Diazotrophic community structure and function in two successional stages of biological soil crusts from the Colorado Plateau and Chihuahuan Desert

The objective of this study was to characterize the community structure and activity of N2-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of nifH sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to Nostoc spp. and that the composition of N2-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of nifH sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a Microcoleus vaginatus-dominated, poorly developed crust to mature crusts harboring a greater percentage of Nostoc and Scytonema spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N2-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by Microcoleus vaginatus.     

MICROENVIRONMENTS AND MICROSCALE PRODUCTIVITY OF CYANOBACTERIAL DESERT CRUSTS1

We used microsensors to characterize physicochemical microenvironments and photosynthesis occurring immediately after water saturation in two desert soil crusts from southeastern Utah, which were formed by the cyanobacteria Microcoleus vaginatus Gomont, Nostoc spp., and Scytonema sp. The light fields within the crusts presented steep vertical gradients in magnitude and spectral composition. Near-surface light-trapping zones were formed due to the scattering nature of the sand particles, but strong light attenuation resulted in euphotic zones only ca. 1 mm deep, which were progressively enriched in longer wavelengths with depth. Rates of gross photosynthesis (3.4–9.4 mmol O₂·m^?2·h^?1) and dark respiration (0.81–3.1 mmol O^?2·m^?2·h^?1) occurring within 1 to several mm from the surface were high enough to drive the formation of marked oxygen microenvironments that ranged from oxygen supersaturation to anoxia. The photosynthetic activity also resulted in localized pH values in excess of 10, 2–3 units above the soil pH. Differences in metabolic parameters and community structure between two types of crusts were consistent with a successional pattern, which could be partially explained on the basis of the microenvironments. We discuss the significance of high metabolic rates and the formation of microenvironments for the ecology of desert crusts, as well as the advantages and limitations of microsensor-based methods for crust investigation.     

The vertical microdistribution of cyanobacteria and green algae within desert crusts and the development of the algal crusts

Substantial amounts of algal crusts were collected from five different desert experimental sites aged 42, 34, 17, 8 and 4 years, respectively, at Shapotou (China) and analyzed at a 0.1 mm microscale of depth. It was found that the vertical distribution of cyanobacteria and microalgae in the crusts was distinctly laminated into an inorganic-layer (ca.0.00–0.02 mm, with few algae), an algae-dense-layer (ca.0.02–1.0 mm) and an algae-sparse-layer (ca.1.0–5.0 mm). It was interesting to note that in all crusts Scytonema javanicum Born et Flah (or Nostoc sp., cyanobacterium), Desmococcus olivaceus (Pers ex Ach., green alga) Laundon and Microcoleus vaginatus Gom. (cyanobacterium) dominated at the depth of 0.02–0.05, 0.05–0.1 and 0.1–1.0 mm, respectively, from the surface. Phormidium tenue Gom. (or Lyngbya cryptovaginatus Schk., cyanobacterium) and Navicula cryptocephala Kutz.(or Hantzschia amphioxys (Ehr.) Grun. and N. cryptocephala together, diatom) dominated at the depth of 1.0–3.0 and 3.5–4.0 mm, respectively, of the crusts from the 42 and 34 year old sites. It was apparent that in more developed crusts there were more green algae and the niches of Nostoc sp., Chlorella vulgaris Beij., M. vaginatus, N. cryptocephala and fungi were nearer to the surface. If lichens and mosses accounted for less than 41.5% of the crust surface, algal biovolume was bigger when the crust was older, but the opposite was true when the cryptogams other than algae covered more than 70%. In addition to detailed species composition and biovolume, analyses of soil physicochemical properties, micromorphologies and mineral components were also performed. It was found that the concentration of organic matter and nutrients, electric conductivity, silt, clay, secondary minerals were higher and there were more micro-beddings in the older crusts than the less developed ones. Possible mechanisms for the algal vertical microdistribtion at different stages and the impact of soil topography on crust development are discussed. It is concluded that biomethods (such as fine species distribution and biovolume) were more precise than mineralogical approaches in judging algal crust development and thus could be a better means to measure the potentiality of algal crusts in desert amelioration.     

Carbon exchange in biological soil crust communities under differential temperatures and soil water contents: implications for global change

Biological soil crusts (biocrusts) are an integral part of the soil system in arid regions worldwide, stabilizing soil surfaces, aiding vascular plant establishment, and are significant sources of ecosystem nitrogen and carbon. Hydration and temperature primarily control ecosystem CO₂ flux in these systems. Using constructed mesocosms for incubations under controlled laboratory conditions, we examined the effect of temperature (5–35 °C) and water content (WC, 20–100%) on CO₂ exchange in light (cyanobacterially dominated) and dark (cyanobacteria/lichen and moss dominated) biocrusts of the cool Colorado Plateau Desert in Utah and the hot Chihuahuan Desert in New Mexico. In light crusts from both Utah and New Mexico, net photosynthesis was highest at temperatures >30 °C. Net photosynthesis in light crusts from Utah was relatively insensitive to changes in soil moisture. In contrast, light crusts from New Mexico tended to exhibit higher rates of net photosynthesis at higher soil moisture. Dark crusts originating from both sites exhibited the greatest net photosynthesis at intermediate soil water content (40–60%). Declines in net photosynthesis were observed in dark crusts with crusts from Utah showing declines at temperatures >25 °C and those originating from New Mexico showing declines at temperatures >35 °C. Maximum net photosynthesis in all crust types from all locations were strongly influenced by offsets in the optimal temperature and water content for gross photosynthesis compared with dark respiration. Gross photosynthesis tended to be maximized at some intermediate value of temperature and water content and dark respiration tended to increase linearly. The results of this study suggest biocrusts are capable of CO₂ exchange under a wide range of conditions. However, significant changes in the magnitude of this exchange should be expected for the temperature and precipitation changes suggested by current climate models.     

The Community and Phylogenetic Diversity of Biological Soil Crusts in the Colorado Plateau Studied by Molecular Fingerprinting and Intensive Cultivation

We studied the bacterial communities in biological soil crusts (BSCs) from the Colorado Plateau by enrichment and cultivation, and by statistically analyzed denaturing gradient gel electrophoresis (DGGE) fingerprinting of environmental 16S rRNA genes, and phylogenetic analyses. Three 500-m-long transects, tens of km apart, consisting of 10 equally spaced samples each, were analyzed. BSC communities consistently displayed less richness (10–32 detectable DGGE bands per sample) and Shannon diversity (2.1–3.3) than typical soil communities, with apparent dominance by few members. In spite of some degree of small-scale patchiness, significant differences in diversity and community structure among transects was detectable, probably related to the degree of crust successional maturity. Phylogenetic surveys indicated that the cyanobacterium Microcoleus vaginatus was dominant, with M. steenstrupii second among phototrophs. Among the 48 genera of nonphototrophs detected, Actinobacteria (particularly Streptomyces spp.) were very common and diverse, with 18 genera and an average contribution to the total 16S rDNA amplificate of 11.8%. β-Proteobacteria and Bacteriodetes contributed around 10% each; Low-GC Gram-positives, α-Proteobacteria, Thermomicrobiales, and Acidobacteria were common (2–5%). However, the second largest contribution was made by deep-branching unaffiliated alleles (12.6%), with some of them representing candidate bacterial divisions. Many of the novel strains isolated are likely new taxa, and some were representatives of the phylotypes detected in the field. The mucoid or filamentous nature of many of these isolates speaks for their role in crust formation.     

Effects of altered temperature and precipitation on desert protozoa associated with biological soil crusts

Biological soil crusts are diverse assemblages of bacteria, cyanobacteria, algae, fungi, lichens, and mosses that cover much of arid land soils. The objective of this study was to quantify protozoa associated with biological soil crusts and test the response of protozoa to increased temperature and precipitation as is predicted by some global climate models. Protozoa were more abundant when associated with cyanobacteria/lichen crusts than with cyanobacteria crusts alone. Amoebae, flagellates, and ciliates originating from the Colorado Plateau desert (cool desert, primarily winter precipitation) declined 50-, 10-, and 100-fold, respectively, when moved in field mesocosms to the Chihuahuan Desert (hot desert, primarily summer rain). However, this was not observed in protozoa collected from the Chihuahuan Desert and moved to the Sonoran desert (hot desert, also summer rain, but warmer than Chihuahuan Desert). Protozoa in culture began to encyst at 37 degrees C. Cysts survived the upper end of daily temperatures (37-55 degrees C), and could be stimulated to excyst if temperatures were reduced to 15 degrees C or lower. Results from this study suggest that cool desert protozoa are influenced negatively by increased summer precipitation during excessive summer temperatures, and that desert protozoa may be adapted to a specific desert's temperature and precipitation regime.     

Nitrogen cycling in desert biological soil crusts across biogeographic regions in the Southwestern United States

Biological soil crusts (BSCs) are thought to be important in the fertility of arid lands as gateways for carbon (C) and nitrogen (N). Studies on the Colorado Plateau have shown that an incomplete internal N cycle operates in BSCs that results in significant exports of dissolved organic N, ammonia and nitrate into the bulk soil through percolating water, thus mechanistically explaining their role as a N gateway. It is not known if this pattern is found in other arid regions. To examine this, we measured rates of major biogeochemical N-transformations in a variety of BSCs collected from the Colorado Plateau and the Mojave, Sonoran and Chihuahuan Deserts. Dinitrogen fixation and aerobic ammonia oxidation were prominent transformations at all sites. We found anaerobic ammonia oxidation (anammox) rates to be below the detection limit in all cases, and at least 50-fold smaller than rates of N₂-fixation, making it an irrelevant process for these BSCs. Heterotrophic denitrification was also of little consequence for the flow of N, with rates at least an order of magnitude smaller than those of N₂-fixation. Thus we could confirm that despite the demonstrable differences in microbial community composition and soil material, BSCs from major biogeographic regions in arid North America displayed a remarkably consistent pattern of internal N cycling. The implications for arid land fertility drawn from previous studies in the Colorado Plateau appear applicable to BSCs across other arid regions of the Southwestern United States.     

Patterns of diversity for fungal assemblages of biological soil crusts from the southwestern United States

Molecular methodologies were used to investigate fungal assemblages of biological soil crusts (BSCs) from arid lands in the southwestern United States. Fungal diversity of BSCs was assessed in a broad survey that included the Chihuahuan and Sonoran deserts as well as the Colorado Plateau. At selected sites samples were collected along kilometer-scale transects, and fungal community diversity and composition were assessed based on community rRNA gene fingerprinting using PCR-denaturing gradient gel electrophoresis (DGGE). Individual phylotypes were characterized through band sequencing. The results indicate that a considerable diversity of fungi is present within crusted soils, with higher diversity being recovered from more successionally mature BSCs. The overwhelming majority of crust fungi belong to the Ascomycota, with the Pleosporales being widespread and frequently dominant. Beta diversity patterns of phylotypes putatively representing dominant members of BSC fungal communities suggest that these assemblages are specific to their respective geographic regions of origin.     

Development of artificially induced biological soil crusts in fields and their effects on top soil

Aims

Biological soil crusts (BSCs) could improve severe environment ecological conditions by increasing soil moisture, soil nitrogen concentration, and so on. In order to control desertification and recover the destroyed soil fertility utilizing a new means using BSCs, the soil surface was artificially inoculated with Microcoleus vaginatus and Scytonema javanicum. Relationships between the development of the artificially induced biological soil crusts and the distribution and dynamic changes of nitrogen and phosphorus in the soil crusts have been analyzed.

Methods

Crusts of different ages were investigated by measuring soil physical and chemical factors, such as moisture, pH, total and available N content, and total and available P, which were correlated with the depths of the crusts.

Results

This study found that the types of color, shape, and species components of the algal crusts increased with crust development. Soil moisture, total N, available N, and available P increased gradually with crust growth. Soil with crusts was wetter than the controlled naked sandy soil, and a significant correlation was observed between biomass and total nitrogen (r?=?0.946, P?=?0.015). Soil pH was lower than that of control. The scytonemin on the soil surface was exceptionally higher than the other pigments, and all the pigments were mainly distributed at the soil surface level. Though the crusts were mainly distributed on soil surface, the available P was mainly stored below the crust layer.

Conclusions

Pearson correlation tests indicated that artificially inoculated biological crusts could improve soil fertility and micro-environment of the top soil: The development of artificially induced BSCs was very well, and this was favorable to inducing the following crust succession.     

UV-B resistance as a criterion for the selection of desert microalgae to be utilized for inoculating desert soils

The adaption capability of microalgae species to intense UV-B radiation is an important feature for their survival under the harsh growth conditions they have to face when used for inoculating unconsolidated sand soils in desert areas. In this study, the responses of photosynthetic activity, reactive oxygen species (ROS) generation, and DNA strand breaks to UV-B radiation in four microalgae isolated from artificially induced biological soil crusts were investigated. It was found that low UV-B doses easily inhibited the photosynthetic activity and induced serious DNA damage in Chlorella vulgaris. Microcoleus vaginatus showed the capability to withstand only moderate doses of UV-B, while Nostoc was capable of facing high doses of UV-B due to its lower generation of ROS and higher capability to repair photosystem II (PSII) and DNA damages. On the other hand, Scytonema javanicum showed additional strategies to survive UV-B irradiance, namely the closure of PSII when ROS generation increased rapidly, in addition to a high repair ability of PSII and DNA damage. The results obtained point out different resistance and defense mechanisms of the four microalgae in response to UV-B irradiance and suggest that the strain of Nostoc sp. tested is the most suitable for surviving under the high UV irradiation levels typical of desertified areas.     

Effect of desert soil algae on the stabilization of fine sands

Four filamentous cyanobacteria, Microcoleusvaginatus, Phormidium tenue,Scytonemajavanicum (Kutz.) and Nostoc sp., and asingle-celled green alga, Desmococcus olivaceus, allisolated from Shapotou (Ningxia Hui Autonomous Region of China), were batchcultured and inoculated onto unconsolidated sand in greenhouse and fieldexperiments. Their ability to reduce wind erosion in sands was quantified byusing a wind tunnel laboratory. The major factors related to cohesion of algalcrusts, such as biomass, species, species combinations, bioactivity, niche,growth phase of algae, moisture, thickness of the crusts, dust accretion(including dust content and manner of dust added) and other cryptogams(lichens,fungi and mosses) were studied. The best of the five species were M.vaginatus and P. tenue, while the best mix wasablend of 80% M. vaginatus and 5% each of P.tenue,S. javanicum,Nostocsp. and D. olivaceus. The threshold friction velocity wassignificantly increased by the presence of all of the cyanobacterial species,while the threshold impact velocity was notably increased only by thefilamentous species. Thick crusts were less easily eroded than thin crusts,while biomass was more effective than thickness. Dust was incorporated bestintoMicrocoleus crust when added in small amounts over time,and appeared to increase growth of the cyanobacterium as well as strengthen thecohesion of the crust. Microbial crust cohesion was mainly attributed to algalaggregation, while lichens, fungi and mosses affected more the soil structureand physico-chemical properties.     

Biological soil crust (BSC) is an effective biofertilizer on Vigna mungo (L.)

Biological soil crusts (BSC) (Cyanobacteria) play an important role in the soil nitrogen fixation and soil stabilization. However, limited researches were carried out about the diversity and distribution of Bio crust in sacred forests. The study aims to identify the distribution of cyanobacteria in biological soil crusts from different sacred groves of Ariyalur and Pudukottai, Tamil Nadu. We identified following microbes of Microcoleus, Scytonema, Anabaena and Nostoc in biological soil crust. A surface experiment was conducted for the efficacy of biological soil crusts on crops seedling growths. The efficacy was assessed by measuring the root & shoot length, dry & fresh weight of the plant, total chlorophyll, protein & carbohydrate content, with reducing sugar. The plant growth was higher in biocrust inoculated pot than the control. In corresponding nitrogenase activity was determined by the acetylene reduction assay, and phytohormones documentation was executed by the high-performance liquid chromatography. The results indicating that the biological soil crusts are the promising factors influencing the plant growth by plant growth-stimulating auxins and nitrogenase activity.     

Desiccation provides photosynthetic protection for crust cyanobacteria Microcoleus vaginatus from high temperature

As the dominant cyanobacterial species in biological soil crusts (BSCs), Microcoleus vaginatus often suffer from many stress conditions, such as desiccation and high temperature. In this study, the activities of light‐harvesting complexes (LHCs) and reaction centers of photosystem II (PS II) in crust cyanobacteria M. vaginatus were monitored under high temperature and desiccation conditions using chlorophyll fluorescence technology. The results showed that all the fluorescence signals were significantly inhibited by high temperature or desiccation treatments. Under high temperature conditions, it was further demonstrated that PS II reaction centers were first destructed within the first hour, then the LHCs gradually dissociated and free phycocyanin formed within 1–5 h, and the activities of all the light harvesting and reaction center pigment proteins were fully suppressed after 24 h of high temperature treatment. Furthermore, the high temperature treated M. vaginatus lost its ability to recover photosynthetic activity. On the contrary, although desiccation also led to the loss of photosynthetic activity in M. vaginatus, after rehydration in the light the fluorescence parameters including Fo, Fv and Fv/Fm could be well recovered within 12 h. It was concluded that desiccation could provide crust cyanobacteria M. vaginatus some protection from other stresses, such as high temperature demonstrated in this experiment. The combine of temperature change and precipitation pattern in the field provide a guarantee for the alternate metabolism and inactivity in crust cyanobacteria. That may be a very important strategy for the survival of crust cyanobacteria in high temperature regions.     

Soil stabilization by a prokaryotic desert crust: Implications for Precambrian land biota

A cyanophyte dominated mat, desert crust, forms the ground cover in areas measuring hundreds of square meters in Utah and smaller patches in Colorado. The algal mat shows stromatolitic features such as sediment trapping and accretion, a convoluted surface, and polygonal cracking. Sand and clay particles are immobilized by a dense network of filaments of the two dominating cyanophyte species,Microcoleus vaginatus andM. chthonoplastes, which secrete sheaths to which particles adhere. These microorganisms can tolerate long periods of desiccation and are capable of instant reactivation and migration following wetting. Migration occurs in two events: 1. immediately following wetting of dry mat, trichomes are mechanically expelled from the sheath as it swells during rehydration, and 2. subsequently, trichomes begin a self-propelled gliding motility which is accompanied by further production of sheath. The maximum distance traveled on solid agar by trichomes ofMicrocoleus vaginatus during a 12 hour period of light was 4.8 cm. This corresponds to approximately 500 times the length of the fastest trichome, and provides a measure of the potential for spreading of the mat in nature via the motility of the trichomes.Dehydration resistence of the sheath modifies the extracellular environment of the trichomes and enables their transition to dormancy. Following prolonged wetting and evaporative drying of the mat in the laboratory, a smooth wafer-like crust is formed by the sheaths ofMicrocleus trichomes that have migrated to the surface. Calcium carbonate precipitates among the algal filaments under experimental conditions, indicating a potential for mat lithification and fossilization in the form of a caliche crust. It is suggested that limestones containing tubular microfossils may, in part, be of such an origin.The formation of mature Precambrian soils may be attributable to soil accretion, stabilization, and biogenic modification by blue-green algal land mats similar to desert crust.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation

Biological soil crusts (BSCs) are key components of ecosystem productivity in arid lands and they cover a substantial fraction of the terrestrial surface. In particular, BSC N₂-fixation contributes significantly to the nitrogen (N) budget of arid land ecosystems. In mature crusts, N₂-fixation is largely attributed to heterocystous cyanobacteria; however, early successional crusts possess few N₂-fixing cyanobacteria and this suggests that microorganisms other than cyanobacteria mediate N₂-fixation during the critical early stages of BSC development. DNA stable isotope probing with ¹⁵N₂ revealed that Clostridiaceae and Proteobacteria are the most common microorganisms that assimilate ¹⁵N₂ in early successional crusts. The Clostridiaceae identified are divergent from previously characterized isolates, though N₂-fixation has previously been observed in this family. The Proteobacteria identified share >98.5% small subunit rRNA gene sequence identity with isolates from genera known to possess diazotrophs (for example, Pseudomonas, Klebsiella, Shigella and Ideonella). The low abundance of these heterotrophic diazotrophs in BSCs may explain why they have not been characterized previously. Diazotrophs have a critical role in BSC formation and characterization of these organisms represents a crucial step towards understanding how anthropogenic change will affect the formation and ecological function of BSCs in arid ecosystems.     

铜陵铜尾矿废弃地生物土壤结皮中的蓝藻多样性

生物土壤结皮是生态系统原生演替过程中的一个早期阶段,在铜陵铜尾矿废弃地自然生态恢复过程中生物土壤结皮在尾矿废弃地表面广泛分布.以生长在铜陵杨山冲和铜官山2处铜尾矿废弃地的生物土壤结皮为研究对象,运用常规培养方法和变性梯度凝胶电泳技术(PCR-DGGE)对不同群落生物土壤结皮中的蓝藻多样性及优势类群进行研究.结果表明2种研究方法所获得的蓝藻种类组成具有明显差异.显微观察结果表明常规培养试验中主要蓝藻类群为微囊藻属(Microcystis)、色球藻属(Chroococcus)、颤藻属(Oscillatoria)、念珠藻属(Nostoc)和浮鞘丝藻属(Planktolyngbya),其中优势种类主要为铜绿微囊藻(Microcystis aeruginosa)、断裂颤藻(Oscillatoria fracta)和细浮鞘丝藻(Planktolyngbya subtilis);提取样品中微生物总DNA,对蓝藻16SrRNA进行PCR-DGGE分析,回收DGGE图谱中24个条带进行测序分析,结果显示,所有序列与GenBank数据库中的近缘蓝藻的相似性系数均在93％以上,其中优势蓝藻类群主要隶属于微鞘藻属(Microcoleus)和细鞘丝藻属(Leptolyngbya),裸地(YL)处和木贼群落下尾矿表面(YM)的生物土壤结皮中优势蓝藻类群主要为微鞘藻属,而黄色真藓-藻类混合结皮(YT)和白茅群落( YB,TG)下的生物土壤结皮中的优势类群主要隶属于细鞘丝藻属.     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.