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Background: Utilization of animal wastes in algal culture has proven to be challenging. The utilization of “free” nutrients has drawn researchers and industry to develop business models that call for the use of these free nutrients, which comes at a cost. Some of these costs include reduced productivity, increased contamination, lower value target markets, and lower treatment capabilities (for wastewater treatment applications). This paper evaluates the impact of dairy lagoon effluent on productivity and wastewater treatment ability. Methods: Screened dairy lagoon wash water was fed to four three square meter outdoor open paddlewheel algal cultivation reactors. The units were operated semi-continuously for one and a half years. Seasonal productivity and nutrient uptake rates, for nitrogen (N) and phosphorous (P) were measured against wastewater dilution requirements. Seasonal algal species dominance was also recorded. Wastewater was added at two levels, and the lower level was supplemented with synthetic fertilizer. Results: Seasonal N uptake rates ranged from 0.5 to 1.2 grams of N uptake per square meter per day, while P uptake ranged from 0.17 to 0.3 grams of P per square meter per day depending on season and Hydraulic Residence Time (HRT). N removal efficiency ranged at 40 to 70% for semicontinuous operation, depending on HRT, season, and dilution of influent wastewater, which was made up from 1.5% to 13% of the daily water exchange. Conclusion: Algal reactors tended to be N limited due to the inability to add enough dairy wastewater to mitigate the high turbidity and dark color. Treatments with lower levels of added dairy wastewater tended to show higher nutrient removal. Algal culture from dairy wash water could benefit from a pretreatment step to reduce turbidity and color, thereby promoting algal growth and productivity.

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was &lt, 10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

Microalgae have been used commercially as a feedstock for the production of high-value compounds, pigments, cosmetics, and nutritional supplements. In addition, because of their rapid growth rates, high photosynthetic efficiency, and high lipid and protein content, commodity products including biodiesel, feed supplements, and polyunsaturated fatty acids derived from algal biomass are of current interest. Since microalgae lack non-photosynthetic structures and float in water, they do not need massive amounts of structural cellulose found in land plants. Thus, under optimal culture conditions, some oleaginous species can allocate up to 70 % of their biomass to lipids. Lipid production and its regulation in microalgae are species-specific and influenced by environmental conditions. Various strategies have been developed to improve lipid productivity and fatty acid composition to meet specific production goals. Manipulation of physiochemical parameters, trophic modes, and nutrient levels, known as process engineering, is a simple approach that leads to desired alterations in the biochemical composition of algal biomass, including lipid quantity and quality. In this paper, we review the effects of manipulating biochemical parameters such as necessary nutrients (C, N, P, S, Fe, and Si), NaCl concentration, and pH of culture medium to optimize lipid content and profile in some algae strains with commercial potential.

The bioconversion of sunlight energy into biofuels can be coupled to nutrient recovery from organic wastes using systems that employ microalgae grown symbiotically with heterotrophic microorganisms. Of interest for algae biofuel production is the high amounts of oil (lipid) produced, primarily as triglycerides. A major cost factor for algal biomass production is the provision of water and nutrients. In this chapter, progress made in microalgae-based systems using primary manure effluents with high biological oxygen demand levels that microbes decompose into inorganic nutrients including CO2, ammonium, and phosphate is reviewed. Anaerobic digesters offer an attractive alternative to direct treatment of agricultural wastes with several advantages from a fuel production perspective. During anaerobic digestion, organic matter is mineralized producing methane for fuel and inorganic C, N, and P, nutrients needed for algal growth. In each case, a resource and economic synergy are possible as algae-based bioremediation generates revenue from the treatment service and improves the economics of biofuel production.

The critical downstream processing steps of harvesting and lipid extraction have seen remarkable technological improvements in recent years which have improved their cost-effectiveness and efficiency. The type of extraction method used greatly influences the quality of lipid, residual biomass coproducts, and the efficiency of biodiesel production. Many different variations on the lipid extraction process have been assessed in recent years which include expellers, organic solvent extraction, supercritical fluid, subcritical water, electrochemical extraction methods, etc. Because the fuel characteristics of biodiesel are impacted by the choice of the extraction process to be employed, proper consideration must be given to the process optimization and technology adaptation for the specifications set for biodiesel by international standards. In this chapter, recent developments in the extraction methods of algae lipids are discussed and compared as well as the advantages and disadvantages of each method is introduced comprehensively in terms of products and economic issues.

In this chapter, biodiesels made from extracted lipids of two microalgae strains (i.e., Botryococcus braunii, Chlorella protothecoides), seed plant oils (i.e., corn oil, sesame oil, and canola oil), commercial biodiesel, BQ9000, and locally produced biodiesel from waste vegetable oil were analyzed. The fatty acid (FA) profiling was performed using gas chromatography analysis of the fatty acid methyl esters (FAMEs) and compared using both flame ionization detector and mass spectrometric detectors. It was determined that the microalgae analyzed contained higher percentages of saturated FAs, while plant biomass sources were rich in unsaturated FAs. The results showed that the FAME content of locally produced biodiesel was at least four times higher than that of the commercially produced biodiesel.

Dynamics of seasonal microbial community compositions in algae cultivation ponds are complex. However, there is very limited knowledge on bacterial communities that may play significant roles with algae in the bioconversion of manure nutrients to animal feed. In this study, water samples were collected during winter, spring, summer, and fall from the dairy lagoon effluent (DLE), high rate algae ponds (HRAP) that were fed with diluted DLE, and municipal waste water treatment plant (WWTP) effluent which was included as a comparison system for the analysis of total bacteria, Cyanobacteria, and microalgae communities using MiSeq Illumina sequencing targeting the 16S V4 rDNA region. The main objective was to examine dynamics in microbial community composition in the HRAP used for the production of algal biomass. DNA was extracted from the different sample types using three commercially available DNA extraction kits; MoBio Power water extraction kit, Zymo fungi/bacterial extraction kit, and MP Biomedicals FastDNA SPIN Kit. Permutational analysis of variance (PERMANOVA) using distance matrices on each variable showed significant differences (P=0.001) in beta-diversity based on sample source. Environmental variables such as hydraulic retention time (HRT; P

This chapter develops the principles and rationale for an algae-based biofuel production coupled to bioremediation of municipal and agricultural wastewaters . A synergistic model for algal wastewater treatment is proposed, which addresses several economic bottlenecks to earlier algal systems and promotes value-added products, including a high-quality effluent in addition to biodiesel to improve the economic feasibility of algal biofuels . Finally, we review candidate species for full-scale algae production ponds based on algal structure, physiology and ecology , and methods for extraction of algal oils for biodiesel production and coproducts. The dominant strains of algae that are commonly found in wastewater ponds, including Euglenia , Scenedesmus , Selenastrum , Chlorella, and Actinastrum , are suggested as candidates for large-scale culturing based on their ability to strip nutrients and organic matter from wastewater , grow rapidly, and produce a significant level of algal oil. Oil extraction by supercritical fluid extraction (SFE) is discussed as an efficient means of isolating algal oil and other commercially important high-value compounds from algal biomass . Together with water and CO2 reclamation, such products may shift the economics of algal biomass production to allow production of low-value commodities including biodiesel and biogas.

Our understanding of the actinorhizal symbiosis, in particular of the Frankia-Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface-sterilized, total DNA was extracted and amplified using prokaryotic-specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep-PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX-PCR fingerprints were also amplified by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus–infective strains.

An 8551-bp plasmid, pFQ11, from Frankia alni strain CpI1 was sequenced. Its sequence was found to be very similar to that presented for pFQ31 from strain ArI3. Six potential protein-encoding open reading frames (ORFs) were identified, and transcriptional activity was shown within four of those regions of the plasmid by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An earlier study reported that ORF E(F) of pFQ31, which is nearly identical to the 3' 45% of ORF1 of pFQ11, is significantly similar to RepF. We found no such similarity. ORF2 and ORF3 predict products that are similar to a repressor protein and a partition protein, respectively. We found inverted repeats within and covering the start codon of ORF3; palindromic sequences and direct repeats between ORF3 and ORF4; and 3' from ORF3, an AT-rich sequence that extensively overlaps the promoter region of a uvrB homolog in strain ArI3.

Mutants ofAnabaena variabilis, unable to fix nitrogen under aerobic conditions, were used to determine whether nitrogenase synthesis is subject to O2 repression, as is the case in some heterotrophic bacteria. Nitrogenase activity in the mutants was induced as heterocysts matured under microaerophilic conditions. However, addition of 5% O2 to the assay system inhibited activity by 95%. Under aerobic conditions, nitrogenase activity in the mutants could not be detected, but an activity-independent, immunological assay showed that the Fe-Mo protein was present at levels similar to those found in wild-type and mutant strains induced microaerophilically. Reducing the O2 tension of an aerobically induced mutant resulted in a rate of nitrogenase activity induction twice the rate under continuous microaerophilic conditions. These results indicate that O2 does not repress Fe-Mo protein synthesis in these mutants.

Frankia, the actinomycete partner in the nitrogenfixing symbiosis of certain woody non-legumes, has been shown to fix nitrogen in pure culture under aerobic conditions. The sensitivity of in vivo nitrogen-fixation (acetylene reduction) to oxygen tension in the gas phase was measured in short-term assays with two Frankia isolates designated ARI3 and CcI3. The carbon source utilized had an effect on the optimum O2 concentration for acetylene reduction. Cells utilizing an organic acid, e.g., propionate or pyruvate had maximum nitrogenase activity at an oxygen concentration of 15 to 20%. In contrast, cells respiring a sugar, e.g., trehalose or glucose, or endogenous reserves (glycogen or trehalose) had maximum acetylene reduction activity at 5 to 10% in the gas phase. Oxygen uptake kinetics showed that respiration in vesicle-containing cells utilizing trehalose had a biphasic response to oxygen concentration with a diffusion limited component at oxygen concentrations of 20 μM to more than 300 μM. These results suggested that trehalose was oxidized in the vesicles as well as in the vegetative hyphae. Oxygen concentration also had an effect on the trehalose-supported growth of cells (non nitrogenfixing, [+NH4Cl]). Cells grown with 5–10% O2 in the gas phase had a doubling time approximately half those grown with 20% O2 (atmospheric). Propionate-grown cells showed similar growth rates at the two oxygen tensions, and grew faster (almost 2x) than the trehalose cells at 5–10% O2. Trehalose also supported approximately 40% lower rates of oxygen uptake than propionate in vesicle-containing cells.

The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O2 for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O2 tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O2 production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.

The mechanism of O 2 protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O 2 inhibition of nitrogenase activity correlated with the O 2 tension of the medium in which heterocyst formation was induced. O 2 resistance also correlated with the apparent K m for acetylene, indicating that O 2 tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O 2 that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O 2 sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O 2 but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O 2 diffusion barrier and active respiration by the heterocyst.

Nitrogenase is known to be irreversibly inactivated by oxygen in vivo and in vitro. In time-course experiments using Anabaena cylindrica, cultures treated with antibiotics and incubated under various O2 tensions, in vivo acetylene reduction activity and immunologically determined Fe–Mo protein (component I) were lost at rates directly related to O2 tension. Activity was lost at a faster rate than cross-reactive material. The half-life of cross-reactive material was 25 h under microaerophilic conditions, 12 h under aerobic conditions, and 6.6 h under an O2 tension of 245% of air saturation. In vitro, cross-reactive material was lost in O2 exposed, but not in anaerobically prepared, crude cell extracts. Loss of cross-reactive material was prevented by freezing and by α-N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of histidine-residue proteases. These results indicate that nitrogenase is continuously inactivated by O2, hydrolyzed, and resynthesized during growth of this heterocystous cyanobacteria under aerobic conditions.

Hemoproteins were localized in the cyanobacteriumAnabaena cylindrica with diaminobenzidine (DAB). Incubation of whole cells in the light with DAB resulted in deposition of oxidized DAB on the lamellae of the vegetative cells and central heterocyst region. This reaction was greatest at pH 7.5, light-dependent, insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, and abolished by glutaraldehyde fixation. A light-independent oxidation of DAB was also observed with light and electron microscopy in the “honeycomb” region and periphery of heterocysts. This reaction was greatest at pH 7.5, enhanced by H2O2, and active in glutaraldehyde-fixed frozen sections. Inhibitors such as sodium cyanide, sulfide, and hydroxylamine severely reduced DAB oxidation and nitrogenase activity under aerobic but not anaerobic conditions. These results indicate that the heme proteins, localized in heterocysts by light-independent DAB oxidation, are involved in the oxygen-protection mechanism of the O2-labile nitrogenase.

The optimum conditions for growth ofFrankia sp. HFPCcI3 isolated fromCasuarina cunninghamiana, were studied in batch culture using defined media. Maximum growth (doubling time was 24 h)_was achieved at 33°C and at pH 6.3 with pyruvate and NH4+ as the sole C and N sources, respectively. Removal of NH4+ from the culture medium resulted in vesicle differentiation which was paralleled by induction of acetylene reduction activity. Growth on atmospheric N2 was optimal with combined pyruvate and glucose as the carbon sources and displayed a doubling time of about 48 h. Comparisons in growth and N2-fixing activity ofFrankia strains grown in a variety of cultural conditions demonstrate the range of behavior among the strains.

Mutants of Anabaena sp. PCC 7120 with O2-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O2 uptake as a function of dissolved pO2 showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant. Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction. Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO2 (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O2 (vesicles absent) or 20 kPa O2 (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO2 at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO2, but the Q10 was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O2-saturated at low pO2 values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (ArI3) was studied in vivo. Acetylene reduction (nitrogenase activity) was correlated with vesicle differentiation following removal of NH4+. Vesicles were broken off from the point of attachment to vegetative filaments by brief sonication. Loss of acetylene reduction was correlated with the increase in detached vesicles. Respiratory protection was suggested by the observation that O2 consumption rates in actively-fixing vesicle-containing cells were 5 times greater than rates in ammonia-grown undifferentiated cells. Furthermore, carbon limitation resulted in a decrease in the optimum O2 tension for nitrogenase activity. In actively-fixing (10 nMol C2H4/min/mg protein), aerobically induced cells, maximum acetylene reduction occurred near 20k PaO2. Acetylene reduction and O2 uptake decreased more than 10-fold in cells that were washed and incubated aerobically without a carbon source. Residual acetylene reduction activity was optimal at 5k PaO2 and nearly eliminated at 20 k PaO2. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo enzyme rates. O2 uptake rates of NH4+-grown cells showed an apparent KmO2 of approximately 1.0 um O2. In N2-fixing cultures, a KmO2 of approximately 160 um O2 was observed. Thus, respiration remains unsaturated by O2 at air saturation levels. In vivo the apparent Km for acetylene was 10-fold greater than reported in vitro values. These data show diffusion-limited kinetics and are interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of ArI3.

The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of [Formula: see text] from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa)) in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 μM O2. In contrast, respiration in [Formula: see text]-grown cells was saturated by O2 between 8 and 10 μM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in [Formula: see text]-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.

Hydrogenase activity in the thermophilic cyanobacterium, Mastigocladus laminosus was studied both in vivo and in vivo hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H 2 -supported acetylene reduction in reductant-limited cultures was a light-dependent, but O 2 -independent reaction. In vitro hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for Anabaena cylindrica .

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O2 uptake and acetylene reduction rates in response to substrate concentration. O2 of NH 4 + -grown cells showed an apparent K m O2 of approximately 1μM O2. In N2-fixing cultures a second K m O2 of about 215 μM O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent K m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.

Kinetics of growth and nitrogenase induction in Frankia sp. ArI3 were studied in batch culture. Growth on defined medium with NH 4 + as the N source displayed typical batch culture kinetics; however, a short stationary phase was followed by autolysis. Removal of NH 4 + arrested growth and initiated vesicle differentiation. Vesicle numbers increased linearly and were paralleled by a rise in nitrogenase (acetylene reduction) activity. Nitrogenase activity (10 mM C2H4 · mg protein−1 · min−1) was sufficient to support growth on N2 and protein levels rose in parallel with nitrogenase induction. Optimal conditions for vesicle and nitrogenase induction were investigated. Maximum rates of acetylene reduction were obtained with 5 to 10 mM K2 HPO4/KH2PO4, 0.1 mM CaCl2 and MgSO4. The optimum pH for acetylene reduction and respiration was around 6.7. The amount (5 to 10µg protein/ml) and stage (exponential) of growth of the ammonium-grown inoculum strongly influenced the subsequent development of nitrogenase activity. Propionate was the most effective carbon source tested for nitrogenase induction. Respiration in propionate-grown cells was stimulated by CO2 and biotin, suggesting that propionate is metabolized via the propionyl CoA pathway.

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in [Formula: see text], undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.Key words: Frankia, hydrogenase, nitrogenase, O2 protection.