. Here, we summarize some remarkable achievements in strains improvement by traditional genetic engineering and discuss common drawbacks for further progress. We present general knowledge on natural microalgal-bacterial mutualistic interactions and discuss the potential of recent developments in genetic engineering of multispecies microbial cell factories. This synthetic biology approach would rely on the assembly of complex metabolic networks from optimized metabolic modules such as photosynthetic or nitrogen-fixing parts.

Bioprospecting for fast growing and biomass characterization of oleaginous microalgae from South-Eastern Buenos Aires, Argentina.

As part of pioneering efforts to assess the potential of native microalgae as biofuel feedstock in South-Eastern Buenos Aires, 34 monoalgal cultures (corresponding to the Phylum Chlorophyta) were established and 21 were selected for further growth and biomass composition characterization. Novel RNA sequences in the ITS1-5.8S-ITS2 region were identified. Some strains showed desirable traits as biodiesel feedstock such as (i) apparent maximal doubling times of 6h, (ii) lipids accumulation of up to 43% of their dry biomass, (iii) high ration of mono-unsaturated to poly-unsaturated fatty acids, (iv) high response to CO(2) supplementation, and (v) complete sedimentation in 4h. Data of the outdoors performance of some strains suggested they might represent valuable resources for future research towards the regional development of the technology for microalgae-based biofuels.

Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae.

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.

NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii.

The major part of biological nitrogen fixation is catalysed by the molybdenum nitrogenase that carries at its active site the iron and molybdenum cofactor (FeMo-co). The nitrogen fixation (nif) genes required for the biosynthesis of FeMo-co are derepressed in the absence of a source of fixed nitrogen. The nifB gene product is remarkable because it assembles NifB-co, a complex cluster proposed to comprise a [6Fe-9S-X] cluster, from simpler [Fe-S] clusters common to other metabolic pathways. NifB-co is a common intermediate of the biosyntheses of the cofactors present in the molybdenum, vanadium and iron nitrogenases. In this work, the expression of the Azotobacter vinelandii nifB gene was uncoupled from its natural nif regulation to show that NifB protein levels are lower in cells growing diazotrophically than in cells growing at the expense of ammonium. A. vinelandii carries a duplicated copy of the ATPase component of the ubiquitous ClpXP protease (ClpX2), which is induced under nitrogen fixing conditions. Inactivation of clpX2 resulted in the accumulation of NifB and NifEN and a defect in diazotrophic growth, especially when iron was in short supply. Mutations in nifE, nifN and nifX or in nifA also affected NifB accumulation, suggesting that NifB susceptibility to degradation might vary during its catalytic cycle.

Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes.

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor.

The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron-molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum-iron-sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex.

Sucrose synthase is involved in the conversion of sucrose to polysaccharides in filamentous nitrogen-fixing cyanobacteria.

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, UDP-glucose: D: -fructose 2-alpha-D: -glucosyl transferase, EC 2.4.1.13) catalyses the synthesis and cleavage of Suc, and in higher plants, it plays an important role in polysaccharides biosynthesis and carbon allocation. In this work, we have studied the functional relationship between SuS and the metabolism of polysaccharides in filamentous nitrogen-fixing cyanobacteria. We show that the nitrogen and carbon sources and light regulate the expression of the SuS encoding gene (susA), in a similar way that they regulate the accumulation of polysaccharides. Furthermore, glycogen content in an Anabaena sp. mutant strain with an insertion inactivation of susA was lower than in the wild type strain under diazotrophic conditions, while both glycogen and polysaccharides levels were higher in a mutant strain constitutively overexpressing susA. We also show that there are soluble and membrane-bound forms of SuS in Anabaena. Taken together, these results strongly suggest that SuS is involved in the Suc to polysaccharides conversion according to nutritional and environmental signals in filamentous nitrogen-fixing cyanobacteria.

In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins.

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe(2+), S(2-), MoO4(2-), and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe(2+), S(2-), MoO4(2-), R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.

Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.

NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.

The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.