Cellulose Decomposition and Associated Nitrogen Fixation by Mixed Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans

Mixed cultures of Cellulomonas gelida plus Azospirillum lipoferum or Azospirillum brasilense and C. gelida plus Bacillus macerans were shown to degrade cellulose and straw and to utilize the energy-yielding products to fix atmospheric nitrogen. This cooperative process was followed over 30 days in sand-based cultures in which the breakdown of 20% of the cellulose and 28 to 30% of the straw resulted in the fixation of 12 to 14.6 mg of N per g of cellulose and 17 to 19 mg of N per g of g straw consumed. Cellulomonas species have certain advantages over aerobic cellulose-degrading fungi in being able to degrade cellulose at oxygen concentrations as low as 1% O₂ (vol/vol) which would allow a close association between cellulose-degrading and microaerobic diazotrophic microorganisms. Cultures inoculated with initially different proportions of A. brasilense and C. gelida all reached a stable ratio of approximately 1 Azospirillum/3 Cellulomonas cells.     

Comparison of Two Cellulomonas Strains and Their Interaction with Azospirillum brasilense in Degradation of Wheat Straw and Associated Nitrogen Fixation

A mutant strain of Cellulomonas sp. CS1-17 was compared with Cellulomonas gelida 2480 as the cellulolytic component of a mixed culture which was responsible for the breakdown of wheat straw to support asymbiotic nitrogen fixation by Azospirillum brasilense Sp7 (ATCC 29145). Cellulomonas sp. strain CSI-17 was more efficient than was C. gelida in cellulose breakdown at lower oxygen concentrations and, in mixed culture with A. brasilense, it supported higher nitrogenase activity (C₂H₂ reduction) and nitrogen fixation with straw as the carbon source. Based on gravimetric determinations of straw breakdown and total N determinations, the efficiency of nitrogen fixation was 72 and 63 mg of N per g of straw utilized for the mixtures containing Cellulomonas sp. and C. gelida, respectively. Both Cellulomonas spp. and Azospirillum spp. exhibited a wide range of pH tolerance. When introduced into sterilized soil, the Cellulomonas sp.-Azospirillum brasilense association was more effective in nitrogen fixation at a pH of 7.0 than at the native soil pH (5.6). This was also true of the indigenous diazotrophic microflora of this soil. The potential implications of this work to the field situation are discussed.     

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp

Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO₂/C₂H₂ ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by ¹⁵N₂ incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.     

Continuous single cell protein production fromCellulomonas sp. andCandida utilis grown in mixture on barley straw

Summary Continuous fermentations with mixed cultures of the cellulolytic bacteriaCellulomonas sp. and the yeastCandida utilis were examined. Fermentations were carried out in an aerated 5-l fermenter with different preparations of wet disintegrated barley straw as the cellulose source (3.6–4.2%). The straw was pretreated with NaOH (3.2–8.5 kg NaOH/100 kg dry straw) under high pressure and temperature in a feedstuff pellet press. The quantity of dry cell mass produced and the breakdown of the straw were measured. Crude protein and ash content in cell dry matter and residual fiber were determined. The experiments showed thatCellulomonas sp. andCandida utilis may be grown together in a continuous culture (dilution rate D=0.12–0.14 h^–1) for at least 3 days without washing out one of the organisms. Highest productivity was 1.39 g cell dry matter/l/h when using straw pretreated with 5.7% NaOH. The dry cell product contained 58–66% crude protein and up to 51% of the organic fiber dry matter was solubilized. The yield constants were 0.32–0.61 g cell dry matter per g solubilized organic fibers.     

N2 Fixation by Azospirillum brasilense and Its Incorporation into Host Setaria italica

Growth and nitrogen fixation were followed during the life cycle of Setaria italica (foxtail millet) inoculated with Azospirillum brasilense in controlled-environment growth chambers. The plants were fertilized at seeding with a limiting amount of combined nitrogen and maintained with an N-free mineral solution. During maturation of the plants, substantial nitrogenase activity, measured by acetylene reduction, developed in the rhizosphere, with total fixation estimated to be equivalent to 20% of the N in the inoculated plants. The peak of this activity coincided with depletion of soluble nitrogen from the system, which in turn was reflected by a sharp decrease in the nitrate reductase activity of the leaves. A. brasilense was found in association with the root populations at 8 × 10⁷ cells per gram of dry weight. An increase in shoot growth occurred at this time, but no significant increase in total plant nitrogen could be demonstrated. ¹⁵N₂ enrichment experiments confirmed that fixation was occurring, but only about 5% of the nitrogen fixed by A. brasilense was incorporated into the plants within 3 weeks. There was thus no evidence of direct bacterium-to-plant transport of fixed nitrogen, but rather a slow transfer suggesting the gradual death of bacteria and subsequent mineralization of their nitrogen, at least under growth-room conditions.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

Characterization of Azospirillum Isolated from Nitrogen-Fixing Roots of Harvested Sorghum Plants

Root segments of harvested sorghum plants had acetylene reduction activity ranging from 11 to 61 nmol of ethylene formed per h per g (dry weight). Five strains of Azospirillum brasilense sp. nov. were isolated from root segments.     

Microbial Fermentation of Rice Straw: Nutritive Composition and In Vitro Digestibility of the Fermentation Products

Rice straw was fermented with Cellulomonas sp. and Alcaligenes faecalis. Microbial cells and undigested residue, as well as chemically treated (NaOH or NH₄OH) and untreated straws, were analyzed for nutrient composition and in vitro digestibility. In a typical fermentation, 75% of the rice straw substrate was digested, and 18.6% of the total substrate weight that disappeared was recovered as microbial protein. The microbial cell fraction was 37% protein and 5% crude fiber; the residue was 12% protein and 45% crude fiber. The microbial protein amino acid profile was similar to alfalfa, except for less cysteine. The microbial cells had more thiamine and less niacin than Torula yeast. In vitro digestibility of the microbial protein was 41.2 to 55%; that of cellulose was 52%.     

Responses of Sorghum and Pennisetum Species to the N2-Fixing Bacterium Azospirillum brasilense

Three field inoculation experiments, two in Florida and one in New Mexico, were conducted with Azospirillum brasilense Cd. Each of the Florida experiments evaluated two crop species. One species in each of the Florida experiments responded to inoculation with a significant dry matter yield increases of 11 to 24% and nitrogen yield increases of 9 to 39%. No inoculation response was noted in the New Mexico experiment. The responding species were Sorghum bicolor (L.) Moench (sorghum) and the interspecific hybrid between Pennisetum americanum (L.) K. Schum. (pearl millet) and P. purpureum Schumach. (napiergrass). Nonresponding species were pearl millet (Florida) and Sorghum sudanense (Piper) Staph. (New Mexico). Acetylene reduction activity of inoculated plots in Florida was low, showing no increase over the natural uninoculated background rates and, in one case, was negatively correlated with yield. Acetylene reduction activity was not measured in New Mexico. In Florida, A. brasilense populations were found to decline from 5 × 10³ to 5 × 10² bacteria g of soil⁻¹ in about 3 weeks (quadratic regressions). Continued decline to less than 10² by week 5 indicated that the inoculated bacteria did not become established in the soil in high numbers. The A. brasilense population declined at about the same rate in the New Mexico experiment. The erractic inoculation responses in these experiments are similar to those observed in earlier work at the University of Florida. The lack of acetylene reduction activity response to inoculation and the rapid population decline of the inoculated bacteria suggest that N₂ fixation is not the major mechanism causing yield responses after inoculation.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.     

Effects of Temperature, Nitrogen Fertilization, and Plant Age on Nitrogen Fixation by Setaria italica Inoculated with Azospirillum brasilense (strain cd)

The association between the nitrogen-fixing bacterium Azospirillum brasilense (strain cd) and the grass Setaria italica was studied under different environmental and soil conditions. Highest acetylene reduction rates in intact plants were observed at the booting stage of Setaria (2350 nmol ethylene produced hour⁻¹ plant⁻¹) at 27 C. Higher temperatures, up to 32 C, enhanced ethylene reduction. Significant increases in shoot dry weight, panicle weight, and length were obtained in inoculated plants fertilized with suboptimal NH₄NO₃ levels. The increase in nitrogen content of plants inoculated with A. brasilense was shown to be due to N₂ fixation. This was demonstrated by growing plants in washed quartz sand with no combined nitrogen. The bacteria also increased branching and development of roots. It was concluded that inoculation of Setaria with A. brasilense may lead both to increases in plant yield and saving of nitrogen fertilizer.     

Effect of oxygen concentration on dinitrogen fixation and volatile fatty acid production by Clostridium butyricum growing in association with fungi on cellulose and on wheat straw

Clostridium butyricum was grown with cellulolytic fungi on the substrates wheat straw and cellulose at a range of oxygen concentrations. The straw was not sterile and was inoculated with Sodaria alcina while the cellulose was sterile and was inoculated with Trichoderma harzianum as examples of cellulolytic organisms. The straw subsequently developed a mixed fungal population. Dinitrogen fixation was only significant at a reduced oxygen concentration: optima being 9.3 and 2.3% O₂ for the straw and cellulose experiments, respectively. The efficiencies of N, fixation were 6 mg N per g straw decomposed and 2.3 mg N per g cellulose decomposed and N, fixation occurred only in the presence of significant cellulolysis. Both acetate and butyrate formation increased as the oxygen concentration decreased. With cellulose as a substrate, their formation correlated with a decrease in pH and an increase in final numbers of Cl. butyricum . The thickness of the anaerobic zone at the aerobic/anaerobic interface was linearly related to the square root of the oxygen concentration at the surface of the interface.     

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N₂-grown cultures than in NH₄⁺-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h⁻¹ mg of protein⁻¹), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C₂H₄ formed h⁻¹ mg of protein⁻¹ when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C₂H₂ reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.     

Cellulose Fermentation: Effect of Substrate Pretreatment on Microbial Growth

The effects of chemical, physical, and enzymatic treatments of rice straw and sugarcane bagasse on the microbial digestibility of cellulose have been investigated. Treatment with 4% NaOH for 15 min at 100 C increased the digestibility of cellulose from 29.4 to 73%. Treatment with 5.2% NH₃ could increase digestibility to 57.0% Treatments with sulfuric acid and crude cellulase preparation solubilized cellulose but did not increase the digestibility. Grinding or high-pressure cooking of the substrate had little effect on increasing the digestibility of cellulosic substrates by the Cellulomonas species.     

Bradyrhizobium japonicum Survival in and Soybean Inoculation with Fluid Gels

The utilization of gels, which are used for fluid drilling of seeds, as carriers of Bradyrhizobium japonicum for soybean (Glycine max (L.) Merr.) inoculation was studied. Gels of various chemical composition (magnesium silicate, potassium acrylate-acrylamide, grafted starch, and hydroxyethyl cellulose) were used, although the hydroxyethyl cellulose gels were more extensively investigated. Gel inocula were prepared by mixing gel powder with liquid cultures of B. japonicum (2% [wt/vol]). The population of B. japonicum USDA 110 did not change in each gel type during 8 days of incubation at 28°C. These fluid gels were prepared with late-exponential-growth-phase cells that were washed and suspended in physiological saline. Mid-exponential-growth-phase B. japonicum USDA 110, 123, and 138 grew in cellulose gels prepared with yeast extract-mannitol broth as well as or better than in yeast extract-mannitol broth alone for the first 10 days at 28°C. Populations in these cellulose gels after 35 days were as large as when the gels had originally been prepared, and survival occurred for at least 70 days. Soybeans grown in sand in the greenhouse had greater nodule numbers, nodule weights, and top weights with gel inoculants compared with a peat inoculant. In soil containing 10³ indigenous B. japonicum per g of soil, inoculation resulted in increased soybean nodule numbers, nodule weights, and top weights, but only nodule numbers were greater with gel than with peat inoculation. The gel-treated seeds carried 10² to 10³ more bacteria per seed (10⁷ to 10⁸) than did the peat-treated seeds.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Enumeration and Relative Importance of Acetylene-Reducing (Nitrogen-Fixing) Bacteria in a Delaware Salt Marsh

Three groups of N₂-fixing bacteria were enumerated from the top 1 cm of the surface in four vegetational areas in a Delaware salt marsh. The results over the 9-month sampling period showed that there were no discernible seasonal patterns for any of the groups enumerated (Azotobacter sp., Clostridium sp., and Desulfovibrio sp.). Azotobacter sp. was present in numbers of 10⁷ per g of dry mud, whereas the two anaerobic fixers were present in much lower numbers (10³ to 10⁴ per g of dry mud). There were no differences in the numbers of each group among the different vegetational areas, indicating that there was a heterogeneous population of N₂ fixers present. Additional studies indicate that the activity of sulfate reducers (Desulfovibrio sp.) may account for as much as 50% of the total observed acetylene reduction activity. Oxygen was found to exert little effect on the observed acetylene reduction activity, indicating that stable aerobic and anaerobic microenvironments exist in the surface layer of marsh sediments.     

NH4+-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH₄⁺. Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH₄⁺. The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O₂) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH₄Cl. Both mutants failed to incorporate [¹⁴C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10⁷ cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a ¹⁵N₂-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of ¹⁵N inside root and shoot material than the wild type did. The results of our nitrogen balance and ¹⁵N enrichment studies indicated that NH₄⁺-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Microcalorimetric study of aerobic growth of Cellulomonas sp. 21399 on various carbohydrates

Summary Microcalorimetry was used to study the energetic aerobic growth of Cellulomonas sp. 21399 on glucose, cellobiose and amorphous and crystalline cellulose. The thermochemical aspect of growth on glucose was established with regard to the anabolic contribution. The results obtained allowed the use of glucose as a reference substrate for cellulose degradation. The experimental enthalpy change and the maximum catabolic activity, calculated from the maximum power evolved by the culture, were, respectively,-1079 kJ/mol and 0.85 mmol glucose per hour per dry weight of cells. The growth response on amorphous cellulose was equivalent to that demonstrated on glucose. However, on crystalline cellulose media, Cellulomonas sp. 21399 exhibited eight times less power and the quantity of heat evolved during growth showed that 50% of the cellulose was degraded. Quantitative results and the shape of power-time curves achieved indicate that the structural features of cellulose strongly influence its microbial degradability.     

Localized Tetrazolium Reduction in Relation to N2 Fixation, CO2 Fixation, and H2 Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N₂ fixation (acetylene reduction), ¹⁴CO₂ fixation, and ³H₂ utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, ¹⁴CO₂ fixation and ³H₂ utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N₂-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.