Nitrogen fixation in submerged soilsand-energy material media and the aerobic-anaerobic interface

Summary Flooded soils usually consist of a surface aerobic phase a few millimeters thick (in contact with the atmosphere or oxygenated solution) underlain by an anaerobic phase. The objective of this research was to study nitrogen fixation in the aerobic-anaerobic interfacial area in flooded, cellulose enriched media and the utilization of the products of anaerobic decomposition of cellulose by nitrogen-fixing organisms when these products are brought under aerobic conditions by processes such as diffusion, mixing, and drying. The medium used for these studies was basically a sand matrix supplemented with a small amount of soil and mineral nutrients. When columns of medium enriched with cellulose were sectioned after incubation in the dark under flooded conditions, the increase in content of nitrogen per gram medium in the top 2 to 3 mm of the column was as much as 10 to 15 times the increase in nitrogen content of the lower portions of the column. Periodic mixing of flooded media, alternation of shaking in nitrogen and air atmospheres, and alternate flooding and drying all enhanced fixation relative to undisturbed, continuously flooded media incubated in air. Fixation during incubation under a nitrogen atmosphere was less than fixation under air atmospheres. The results of numerous experiments are consistent with the hypothesis that nitrogen fixation is enhanced when the products of anaerobic decomposition of cellulose are brought under aerobic conditions by any of several processes. Portions of a Ph.D. thesis submitted by the senior author. For more details refer to: Magdoff, F. R. Nitrogen fixation in submerged soil-sand-energy material media and the aerobic-anaerobic interface. Unpublished Ph.D. thesis. Cornell University Library. Ithaca, New York, 1969. Agronomy Department Paper No. 868.     

Nitrogen-fixing Enterobacter agglomerans isolated from guts of wood-eating termites.

Two strains of facultatively anaerobic, N2-fixing bacteria were isolated from guts of Coptotermes formosanus and identified as Enterobacter agglomerans. The deoxyribonucleic acid base composition of isolates was 52.6 and 53.1 mol% guanine plus cytosine. Both isolates and a known strain of E. agglomerans carried out a mixed acid type of glucose fermentation. N2 fixation by E. agglomerans was inhibited by O2; consequently, N2 served as an N source only for cells growing anaerobically in media lacking a major source of combined N. However, peptone, NH4Cl, or KNO3 served as an N source under either aerobic or anaerobic conditions. It was estimated that 2 x 10(2) cells of E. agglomerans were present per termite gut. This value was 100-fold lower than expected, based on N2 fixation, low recoveries of E. agglomerans may be related to the marked decrease in N2 fixation rates observed when intact termites or their extracted guts were manipulated for the isolation of bacteria. It was concluded that the N2-fixing activity of E. agglomerans may be important to the N economy of C. formosanus.     

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.     

Acetylene reduction (nitrogen fixation) by enterobacteriaceae isolated from paper mill process waters

Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C(2)H(4) per minute per milligram of protein) and the activities (nanomoles of C(2)H(4) per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.     

A Note on the Microflora of Beef Muscle Stored in Nitrogen at 0°

Fresh beef stored in nitrogen at 0° was spoiled by Gram positive rods. Almost all the organisms isolated were Microbacterium spp., although in one experiment about 10% of the organisms isolated were Lactobacillus spp. The numbers and types of organisms isolated were similar on each of two media incubated under aerobic and anaerobic conditions.     

Presence of nitrogen fixing Klebsiella pneumoniae in the gut of the Formosan subterranean termite (Coptotermes formosanus)

A gram-negative facultative anaerobic enteric bacterium, Klebsiella pneumoniae was isolated from the hindgut of the Formosan subterranean termite (FST). It was characterized using, fatty acid methyl ester (FAME) analysis, BIOLOG assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and biochemical studies. The role of this isolate seems to be nitrogen fixation because the termite's diet is nitrogen deficient and the isolate produced significant amounts of ammonia when it was grown on nitrogen deficient medium under anaerobic condition with nitrogen gas in the headspace.     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.     

Factors influencing the production of hydrogen by the purple non‐sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen_. L‐tyrosine was the least preferred nitrogen source by both growing and resting cells.     

Occurrence of nitrogen fixation among Vibrio spp.

Virtually all Vibrio spp. known and available in culture collections and several newly isolated Vibrio sp. were tested for their ability to fix molecular nitrogen, using the acetylene reduction technique, the fixation of the heavy isotope ¹⁵N, and by growth on media devoid of combined nitrogen. Among the 27 species tested, four, including V. diazotrophicus, proved to be nitrogenase-positive. The potential of nitrogen fixation was now also discovered in V. natriegens, V. pelagius and V. cincinnatiensis. Among the 9 newly isolated strains, 4 were nitrogenase-positive. These strains were classified as V. diazotrophicus on the basis of DNA homology studies. Nitrogenase was only induced during growth under anaerobic conditions. Dissolved oxygen as low as 1 M inhibited nitrogenase completely. This inhibition at low oxygen concentration, however, was reversible. 50–100 M dissolved oxygen inhibited nitrogenase irreversibly.This work was carried out at Geomicrobiology Division, the University of Oldenburg, FRG     

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA: EVIDENCE FOR A CIRCADIAN RHYTHM1

Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert-Bebout and colleagues and maintained in seawater-based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater-based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.     

Perspectives in biological nitrogen fixation research

Nitrogen fixation, along with photosynthesis is the basis of all life on earth. Current understanding suggests that no plant fixes its own nitrogen. Some plants (mainly legumes) fix nitrogen via symbiotic anaerobic microorganisms (mainly rhizobia). The nature of biological nitrogen fixation is that the dinitrogenase catalyzes the reaction-splitting triple-bond inert atmospheric nitrogen (N2) into organic ammonia molecule (NH3). All known nitrogenases are found to be prokaryotic,multi.complex and normally oxygen liable. Not surprisingly, the engineering of autonomous nitrogen-fixing plants would be a long-term effort because it requires the assembly of a complex enzyme and provision of anaerobic conditions. However,in the light of evolving protein catalysts, the anaerobic enzyme has almost certainly been replaced in many reactions by the more efficient and irreversible aerobic version that uses O2. On the other hand, nature has shown numerous examples of evolutionary convergence where an enzyme catalyzing a highly specific, O2-requiring reaction has an oxygen-independent counterpart, able to carry out the same reaction under anoxic conditions. In this review, I attempt to take the reader on a simplified journey from conventional nitrogenase complex to a possible simplified version of a yet to be discovered Ilght-utilizing nitrogenase.     

Inhibition of cell division blocks the synthesis of the second nitrogenase (Nif2) in the cyanobacterium Anabaena variabilis

Anabaena variabilis ATCC 29413 belongs to the cyanobacteria that use a specific cell type, heterocysts, for fixation of atmospheric nitrogen under aerobic conditions. Nitrogen fixation under anaerobic conditions is catalyzed by a Mo-dependent nitrogenase (Nif2) that is expressed in the vegetative cells. We demonstrate here using immunolocalization/light microscopy (LM) that the synthesis of NifH2 is mainly initiated in dividing vegetative cells along the trichomes. Blocking cell division by cephalexin abolished nitrogenase synthesis under anaerobic conditions.     

Hormone independent activation of rat uterine estrogen receptor by exposure of isolated uteri to anaerobic conditions

Incubation of isolated rat uteri under anaerobic conditions, which consisted of either an atmosphere of carbon monoxide or nitrogen, caused an increase in nuclear estrogen binding which was not dependent on added estrogen. The incubation of uteri in the absence of added estrogen under aerobic conditions (atmosphere of oxygen or oxygen-carbon dioxide [95-5%]) did not increase uterine nuclear estrogen binding levels. High salt (0.5-M KCl) extracts of the nuclear estrogen binding moiety induced by anaerobiosis were shown to possess a sedimentation coefficient on sucrose-glycerol gradients of 4.8S, a binding specificity restricted to estrogens and an apparent affinity constant of 1.35 nM. These data confirm that the nuclear binding moiety induced by anaerobiosis possesses the characteristics of an estrogen receptor. The enhanced nuclear estrogen receptor retention induced under anaerobic conditions could be accounted for by a significant increase in nuclear receptor extracted by high salt (0.5 M KCl) and by ethanol (salt resistant fraction). Furthermore, sequential extraction of nuclear estrogen receptor from uteri exposed to aerobic conditions in the presence of added estradiol paralleled the results obtained with anaerobiosis. Total receptor retained under anaerobiosis represented 25% of that observed under aerobic conditions in the presence of estrogen. These results indicate that anaerobic conditions can cause an activation of uterine estrogen receptor. This activation process represents a pathway for receptor activation which does not require steroid.     

Effect of growth conditions on the activity of the enzymes of cyclic 3':5'-AMP synthesis and decay in phototrophic bacteria]

Activity, ratio and summary content of cyclic AMP enzymes, adenylate cyclase and phosphodiesterase varied depending on growth conditions of phototrophic bacteria (Rhodospirillum rubrum and Rhodopseudomonas palustris). It suggests, that membrane-bound and soluble enzymes carry different functions. The increase of adenylate cyclase under chaning growth conditions was usually accompanied by the increase of phosphodiesterase. Sharp increase of both enzymes activity was observed when bacteria were growth in aerobic conditions. The activity of both enzymes in chromatophores was 2.8-fold higher when bacteria were grown in the light in anaerobic conditions, than in chromatophores of bacteria grown under stationary aerobic conditions in the light. It is suggested that 3':5' AMP can participate in autotrophic carbon assimilation or in the synthesis of pigments and other components of bacterial photosynthetizing apparatus. Substitution of NH4+ into NO3- and glutamate under the growing of R. rubrum in anaerobic conditions in the light resulted in the increase of the enzymes activities, which is the evidence of possible role of 3':5' AMP in mineral nitrogen uptake and nitrogen fixation. Glutamate concentration of 4 g/l stimulated the enzymes both in vivo and in vitro. The data obtained suggest that 3':5' AMP can carry multiple functions, participating in regulation of a number of metabolic processes in photorophic bacteria.     

Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions

Forty-four differentially expressed proteins have been identified in the photosynthetic diazotroph Rhodospirillum rubrum grown anaerobic and photoheterotrophically, with different nitrogen sources, using 2D-PAGE and MALDI-TOF, from gels containing an average of 679 +/- 52 (in N(+)) and 619 +/- 37 (in N(-)) protein spots for each gel. A higher level of expression was found under nitrogen-rich growth, for proteins involved in carbon metabolism (reductive tricarboxylic acid cycle, CO(2) fixation, and poly-beta-hydroxybutyrate metabolism) and amino acid metabolism. The key enzymes RuBisCO and alpha-ketoglutarate synthase were found to be present in higher amounts in nitrogen-rich conditions. Ntr and Nif regulated proteins, such as glutamine synthetase and nitrogenase, were, as expected, induced under nitrogen-fixing conditions and glutamate dehydrogenase was down regulated. A novel 2Fe-2S ferredoxin with unknown function was identified from nitrogen-fixing cultures. In addition to differential expression, two of the identified proteins revealed variable p I values in response to the nitrogen source used.     

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H₂, CO₂) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N₂ was rather low (0.14 mg O₂/l) compared with cells grown in the presence of NH ₄ ⁺ (4.45 mg O₂/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30°C with N₂ as N-source and sucrose as carbon source and at optimal pO₂. Acetylene reduction reached values of 12 nmoles of ethylene produced/minxmg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO₂-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.     

Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.     

Denitrification with Methanol: a Selective Enrichment for Hyphomicrobium Species

Hyphomicrobium species were enriched in media with methanol as sole carbon source under conditions supporting denitrification. Pure cultures of Hyphomicrobium species were isolated which denitrified vigorously with methanol. Hyphomicrobium B522, isolated by aerobic enrichment, was adapted to anaerobic growth and denitrification. Hyphomicrobium B522 and a new isolate were surveyed for anaerobic growth and denitrification on a number of simple organic compounds. Cell suspensions were tested for denitrifying activity. Nitrogen production from nitrate and nitrite and carbon dioxide production from methanol were stoichiometric.     

A Photobacterium-like bacterium able to fix nitrogen

A Photobacterium-like bacterium isolated from the roots of eelgrass (Zostera marina) was shown to fix nitrogen under anaerobic conditions. Nitrogen fixation by Photobacterium spp. has not been reported previous to this.Abbreviation PHB Poly--hydroxybutyrate