Phototrophic Purple Bacteria Research Articles

Continuous H2S removal from biogas using purple phototrophic bacteria

Biogas from anaerobic digestion requires desulfurisation before its intended use due to the corrosive nature of H2S. Anaerobic digesters produce biogas continuously and require a continuous process for desulfurisation. Commonly, H2S is removed using adsorption to solid media (iron sponge) or absorption and removal through chemical processes (scrubbing), requiring regeneration or replacement of sorbents. As an alternative, a continuous, anaerobic H2S removal process, utilising purple phototrophic bacteria was developed. The removal is driven by light as an energy source and potentially reduces the requirement for caustic. A desulfurisation bubble column was fed continuously with a synthetic biogas mixture (2000/5000 ppmV H2S, 30% CO2, CH4 balance) at sulfide loading rates ranging from 3.06 to 11.88 mg-S d−1. The liquid was inoculated with purple phototrophic bacteria, nutrients supplied via centrate, an anaerobic digestion residue, and externally irradiated with IR-light. The maximum sulfide removal rate was 10.13 ± 0.06 mg-S (Lh)−1 at a loading rate of 11.83 mg-S (Lh)−1, and the biomass oxidised the sulfide to sulfate utilising electrons for growth. Initially, caustic was dosed to control pH, which was replaced by hydraulic flushing (HRT 4 days), resulting in a mean pH of 6.8 at applied sulfide loading rate of 9.44 mg-S (Lh)−1. A reduction in HRT to 2 days resulted in performance loss. Thus, controlling the biomass inventory was found to be the major challenge of the process. Overall, the process can run caustic-free, and integrate with anaerobic digestion using the centrate as a nutrient source, with light supply limiting feasibility for night-time operation in a scaled process.

Exploring the metabolic capabilities of purple phototrophic bacteria during piggery wastewater treatment

Purple phototrophic bacteria (PPB) are receiving an increasing attention due to their extraordinary metabolic capabilities for nutrient and carbon recovery from wastewaters. Batch experiments were herein performed to assess the influence of the type of radiation (photosynthetic active radiation (PAR), near-infrared radiation (NIR) and PAR using an UV-VIS absorbing filter), temperature (13 °C and 30 °C), type of metabolism (photoheterotrophic and chemoheterotrophic), type of inoculum (mixed culture and pure strain Rhodopseudomonas palustris) and wastewater load (1:5 and 1:10 dilutions) during piggery wastewater (PWW) treatment by PPB. The use of UV-VIS filtered PAR supported both a high content of bacteriochlorophyll in PPB and the highest total organic carbon (TOC) and total nitrogen (TN) removal efficiency (RE) (74 % and 37 %, respectively), but at lower maximum removal rates. Interestingly, PPB exhibited similar TOC-REs and TN-REs (73 % and 37 %, respectively) at 13 °C than at 30 °C (71 % and 45 %, respectively), at similar removal rates. Mixed cultures of PPB achieved a higher nutrient assimilation rate than R. palustris, supporting a total assimilation of the volatile fatty acids present in 10 folds diluted PWW. In brief, mixed cultures of PPB were highly efficient during PWW treatment, regardless of the type of radiation and temperature under photoheterotrophic growth.

Polyhydroxybutyrate production in one-stage by purple phototrophic bacteria: Influence of alkaline pH, ethanol, and C/N ratios

This work aimed to evaluate the effect of alkaline pH, nutrient limitation, nitrogen source, C/N ratio, and ethanol addition on polyhydroxybutyrate (PHB) production by a photoheterotrophic mixed culture enriched in Rhodopseudomonas palustris. The results demonstrated the natural alkalinization of the culture medium was enough to induce PHB accumulation in the mixed culture. The combination of alkaline pH, nitrogen, sulfur, or phosphate deficiency negatively affected polymer production. The incorporation of glutamate for biomass synthesis was better than that of ammonium, which had a positive effect on PHB production. Interestingly, ethanol addition improved the PHB accumulation by 25 %, but without consumption. PHB production improved at low C/N ratios, where 27 ± 0.9% of the carbon source was transformed into biopolymers. The evaluated factors allowed the biomass PHB content to increase by 60%, reaching PHB production of 219 ± 5 mg/L. The PHB production by the photoheterotrophic mixed culture is associated with growth, allowing the PHB production in one stage. That has significant advantages in technical-practical terms, such as reducing the time of the process, dispensing with the separation of biomass from the feast phase to the famine phase, and requiring a single reactor.

Improvement of biogas production and nitrogen recovery in anaerobic digestion of purple phototrophic bacteria by thermal hydrolysis

Purple phototrophic bacteria (PPB) are a novel driver to recover organics and nutrients from wastewater by assimilative growth. Depending on the source, assimilated resources from the PPB biomass can still be recovered after a releasing step. Anaerobic digestion (AD) releases carbonand nutrients, but the release is incomplete. Thermal hydrolysis (TH) as a pretreatment before AD improves the digestibility, release, and subsequent recovery potentials. This work determines the effects of TH in batch and continuous modes regarding methane potential, nutrients' release efficiencies, volatile solids destruction, degradability, and hydrolysis rates. Continuous runs over 165 days (d) confirmed enhanced recovery potentials, achieving up to 380 LCH4/kgVS (83 % solids destruction) and 73 % N release, respectively. The TH pretreatment is energy-intensive, but with appropriate heat recovery and increased methane production in the AD of the pretreated biomass, a combined configuration is energy positive.

Exploring the role of antimicrobials in the selective growth of purple phototrophic bacteria through genome mining and agar spot assays.

Purple non-sulphur bacteria (PNSB) are an emerging group of microbes attractive for applied microbiology applications such as wastewater treatment, plant biostimulants, microbial protein, polyhydroxyalkanoates and H2 production. These photoorganoheterotrophic microbes have the unique ability to grow selectively on organic carbon in anaerobic photobioreactors. This so-called selectivity implies that the microbial community will have a low diversity and a high abundance of a particular PNSB species. Recently, it has been shown that certain PNSB strains can produce antimicrobials, yet it remains unclear whether these contribute to competitive inhibition. This research aimed to understand which type of antimicrobial PNSB produce and identify whether these compounds contribute to their selective growth. Mining 166 publicly-available PNSB genomes using the computational tool BAGEL showed that 59% contained antimicrobial encoding regions, more specifically biosynthetic clusters of bacteriocins and non-ribosomal peptide synthetases. Inter- and intra-species inhibition was observed in agar spot assays for Rhodobacter blasticus EBR2 and Rhodopseudomonas palustris EBE1 with inhibition zones of, respectively, 5.1 and 1.5-5.7 mm. Peptidomic analysis detected a peptide fragment in the supernatant (SVLQLLR) that had a 100% percentage identity match with a known non-ribosomal peptide synthetase with antimicrobial activity.

Light intensity defines growth and photopigment content of a mixed culture of purple phototrophic bacteria.

Purple bacteria (PPB), anoxygenic photoorganoheterotrophic organisms with a hyper-versatile metabolism and high biomass yields over substrate, are promising candidates for the recovery of nutrient resources from wastewater. Infrared light is a pivotal parameter to control and design PPB-based resource recovery. However, the effects of light intensities on the physiology and selection of PPB in mixed cultures have not been studied to date. Here, we examined the effect of infrared irradiance on PPB physiology, enrichment, and growth over a large range of irradiance (0 to 350 W m-2) in an anaerobic mixed-culture sequencing batch photobioreactor. We developed an empirical mathematical model that suggests higher PPB growth rates as response to higher irradiance. Moreover, PPB adapted to light intensity by modulating the abundances of their phototrophic complexes. The obtained results provide an in-depth phylogenetic and metabolic insight the impact of irradiance on PPB. Our findings deliver the fundamental information for guiding the design of light-driven, anaerobic mixed-culture PPB processes for wastewater treatment and bioproduct valorization.

Fluid-like electrodes and Purple Phototrophic Bacteria: bridging the gap in wastewater biorefineries

Wastewater biorefineries aim to generate value-added products in an economically viable process while removing pollutants. In this scenario, Purple phototrophic bacteria (PPB), the most versatile microorganisms on earth, are highly effective for sustainable wastewater treatment and nutrient recovery as cell protein. One of the most innovative approaches for applying PPB in the wastewater sector is their capacity for interchanging electrons with electroconductive materials. In contrast with classical biofilm-based techniques, we have demonstrated that a fluid-like electrode can accept electrons from planktonically grown PPB. We anticipate that such findings will impact in wastewater electrobioremediating capacity of PPB. Moreover, controlling the electrochemical nature of the extracellular electron acceptor (fluid-like electrode) allows for fine-tuning the metabolism of a planktonic PPB-dominated community to enhance their biodegradation rate (2-fold) while growing on brewery wastewater. For this purpose, a twin set of microbial electrochemical fluidized bed reactors (ME-FBR) were operated in identical conditions, except for illumination conditions (dark vs. infrared), to promote the development of PPB. Illumina sequencing revealed that both infrared radiation and polarization led to changes in the microbial population while producing an electrical current of 7 A·m-3. Indeed, the Geobacter genus was the electroactive bacteria outcompeting under dark conditions. In contrast, electroactive PPB genera like the Rhodopseudomonas and Rhodobacter outcompeted others under infrared illumination and electrostimulation. In this work, we have demonstrated how microbial selection can contribute to the sustainability of an electrobioremediation wastewater treatment by avoiding emissions of greenhouse gases such as methane. In addition, fluid-like bed bioreactors have shown their usefulness in recovering nutrients as PPB biomass, favoring planktonic growth and thus facilitating the recovery of a valuable product: the biomass of PPB.

Diversity of anoxygenic phototrophic purple bacteria from India

Diversity of anoxygenic phototrophic purple bacteria from India

Taxonomic Re-Evaluation and Genomic Comparison of Novel Extracellular Electron Uptake-Capable Rhodovulum visakhapatnamense and Rhodovulum sulfidophilum Isolates

Rhodovulum spp. are anoxygenic phototrophic purple bacteria with versatile metabolisms, including the ability to obtain electrons from minerals in their environment to drive photosynthesis, a relatively novel process called phototrophic extracellular electron uptake (pEEU). A total of 15 strains of Rhodovulum sulfidophilum were isolated from a marine estuary to observe these metabolisms in marine phototrophs. One representative strain, Rhodovulum sulfidophilum strain AB26, can perform phototrophic iron oxidation (photoferrotrophy) and couples carbon dioxide fixation to pEEU. Here, we reclassify two R. sulfidophilum isolates, strainAB26 and strain AB19, as Rhodovulum visakhapatnamense using taxonomic re-evaluation based on 16S and pufM phylogenetic analyses. The strain AB26 genome consists of 4,380,746 base-pairs, including two plasmids, and encodes 4296 predicted protein-coding genes. Strain AB26 contains 22 histidine kinases, 20 response regulators, and dedicates ~16% of its genome to transport. Transcriptomic data under aerobic, photoheterotrophy, photoautotrophy, and pEEU reveals how gene expression varies between metabolisms in a novel R. visakhapatnamense strain. Genome comparison led by transcriptomic data under pEEU reveals potential pEEU-relevant genes both unique to R. visakhapatnamense strains and shared within the R. sulfidophilum genomes. With these data we identify potential pEEU-important transcripts and how speciation may affect molecular mechanisms of pEEU in Rhodovulum species from the same environment.

Unraveling PHA production from urban organic waste with purple phototrophic bacteria via organic overload

The production of polyhydroxyalkanoates (PHA) with purple phototrophic bacteria (PPB) has been limited due to low yields and limited knowledge regarding the diverse routes used for carbon biosynthesis. The present study increases PHA accumulation yields using urban organic waste pretreated by steam explosion and acidogenic fermentation as substrate. Throughout the PPB-based photoheterotrophic process in an anaerobic membrane photobioreactor, the organic loading rate (OLR) was modified to increase the amount of PHA and biomass in the reactor. A maximum PHA accumulation of 42% (gPHA gBiomass−1) on a dry basis was achieved and maintained for 10 d for an OLR of 1 gCOD L−1 d−1, and hydraulic and sludge retention times of 2 and 6 d, respectively. This PHA accumulation capacity is the maximum obtained using a mixed culture of PPB fed with waste. Also, a medium-chain PHA (polyhydroxyhexanoate) has been quantified, enhancing the physicochemical properties and diversifying their industrial applications. Furthermore, we show novel alternatives to PHA accumulation: carbon storage as glycogen and extracellular polymers while deriving the excess electrons into hydrogen. Finally, a statistical study of microbial communities has settled the environmental variables with the most significant influence on these communities' variability. This work demonstrates the importance of acquiring a thorough understanding of carbon accumulation and electron allocation strategies of PPB under stressful conditions and shows promising results for a larger scale implementation of a PPB-based photobiorefinery, which could valorize urban organic waste to produce different high added-value products within the context of the circular bioeconomy.

Light attenuation in enriched purple phototrophic bacteria cultures: Implications for modelling and reactor design

Light attenuation in enriched purple phototrophic bacteria (PPB) cultures has not been studied, and its understanding is critical for proper process modelling and reactor design, especially for scaled systems. This work evaluated the effect of different biomass concentrations, reactor configurations, wastewater matrices, and growth conditions, on the attenuation extent of near infra-red (NIR) and ultraviolet-visible (UV–VIS) light spectra. The results show that increased biomass concentrations lead to higher light attenuation, and that PPB absorb both VIS and NIR wavelengths, with both fractions of the spectrum being equally absorbed at biomass concentrations above 1,000 g COD·m−3. A flat plate configuration showed less attenuation compared with cylindrical reactors illuminated from the top, representative for open ponds. Neither a complex wastewater matrix nor the presence of polyhydroxyalkanoates (under nutrient limited conditions) affected light attenuation significantly. The pigment concentration (both bacteriochlorophyll and carotenoids) however, had a strong effect, with significant attenuation in the presence of pigments. Attenuation predictions using the Lambert-Beer law (excluding scattering) and the Schuster model (including scattering) indicated that light scattering had a minimal effect. A proposed mathematical model, based on the Lambert-Beer law and a Monod function for light requirements, allowed effective prediction of the kinetics of photoheterotrophic growth. This resulted in a half saturation coefficient of 4.6 W·m−2. Finally, the results showed that in dense outdoor PPB cultures (≥1,000 g COD·m−3), effective light penetration is only 5 cm, which biases design away from horizontal lagoons, and towards non-incident multi-panel systems such as flat plate reactors.

Photosynthetic treatment of piggery wastewater in sequential purple phototrophic bacteria and microalgae-bacteria photobioreactors

Nowadays, piggery wastewater (PWW) management still represents an unsolved global environmental problem. Photosynthetic processes have emerged as an innovative biological platform capable of performing a cost-effective treatment of wastewater with a concomitant assimilation of nutrients into biomass. In this work, the performance of a purple phototrophic bacteria photobioreactor (PPB-PBR) coupled with a microalgae-bacteria photobioreactor (MB-PBR) was assessed during the treatment of PWW at an hydraulic retention time (HRT) of 12.2 (stage I) and 6.2 days (stages II–VI) and intensities of near-infrared radiation in the PPB-PBR of 30 W m−2 (stages I–II) and 114 W m−2 (stages III–IV). Maximum removal efficiencies of total dissolved organic carbon (TOC-RE) and total dissolved nitrogen (TN-RE) of 91% and 82%, respectively, were recorded at an HRT of 12.2 days. The decrease in HRT to 6.2 days reduced the TOC-RE and TN-RE in both photobioreactors, but the increase in near-infrared radiation enhanced TOC-RE in the PPB-PBR, contributing to a global carbon recovery of 67% via assimilation in the form of PPB biomass. PPB-PBR was highly efficient in carbon assimilation, while MB-PBR enhanced nitrogen and total suspended solids removals, with a contribution to TN-RE of 63% and a global decrease in TSS of 76%. The culture broth of PPB-PBR was dominated by Rhodopseudomonas sp. up to 54%, supported by the high HRT and the increase in near-infrared radiation, while the sequential MB-PBR favoured the dominance of Mychonastes homosphaera. This work demonstrated, for the first time, the high efficiency of sequentially coupling PPB and microalgae for the treatment of PWW.

A Ca2+-binding motif underlies the unusual properties of certain photosynthetic bacterial core light-harvesting complexes

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca2+-dependent. However, it is unknown what structural determinants might contribute to these properties. Here, we present a cryo-EM structure of the reaction center–associated LH1 complex at 2.81 Å resolution, in which we identify multiple pigment-binding α- and β-polypeptides within an LH1 ring. Of the 16 α-polypeptides, we show that six (α1) bind Ca2+ along with β1- or β3-polypeptides to form the Ca2+-binding sites. This structure differs from that of fully Ca2+-bound LH1 from Thermochromatium tepidum, enabling determination of the minimum structural requirements for Ca2+-binding. We also identified three amino acids (Trp44, Asp47, and Ile49) in the C-terminal region of the A. tepidum α1-polypeptide that ligate each Ca ion, forming a Ca2+-binding WxxDxI motif that is conserved in all Ca2+-bound LH1 α-polypeptides from other species with reported structures. The partial Ca2+-bound structure further explains the unusual phenotypic properties observed for this bacterium in terms of its Ca2+-requirements for thermostability, spectroscopy, and phototrophic growth, and supports the hypothesis that A. tepidum may represent a “transitional” species between mesophilic and thermophilic purple sulfur bacteria. The characteristic arrangement of multiple αβ-polypeptides also suggests a mechanism of molecular recognition in the expression and/or assembly of the LH1 complex that could be regulated through interactions with reaction center subunits.

Structural basis for the assembly and quinone transport mechanisms of the dimeric photosynthetic RC\u2013LH1 supercomplex

The reaction center (RC) and light-harvesting complex 1 (LH1) form a RC–LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple phototrophic bacteria. Some species possess the dimeric RC–LH1 complex with a transmembrane polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC–LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC–LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC–LH1 dimer, interlocking association between the components and mediating RC–LH1 dimerization. Moreover, we identify another transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations provide a mechanistic understanding of the assembly and electron transport pathways of the RC–LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex.

Outdoor demonstration-scale flat plate photobioreactor for resource recovery with purple phototrophic bacteria

To make purple phototrophic bacteria (PPB)-based technologies a reality for resource recovery, research must be demonstrated outdoors, using scaled reactors. In this study, a 10 m long PPB-enriched flat plate photobioreactor (FPPBR) with a volume of 0.95 m3 was operated for 253 days, fed with poultry processing wastewater. Different operational strategies were tested, including varying influent types, retention times, feeding strategies, and anaerobic/aerobic conditions in a novel mixed metabolic mode concept. The overall results show that regardless of the fermented wastewater fed (raw or after solid removal via dissolved air flotation) and the varying environmental conditions (e.g., light exposure and temperatures), the FPPBR provided effective volatile fatty acids (VFAs), N, and P removals (average efficiencies of >90%, 34–77%, and 28–45%, respectively). The removal of N and P was limited by the availability of biodegradable COD. Biomass (C, N and P) could be harvested at ∼90% VS/TS ratio, 58% crude protein content and a suitable amino acid profile for potential feed applications. During fully anaerobic operation with semicontinuous/day-only feeding, the FPPBR showed biomass productivities between 25 and 84 g VS m−2 d−1 (high due to solid influx; the productivities estimated from COD removal rates were 6.0–24 g VS•m−2•d−1 (conservative values)), and soluble COD removal rates of up to 1.0 g•L−1•d−1 (overall average of 0.34 ± 0.16 g•L−1•d−1). Under these conditions, the relative abundance of PPB in the harvested biomass was up to 56%. A minimum overall HRT of 2–2.4 d (1.0–1.2 d when only fed during the day) is recommended to avoid PPB washout, assuming no biomass retention. A combined daily-illuminated-anaerobic/night-aerobic operation (supplying air during night-time) exploiting photoheterotrophy during the day and aerobic chemoheterotrophy of the same bacteria at night improved the overall removal performance, avoiding VFA accumulation during the night. However, while enabling enhanced treatment, this resulted in a lower relative abundance of PPB and reduced biomass productivities, highlighting the need to balance resource recovery and treatment goals.

Naturally illuminated photobioreactors for resource recovery from piggery and chicken-processing wastewaters utilising purple phototrophic bacteria

Resource recovery from wastewater, preferably as high value products, has become an integral part of modern wastewater treatment. This work presents the potential to produce single cell protein (SCP) from pre-settled piggery wastewater (PWW) and meat chicken processing wastewater (CWW), utilising anaerobic purple phototrophic bacteria (PPB). PPB were grown as biofilm in outdoors 60 L, 80 L and 100 L flat-plate reactors, operated in sequential batch mode. PPB biofilm was recovered from reactor walls at a total solid (TS) content ∼90 g•L − 1, and the harvested biomass (depending on the wastewater) had a consistent quality, with high protein contents (50–65%) and low ash, potentially applicable as SCP. The COD, N and P removal efficiencies were 71±5.3%, 22±6.6%, 65±5.6% for PWW and 78±1.8%, 67±2.7% and 37±4.0% for CWW, respectively, with biofilm areal productivities up to 14 g TS•m − 2•d − 1. This was achieved at ammonium-N concentrations over 1.0 g•L − 1 and temperatures up to 55 °C and down to 6 °C (daily fluctuations of 20–30 °C). The removal performances and biomass productivities were mostly dependent on the bioavailable COD in the form of volatile fatty acids (VFA). At sufficient VFA availability, the irradiance became limiting, capping biofilm formation. Harvesting of the suspended fraction resulted in increased productivities and recovery efficiencies, but lowered the product quality (e.g., containing undesired inerts). The optimum between quantity and quality of product is dependent on the wastewater characteristics (i.e., organic degradable fraction) and potential pre-treatment. This study shows the potential to utilise sunlight to treat agri-industrial wastewaters while generating protein-rich PPB biomass to be used as a feed, feed additive or feed supplement.

Continuous H2s Removal from Biogas Using Purple Phototrophic Bacteria

Continuous H2s Removal from Biogas Using Purple Phototrophic Bacteria

Engineering purple bacterial carotenoid biosynthesis to study the roles of carotenoids in light-harvesting complexes.

Carotenoids are important photosynthetic pigments that play key roles in light harvesting and energy transfer, photoprotection, and in the folding, assembly, and stabilization of light-harvesting pigment-protein complexes. The genetically tractable purple phototrophic bacteria have been useful for investigating the biosynthesis and function of photosynthetic pigments and cofactors, including carotenoids. Here, we give an overview of the roles of carotenoids in photosynthesis and of their biosynthesis, focusing on the extensively studied purple bacterium Rhodobacter sphaeroides as a model organism. We provide detailed procedures for manipulating carotenoid biosynthesis, and for the preparation and analysis of the light-harvesting and photosynthetic reaction center complexes that bind them. Using appropriate examples from the literature, we discuss how such approaches have enhanced our understanding of the biosynthesis of carotenoids and the photosynthesis-related functions of these fascinating molecules.

Exploring the Metabolic Capabilities of Purple Phototrophic Bacteria During Piggery Wastewater Treatment

Exploring the Metabolic Capabilities of Purple Phototrophic Bacteria During Piggery Wastewater Treatment

Potential of Phototrophic Purple Nonsulfur Bacteria to Fix Nitrogen in Rice Fields.

Biological nitrogen fixation catalyzed by Mo-nitrogenase of symbiotic diazotrophs has attracted interest because its potential to supply plant-available nitrogen offers an alternative way of using chemical fertilizers for sustainable agriculture. Phototrophic purple nonsulfur bacteria (PNSB) diazotrophically grow under light anaerobic conditions and can be isolated from photic and microaerobic zones of rice fields. Therefore, PNSB as asymbiotic diazotrophs contribute to nitrogen fixation in rice fields. An attempt to measure nitrogen in the oxidized surface layer of paddy soil estimates that approximately 6–8 kg N/ha/year might be accumulated by phototrophic microorganisms. Species of PNSB possess one of or both alternative nitrogenases, V-nitrogenase and Fe-nitrogenase, which are found in asymbiotic diazotrophs, in addition to Mo-nitrogenase. The regulatory networks control nitrogenase activity in response to ammonium, molecular oxygen, and light irradiation. Laboratory and field studies have revealed effectiveness of PNSB inoculation to rice cultures on increases of nitrogen gain, plant growth, and/or grain yield. In this review, properties of the nitrogenase isozymes and regulation of nitrogenase activities in PNSB are described, and research challenges and potential of PNSB inoculation to rice cultures are discussed from a viewpoint of their applications as nitrogen biofertilizer.