Production of arachidonic acid byMortierella alpina ATCC 32222

Summary WhenMortierella alpina ATCC 32222 was incubated in a glucose salts medium at 25°C the biomass (17.5 g/l) contained 9.62% arachidonic acid which amounted to 54% (w/w) of total biomass lipids. When the glucose concentration in the medium was varied from 0 to 150 g/l, the percentage of arachidonic acid in biomass and in lipids was highest at a glucose concentration of 30 g/l, but highest yield of arachidonic acid per litre of culture broth was observed at a glucose concentration of 100 g/l. While production of biomass reached a plateau of 17 g/l after a 3-day incubation at 25°C, the percentage of arachidonic acid in lipids and biomass increased dramatically from 3 to 6 days with a concurrent arachidonic acid yield increase from 0.89 to 1.63 g/l. Optimum initial culture pH for arachidonic acid production was in the range 6.0–6.7. By increasing the concentration of the glucose salts medium three-fold, yields of biomass and arachidonic acid were increased to 35.8 g/l and 3.73 g/l, respectively.     

Effect of cultural conditions on production of eicosapentaenoic acid byPythium irregulare

Summary The effect of culture conditions upon lipid content and fatty acid composition of mycelia ofPythium irregulare was investigated with particular attention to increasing the yield of 5,8,11,14,17-eicosapentaenoic acid (205; –3) (EPA). All experiments were done by shake flask culture using a yeast extract + malt extract medium. The maximum growth rate was obtained at 25°C, but maximum EPA production was obtained at 12°C. The highest EPA production was 76.5 g EPA/ml 13 days fermentation at 12°C. Addition of glucose during fermentation increased the yield considerably. The highest yield was 112 g/ml, obtained at 13 days fermentation with spiking on day 11. Fermentation time could be shortened by initial incubation at 25°C for 2 days, followed by incubation at 12°C for 6 days. The culture also produced arachidonic acid and other -6 polyunsaturated fatty acids. EPA production was also obtained with lactose or sweet whey permeate, a by-product of cheese manufacture that contains lactose as the main carbohydrate.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH₄C1, -ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 moles/ml and pH 5.8 at 48 h to 10.6 moles/ml and pH 7.2 at 72 h, and to 23.0 moles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.     

Impact of acute temperature transition on enzyme activity levels,oxygen consumption,and exogenous fuel utilization in sea raven (Hemitripterus americanus) hearts

Summary The impact of an acute temperature transition between 5 °C and 15 °C on energy metabolism and performance of sea raven (Hemitripterus americanus) heart was assessed. Maximal in vitro activity of hexokinase was 1.2 and 3.7 mol · min^-1 · g^-1 at 5 °C and 15 °C, respectively. Carnitine palmitoyl transferase and carnitine palmitoleoyl transferase activities were 0.07 mol · min^-1 · g^-1 at 15 °C and declined substantially at 5 °C. Oxygen consumption and power output of perfused isolated hearts offered glucose alone as a metabolic fuel decreased significantly between 15 °C and 5 °C. When palmitoleate was included in the perfusion medium, oxygen consumption and power development remained constant between 15 °C and 5 °C, suggesting that glucose alone was not an adequate metabolic fuel at low temperature. However, maximal in vitro activity of HK implied that the catalytic potential at this locus was quite capable of meeting demands of carbon flow, while the maximal in vitro activity of the carnitine acyl CoA transferases implied that fatty acid metabolism should be greatly compromised at low temperatures. In an effort to resolve the contradiction, hearts were perfused with medium containing ¹⁴C-glucose or ¹⁴C-palmitate. Rates of ¹⁴CO₂ production from labelled metabolic fuels could account for only about 2% of the oxygen consumption rates. Most of the label from ¹⁴C-glucose was incorporated into the glycogen and lipid fractions and label from ¹⁴C-palmitate was incorporated into the lipid fraction. The net incorporation rates of label into intracellular pools were temperature insensitive over the range 5–15 °C. The incorporation of exogenous glucose into the lipid fraction suggests that activity of the entire glycolytic pathway was maintained over the temperature range. Thus, the relatively low rate of oxygen consumption of hearts perfused with glucose alone as an exogenous substrate cannot be attributed to a limitation of glucose catabolism. The alternative explanation is that the presence of fatty acids induces an increase in oxygen consumption, especially at 5 °C. It is speculated that this is due to alterations in Ca²⁺ balance.Abbreviations ATPase adenosine triphosphatase - BSA bovine serum albumin - CoA coenzyme A - C palmitoyl T carnitine palmitoyl transferase - CS citrate synthase - HK hexokinase - MO oxygen consumption - PFK phosphofructokinase - PO ₂ oxygen partial pressure     

Micropropagation of garlic through in vitro bulblet formation

We developed a novel micropropagation method for garlic (Allium sativum L.) by the combination of initial shoot-tip culture, shoot multiplication and in vitro bulblet formation. Garlic shoot-tips were cultured on LS medium containing 1 M indole-3-acetic acid (IAA) and 1 M 6-benzyladenine (BA) to regenerate proliferative shoots. These shoot-tips produced multiple shoots when transferred to modified LS medium containing 5 M 1-naphthaleneacetic acid (NAA) and 10 M BA, and cultured at 20°C under 12-h light conditions. Higher ratios of KNO₃/NH₄Cl in the media promoted multiple shoot formation, together with suppressing vitrification of these shoots. The proliferated shoots of early maturing cultivars produced bulblets by culture on LS growth regulator-free medium at 25°C under 16-h light. On the other hand, the late maturing cultivar, Howaito-roppen, formed bulblets after a low temperature treatment of the proliferated shoots for 6 months followed by culture on LS medium containing 6 to 12% sucrose for two months. The dormancy of the bulblets of cv. Howaito-roppen was broken by successive treatments at a high (35°C), a middle (20°C), and then a low (5°C) temperature.Abbreviations IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - LS Linsmaier and Skoog macro- and microelements     

L-malic acid production by an albino strain of Monascus araneosus

An albino mutant was isolated after treating Monascus araneosus AHU9087 with N-methyl-N-nitro-N-nitrosoguanidine. All other physiological and biochemical characteristics were retained. The mutant did not produce any pigment but produced L-malic acid at 28 g/l, compared with 20 g/l by the parent strain, in media containing 10% (w/v) glucose after incubation under aerobic conditions for 5 days at 37°C.S. Lumyong is with the Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50002, Thailand. F. Tomita is with the Laboratory of Applied Microbiology, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan.     

Growth characteristics of an obligately psychrophilic Vibrio sp.

The growth characteristics of an obligately psychrophilic Vibrio sp. have been studied in a chemostat with glucose or lactose as the limiting substrate over a temperature range 0–23°C. Vibrio AF-1 has an optimum growth temperature of 15°C and maximum growth temperature which is dependent upon the carbon source. On glucose growth ceases at 20°C whereas on lactose growth continues to 23°C. Growth rate is also a function of the carbon source provided. When grown on glucose, fructose, sucrose, maltose and galactose _max values of 0.046 h^-1 at 15°C were recorded whereas on lactose, mannose, ribose and xylose _max values of 0.020 h^-1 were obtained. Substrate affinities (K _s ) for the 9 sugars also fall into 2 divisions as for _max and are temperature dependent. Those sugars which support a high growth rate have highest K _s values at 0°C whereas these which give a low growth rate show maximum affinities at 15°C. Vibrio AF-1 produces the maximum cell yield (0.6 g/g sugar consumed) at temperature <8°C irrespective of the carbon source utilised and correlated with maximum rates of sugar uptake and minimum O₂ consumption. Maintenance energy determination on glucose grown cells show that at 2° C 2% of the carbon input is used for maintenance whereas at 20°C the requirement increases to 10% of the carbon input.     

Thermoanaerobium brockii gen. nov. and sp. nov., a new chemoorganotrophic,caldoactive, anaerobic bacterium

The isolation of a new anaerobic thermophilic bacterium, Thermoanaerobium brockii, from volcanic features is described. Successful enrichment required a complex medium containing glucose or other fermentable sugars and incubation temperatures of 55–80° C. Strains of T. brockii were gram positive, rods of uneven length that existed singly, in pairs, chains or filaments. Electron micrographs of thin sections of cell revealed a monolayered cell wall and a constrictive or pinching off cell division process. The organism was nonsporeforming, obligately anaerobic and chemoorganotrophic. The optimal temperature for growth was 65–70° C, the maxium was below 85° C and the minimum above 35° C. The doubling time at the optimal temperature for growth was about 1 h. The DNA base composition of three strains of T. brockii varied from 30.0–31.4 mol % guanosine plus cytosine. Fermentable carbohydrates included glucose, sucrose, maltose, lactose, cellobiose and insoluble starch. The fermentation products of cells grown on glucose were ethanol, lactic acid, acetic acid, hydrogen and carbon dioxide. Growth of all strains tested was inhibited by fairly low concentrations of cycloserine, penicillin, streptomycin, tetracycline and chloramphenicol. The possible ecological, evolutionary, and industrial significance, and taxonomic relationships of Thermoanaerobium are discussed.Abbreviations TYEG complex medium containing mineral salts, 0.3% yeast extract, 1.0% tryptone and 0.5% glucose - O.D. optical density - G+C guanosine plus cytosine     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.     

H<Subscript>2</Subscript>-Producing bacterial communities from a heat-treated soil inoculum

Hydrogen gas (60% H₂) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l^–1). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H₂ by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30°C and 37°C). At 30-h HRT, the H₂ production rate was 80 ml h^–1 and yield was 0.91 mol H₂/mol glucose. At 10-h HRT, the H₂ production rate was more than 5 times higher at 436 ml h^–1, and yield was 1.61 mol H₂/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30°C to 37°C, the steady-state H₂ production rate increased slightly to 463 ml h^–1 and yield was 1.8 mol H₂/mol glucose. Compared to 30°C, RISA fingerprints at 37°C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).     

Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method

Factors influencing in vitro growth rates of soybean embryos were investigated using embryos isolated in the cotyledon stage. The influence of these factors on final plant recovery from the embryos cultured was tested. Sucrose and glucose could serve as carbon sources with final plant yields being higher with sucrose than with glucose. A culture medium containing only KNO₃ (25 mM) as the nitrogen source supported embryo growth. Adding glutamine (10 mM) to the medium containing KNO₃ increased final plant recovery to 25%. Of several vitamin supplements tested a combination of pyridoxine-HCl, nicotinic acid and pantothenic acid (0.5; 1.0; 0.5 mg l^-1) provided the best growth and plant yield. Of the plant growth regulators tested IAA, BAP and GA₃ stimulated embryo growth and plant development when added to the medium at a low concentration (0.1 M). The optimal temperature for in vitro growth of cotyledon stage embryos was 27°C. Temperatures above 30°C caused growth retardation and reduced plant yield. A protocol for culturing soybean cotyledon stage embryos under conditions ensuring high plant recovery is proposed.Abbreviations IBA indole-3-butyric acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Production of arachidonic acid by Mortierella fungi

Summary Various Mortierella fungi were assayed for their productivity of arachidonic acid (ARA). Only strains belonging to the subgenus Mortierella accumulated detectable amounts of ARA together with dihomo--linolenic acid. None of the strains belonging to the subgenus Micromucor tested accumulated these C-20 fatty acids, although they produced a C-18 fatty acid, -linolenic enic acid. A soil isolate, M. alpina 1S-4, was found to grow well in a liquid medium containing glucose and yeast extract as carbon and nitrogen sources, respectively. Addition of several natural oils such as olive and soybean oils to the medium increased the accumulation of ARA. Under optimal culture conditions in a 5-1 bench-scale fermentor, the fungus produced 3.6 g/l of ARA in 7 days. On cultivation for 10 days at 28°C in a 2000-1 fermentor, the same fungus produced 22.5 kg/kl mycelia (dry weight) containing 9.9 kg lipids, in which ARA comprised 31.0% of the total fatty acids. On standing the harvested mycelia for a further 6 days, major mycelial fatty acids (i.e. palmitic acid, oleic acid, linoleic acid, etc.) other than ARA rapidly decomposed and the ARA content of the total fatty acids reached nearly 70%.     

β-Xylanase produced by Aureobasidium pullulans CBS 58475

Summary Aureobasidium pullulans CBS 58475 produced -xylanase with an activity of 5 units/ml culture filtrate. Xylose, xylan and complex substrates containing xylose served as strong inducers. Purification of the enzyme was achieved by two sets of gel permeation chromatography. The enzyme has an pH optimum at 4.25 and a temperature optimum at 60°C. At slightly acid pH and temperatures up to 60°C -xylanase showed good stability. The analysis of cleavage products classified the -xylanase as an endoenzyme. Together with an endopolygalacturonase, the -xylanase enhanced the maceration of carrots compared to endopolygalacturonase alone.     

Cellulolytic and physiological properties of Clostridium thermocellum

Three strains of Clostridium thermocellum obtained from various sources were found to have nearly identical deoxyribonucleic acid guanosine plus cytosine contents that ranged from 38.1–39.5 mole-%. All strain examined fermented only cellulose and cellulose derivatives, but not glucose, or xylose or other sugars. The principal cellulose fermentation products were ethanol, lactate, acetate, hydrogen and carbon dioxide. Growth of C. thermocellum on cellulose resulted in the production of extracellular cellulase that was non-oxygen labile, was thermally stable at 70° C for 45 min and adsorbed strongly on cellulose. Production of cellulase during fermentation correlated linearly with growth and cellulose degradation. Both the yield and specific activity of crude cellulase varied considerably with the specific growth substrates. Highest cellulase yield was obtained when grown on native cellulose, -cellulose and low degree of polymerization cellulose but not carboxymethylcellulose or other carbohydrate sources. Cellulase activity was not detected when cells were grown on cellobiose. Crude extracellular protein preparations lacked proteolytic and cellobiase activity. The pH and temperafure optima for endoglucanase activity were 5.2 and 65° C, respectively, while that of the exoglucanase activity were 5.4 and 64° C, respectively. The specific activity at 60° c for exoglucanase and endoglucanase of crude cellulase obtained from cells grown on cellulose (MN 300) was 3.6 moles reducing sugar equivalents released per h (unit)/mg of protein and 1.5 mole reducing sugar equivalent released per min (unit)/mg of protein, respectively. The yield of endoglucanase was 125 units per g of cellulose MN 300 degraded and that of exoglucanase was 300 units per g of cellulose MN 300 degraded. Glucose and cellobiose were the hydrolytic end products of crude cellulase action on cellulose, cellotraose and cellotriose in vitro.     

Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.     

Production of 2,3-butanediol from glucose byBacillus licheniformis

Bacillus licheniformis produced 2,3-butanediol from glucose with an optimum yield of 47 g/100 g glucose after 72 h of growth on a peptone/beef extract medium containing 2% (w/v) glucose at pH 6.0 and 37°C. This yield of 2,3-butanediol was higher than those previously reported forKlebsiella oxytoca (37 g/100 g glucose) andBacillus polymyxa (24 g/100 glucose).     

Effect of growth temperature on the morphology and performance ofZymomonas mobilis ATCC 29 191 in batch and continuous culture

Summary Increasing the temperature in chemostat culture ofZymomonas mobilis ATCC 29 191 with low and high glucose concentrations was found to result in a decreasing frequency of septation leading to the formation of long filaments and in increasing outer membrane blebbing. Whether this effect is strain specific or universal inZymomonas is, unknown. Improvements in the fermentation kinetics could be achieved at elevated temperatures, with an optimum at 33°C. Temperatures >30°C induced uncoupled growth in chemostat cultures ofZ. mobilis ATCC 29 191. The results of this study emphasize the importance of temperature regulation in optimizing the performance of continuous fermentations withZymomonas.Nomenclature D Dilution rate, 1/h - _max Maximum specific growth rate, 1/h - S _R Initial substrate concentration, g glucose/1 - S Amount of glucose consumed, g glucose/1 - S ₀ Effluent substrate concentration, g glucose/1 - X Biomass concentration - g cells/1 - [P] Amount of product formed, g ethanol/1 - [P] Product concentrations, g ethanol/l - Y _x/s Growth yield, g cells/g glucose used - Y _p/s Product yield, g ethanol/g glucose used - O _s Specific rate of glucose uptake, g glucose/g cells/h - Q _p Specific rate of ethanol formation, g ethanol/g cells/h - VP Volumetric productivity, g ethanol/1/h - t Fermentation time, h Corresponding author     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

L-Arginine inhibits in vitro nonenzymatic glycation and advanced glycosylated end product formation of human serum albumin

Summary L-Arginine (Arg) has a structure similar to that of aminoguanidine (AG) and may inhibit glycation and advanced glycosylated end product (AGE) formation. Human serum albumin (HSA) (100mg/ml) was incubated for 2 weeks with glucose (200mM) at 37°C or with glucose and equimolar concentrations of Arg, N--acetyl Arg, or AG with or without 25mM diethylenetriaminepentaacetic acid (DTPA). In the absence of DTPA, electrospray ionization mass spectrometry showed a 70% reduction of covalently bound glucose in the presence of Arg and a 30% reduction with AG. Digestibility by trypsin of HSA incubated with glucose and Arg was similar to that of HSA incubated alone. This suggests less covalent modification of HSA in the presence of Arg as compared with the absence of Arg. When incubations contained DTPA, autoradiography showed less¹⁴C labeling of HSA subunits in the presence of Arg and AG. When the-amino group of Arg was blocked with an acetyl group, labeling was similar to that of HSA incubated with glucose, suggesting involvement of the-amino group in the inhibition. Fluorescence of HSA at ex₃₇₀ and em₄₄₀ was reduced with Arg, but AG was more effective than Arg. These results suggest that Arg, like AG, can inhibit glycation and AGE formation.Presented in part at the FASEB meeting, Atlanta, GA, 1991.     

The combined relationship of temperature and molybdenum concentration to nitrogen fixation byAnabaena cylindrica

The joint effects of growth temperature, incubation temperature, and molybdenum concentration on the nitrogen fixation rate ofAnabaena cylindrica were determined using the acetylene-reduction technique. The nitrogen-fixation response to increased molybdenum concentration varied among three growth temperatures (15°, 23°, and 30° C). The pattern of rate change was similar within a growth temperature but increased overall in magnitude with the three incubation temperatures (also 15°, 23°, and 30° C). The maximum rate of nitrogen fixation occurred at 30°C regardless of previous growth temperature. The minimum molybdenum concentration necessary to yield substantial acetylene reduction varied with growth temperature: at 15°C, 15g 1^–1 was effective; at 23°C, less than 5g 1^–1 was effective; and at 30°C, 50g 1^–1 was effective. At all three growth temperatures, increases in molybdenum concentration above the minimum effective concentration produced increases in acetylene reduction. However, at higher molybdenum concentrations inhibition of nitrogen fixation occurred.