The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10⁻⁶ M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.     

Biogenic production of cyanide and its application to gold recovery

Chromobacterium violaceum is a cyanogenic (cyanide-producing) microorganism. Cyanide is used on an industrial scale to complex and recover gold from ores or concentrates of ores bearing the precious metal. A potentially useful approach in gold mining operations could be to produce cyanide biologically in relatively small quantities at the ore surface. In this study, C. violaceum grown in nutrient broth formed a biofilm and could complex and solubilize 100% of the gold on glass test slides within 4–7 days. Approximately 50% of the cyanide-recoverable gold could be mobilized from a biooxidized sulfidic-ore concentrate. Complexation of cyanide in solution by gold appeared to have a beneficial effect on cell growth — viable cell counts were nearly two orders of magnitude greater in the presence of gold-coated slides or biooxidized ore substrates than in their absence. C. violaceum was cyanogenic when grown in alternative feedstocks. When grown in a mineral salt solution supplemented with 13.3% v/v swine fecal material (SFM), cells exhibited pigmentation and suspended cell concentrations comparable to cultures grown in nutrient broth. Glycine supplements stimulated production of cyanide in 13.3% v/v SFM. In contrast, glycine was inhibitory when added at the time of inoculation in the more concentrated SFM, decreasing cell numbers and reducing ultimate bulk-solution cyanide concentrations. However, aeration and addition of glycine to stationary phase cells grown on 13.3% v/v SFM anaerobically resulted in rapid production and high concentrations (up to 38 mg l⁻¹) of cyanide. This indicates that biogenesis of cyanide may be supported in remote areas using locally produced and inexpensive agricultural feedstocks in place of commercial media. Journal of Industrial Microbiology & Biotechnology (2001) 26, 134–139. Received 06 June 2000/ Accepted in revised form 30 September 2000     

Respiration and cyanogenesis inPseudomonas aeruginosa

The respiratory ability of batch cultures ofPseudomonas aeruginosa strain 9-D2 peaks during midlog phase at 3.8 nmol O₂/min/10⁸ cells. This ability declines in late log phase, just prior to the time the culture begins to produce cyanide. The respiration of this organism is particularly sensitive to cyanide inhibition during midlog-phase growth, but is extremely resistant to this compound in stationary phase. These inhibition patterns are biphasic for each of these situations and indicate several respiratory responses to HCN. Addition of cyanide to midlog-phase cells resulted in the production of a stationary-phase type of cyanide respiration pattern in 2 h. A non-cyanideproducing mutant of this organism produced significantly less of the cyanide-resistant respiration components.     

Cyanide assimilation in Rhizobium ORS 571: influence of the nitrogenase catalyzed hydrogen production on the efficiency of growth

When cyanide is gradually added to a nitrogenfixing culture, Rhizobium ORS 571 is capable of assimilating large amounts of cyanide using its nitrogenase. Under these conditions the molar growth yield on succinate (Y _succ) increases from 27 at the start of cyanide addition to 38 at the end. The respiratory chain of cells grown at a concentration of 7 mM cyanide is still very sensitive to cyanide. The increase in growth yield is explained by a decrease in hydrogen production by nitrogenase as soon as cyanide is assimilated. This is confirmed by calculating the influence of hydrogen production on Y _succ. Hydrogen production by nitrogenase has a greater influence on growth yields than the presence or absence of hydrogenase activity. At the end of cyanide addition when all cell nitrogen is synthesized from cyanide and no nitrogen fixation occurs, nitrogenase will be in a very oxidized state.     

Effect of aeration on ethylene production by soil bacteria and soil samples cultivated in a closed system

Summary Ethylene production was studied in shaken cultures ofPseudomonas putida andPseudomonas fluorescens isolated from soil and in unsterile garden soil samples moistened to 60% of the water holding capacity. The highest ethylene accumulation in bacterial cultures was reached under conditions of delayed aeration,i.e. when the culture was closed and the aeration started after the oxygen content decreased to 4%. The ethylene production rose immediately after the beginning of aeration. Under these conditions ethylene production was inP. fluorescens 2–3 times and in glucosecultivatedP. putida 6 times higher than in the fully aerated cultures. Methionine stimulated ethylene production byP. fluorescens, whereas glucose proved to be more suitable forP. putida. This strain was incapable of growth on methionine as the sole carbon source. Samples of nonsterile garden soil produced the highest amounts of ethylene under anaerobic conditions. Artificial inoculation of soil samples byP. putida resulted in an increase of ethylene formation in samples with delayed aeration. Addition of glucose or glucose with methionine stimulated ethylene production in all soil samples.     

Antioxidant property and production of exopolysaccharide from Armillaria mellea in submerged cultures: Effect of culture aeration rate

Effects of culture aeration rate on production and antioxidant property of exopolysaccharide (EPS) by Armillaria mellea were investigated in a 5‐L stirred‐tank bioreactor where an optimal biomass aeration rate of 1.2 vvm with 0.22 g/g cell yield and 0.6 vvm EPS formation rate with 7.66 mg/g product yield were achieved. A two‐stage aeration process to maximize the biomass and EPS productions proceeded with a 1.55‐fold enhancement (from 4.28 to 6.65 g/L) in biomass formation and a 2.68‐fold enhancement (from 86.9 to 233.2 mg/L) in the EPS production, as compared with those from the aeration rate of 0.3 vvm. The molecular weights of EPS in cultures of different aeration rates are closely correlated with their protein/polysaccharide ratios (R²=0.830) and EC₅₀ (EC₅₀, the effective concentration where the antioxidant property is 50%) values in antioxidant activity (R²=0.960), reducing power (R²=0.894) and chelating ability (R²=0.954). EPS from the two‐stage aeration rate culture shows a strong antioxidant property by the conjugated diene method, reducing power and chelating ability on ions. Therefore, we present results to regulate and to optimize A. mellea cultures to efficiently produce biomass and EPS. The fermented EPS has the potential to be used as for antioxidant‐related functional foods and pharmaceutical industries.     

Biodegradation of cyanides, cyanates and thiocyanates to ammonia and carbon dioxide by immobilized cells of Pseudomonas putida

Pseudomonas putida utilizes cyanide as the sole source of carbon and nitrogen. Agar, alginate, and carrageenan were screened as the encapsulating matrices for P. putida. Alginate-immobilized cells of P. putida degraded sodium cyanide (NaCN) more efficiently than non-immobilized cells or cells immobilized in agar or carrageenan. The end products of biodegradation of cyanide were identified as ammonia (NH₃) and carbon dioxide (CO₂). These products changed the medium pH. In bioreactors, the rate of cyanide degradation increased with an increase in the rate of aeration. Maximum utilization of cyanide was observed at 200 ml min⁻¹ of aeration. Immobilized cells of P. putida degraded cyanides, cyanates and thiocyanates to NH₃ and CO₂. Use of Na[¹⁴C]-CN showed that 70% of carbon of Na[¹⁴C]-CN was converted into ¹⁴CO₂ and only 10% was associated with the cell biomass. The substrate-dependent kinetics indicated that the K _m and V _max values of P. putida for the substrate, NaCN were 14 mM and 29 nmol of oxygen consumed mg protein⁻¹ min⁻¹ respectively. Received 29 January 1996/ Accepted in revised form 19 September 1997     

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

Outdoor cultivation ofArthrospira platensis during autumn and winter in temperate climates

Arthrospira (Spirulina) platensis M2 was grown outdoors in 50-mm diameter tubular reactors under the climatic conditions of central Italy (Florence) from September to December 1995 and in March 1996. Except for September, the cultures temperature was regulated. Mean productivities of 0.83, 0.44 and 0.61 g dry wt L^–1 d^–1 were achieved in autumn (September–October), winter (November–December) and March, respectively. In autumn and winter, the photosynthetic efficiency of the cultures and the degree of correlation between productivity and solar irradiance were significantly greater than in summer. The effect of cell density and aeration rate on productivity was evaluated in September. The productivity of cultures operated at high supra-optimal population density was about 30% less at high aeration rate (1.0 LL^–1 min^–1), and 50% less at standard aeration rate (0.17 LL^–1 min^–1), than that of control cultures kept at optimal population density and standard aeration rate. The reduction of productivity in high-density cultures was due to lower daylight output rates and higher night biomass losses (the latter were particularly relevant under standard aeration conditions). The main factor limiting productivity in closed reactors during autumn was the night temperature. Heating the cultures during daylight hours on sunny days did not cause any significant increase of the yields, since under sunlight the unheated cultures also reached the optimal temperature for growth early in the morning. On cloudy days, the day-time temperature of the unheated cultures remained well below the optimum, however this had only a limited effect on productivity since algal growth was mainly light-limited.     

Oxygen supply strongly influences metabolic fluxes,the production of poly(3-hydroxybutyrate) and alginate,and the degree of acetylation of alginate in Azotobacter vinelandii

The aim of this study was to evaluate carbon flux in Azotobacter vinelandii using metabolic flux analysis (MFA) under high and low aeration conditions to achieve an improved understanding of how these changes could be related to alginate acetylation and PHB production. Changes in oxygen availability had a considerable impact on the metabolic fluxes and were reflected in the growth rate, the specific glucose consumption rate, and the alginate and PHB yields. The main differences at the metabolic flux level were observed in three important pathways. The first important difference was consistent with respiratory protection; an increase in the flux generated through the tricarboxylic acid (TCA) cycle for cultures grown under high aeration conditions (up to 2.61 times higher) was observed. In the second important difference, the fluxes generated through pyruvate dehydrogenase, phosphoenol pyruvate carboxykinase and pyruvate kinase, all of which are involved in acetyl-CoA metabolism, increased by 10, 43.9 and 17.5%, respectively, in cultures grown under low aeration conditions compared with those grown under high aeration conditions. These changes were related to alginate acetylation, which was 2.6 times higher in the cultures with limited oxygen, and the changes were also related to a drastic increase in PHB production. Finally, the glyoxylate shunt was active under both of the conditions that were tested, and a 2.79-fold increase was observed in cultures that were grown under the low aeration condition.     

Anaerobic and Aerobic Hydrogen Gas Formation by the Blue-Green Alga Anabaena cylindrica

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5′-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Physiological regulation of the properties of alcohol dehydrogenase II (ADH II) of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>: NADH renders ADH II resistant to cyanide and aeration

The variable cyanide-sensitivity of the iron-containing alcohol dehydrogenase isoenzyme (ADH II) of the ethanol-producing bacterium Zymomonas mobilis was studied. In aerobically grown permeabilized cells, cyanide caused gradual inhibition of ADH II, which was largely prevented by externally added NADH. Cyanide-sensitivity of ADH II was highest in cells grown under conditions of vigorous aeration, in which intracellular NADH concentration was low. Anaerobically grown bacteria, as well as those cultivated aerobically in the presence of cyanide, maintained higher intracellular NADH levels along with a more cyanide-resistant ADH II. It was demonstrated that cyanide acted as a competitive inhibitor of ADH II, competing with nicotinamide nucleotides. NADH increased both cyanide-resistance and oxygen-resistance of ADH II.     

Effect of aerobically generated inoculum on xylose fermentation by an ethanologenic recombinantE. coli

Summary The anaerobic conversion of xylose to ethanol by a genetically engineredE. coli B (pLOI297) was investigated using anaerobically and aerobically grown cultures as inocula. Using anaerobically grown cells, an increase in the inoculation density from 50 to 340 mg dry wt. cells/L resulted in an increase in the overall volumetric productivity from 0.57 to 0.71 g/L/h. At the higher inoculation density, substitution of the anaerobic inoculum by aerobically grown cells resulted in a 15% reduction in volumetric productivity (0.61 g/L/h) that was caused by the introduction of a lag period during which the aerobic inoculum adapted to the anaerobic environment. In all cases, the ethanol yield from xylose approached the theoretical maximum and seemed unaffected by the physiological history of the inoculum with respect to aeration. It is concluded that aeration should be avoided in the production of high performance starter cultures.     

Glutathione transferases catalyze recycling of auto‐toxic cyanogenic glucosides in sorghum

Cyanogenic glucosides are nitrogen‐containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin‐derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant‐specific lambda class (GSTLs) to produce p‐hydroxyphenyl acetonitrile. This is further metabolized to p‐hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p‐glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co‐localized with expression of the genes encoding the p‐hydroxyphenyl acetonitrile‐metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.     

Antibiotic production by a marine isolate (MS 310) in an ultra-low-speed rotating disk bioreactor

Actinomycin-D production by a biofilm-forming estuarine isolate viz Streptomyces sp. MS 310 is studied in small-scale shaken cultures, as well as in a 25 L rotating disk bioreactor, (RDBR) which, when operated at a disk rotational speed of 1 revolution/day with 50% disk submergence, mimics the intertidal conditions of the microbe’s niche estuarine habitat-alternating 12 h periods of inundation and exposure. The ideal pH and temperature for antibiotic production are determined (pH 10, 30°C) through a designed experimental study using shaken flasks. Subsequently, operating conditions in the RDBR are investigated employing a 3ⁿ experimental design wherein each of two (n = 2) parameters viz. aeration and disk submergence are considered at three levels viz high, medium, and low: 9.0, 6.0, and 3.0 L/min for aeration rate; and 75, 50, and 25% for disk submergence, (while maintaining the rotational speed at 1.0 rev/day). The niche-mimic condition along with maximum permissible aeration is found to be most favorable for antibiotic production — peak antibiotic activity (PAA) and peak activity attainment rate (PAAR) simultaneously attaining their highest values: 40 mm and 2.13 mm/h, respectively. Both PAA and PAAR increase with increasing aeration at all operating conditions examined — particularly, at the niche-mimic condition, a threefold increase in aeration rate (3∼9 L/min) causes PAA to increase by 33%, whereas PAAR increases by 2.5 times, thus pointing to the strong aeration dependence of this actinomycin-D producer. Again, compared to the best values obtained in the 500 mL shaken flask experiments, corresponding RDBR values are 16% higher for PAA and more than 5 times higher for PAAR — strong evidence for employing these novel bioreactors for cultivation of antibiotic-producer marine microbes.     

Untersuchungen zur Einsatzwürdigkeit von Methanol-Bakterienmasse bei Mastputen und Mastcairina

The caecal fermentation pattern was studied in four litters of rabbits. Rabbits were sequentially slaughtered at the age of 4 (before weaning), 6, 8 and 11 weeks. Their caecal contents were analyzed and incubated in vitro at 39°C for 6 and 12h. Net productions of short‐chain fatty acids (SCFA), hydrogen and methane were determined. The average caecal weight increased three‐times within two weeks after weaning, from 31.4 to 93.7g. At the end of the experiment, the caecal weight was on average 134.4g. A large variability of the SCFA concentration observed before weaning decreased after weaning. Measurements of caecal metabolite profiles and results of in vitro experiments indicated a certain decrease of propionate in favour of butyrate, associated with the weaning. The establishment of methanogens in rabbits was slow. Methanogenesis started in one out of four rabbits at the age of 6 weeks. Five weeks later, one of four rabbits still did not produce methane. The hydrogen recovery decreased between the 4th and the 6th week of age, due to the increase of the butyrate/propionate ratio. After the 6th week, the hydrogen recovery increased with age, apparently because of the increase in methane production. Hydrogen recovery tended to increase during incubation, suggesting a decrease of reductive acetogenesis. This increase was observed both in methanogenic and non‐methanogenic rabbit caecal cultures. In former cultures, the ratio CH4/SCFA rose with time of incubation.     

Anaerobic and aerobic hydrogen gas formation by the blue-green alga Anabaena cylindrica.

An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5'-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.     

Acetate synthesis from 2 CO2 in acetogenic bacteria: is carbon monoxide an intermediate?

Cultures of Acetobacterium woodii and Clostridium thermoaceticum growing on fructose or glucose, respectively, were found to produce small, but significant amounts of carbon monoxide. In the gas phase of the cultures up to 53 ppm CO were determined. The carbon monoxide production was completely inhibited by 1 mM cyanide. Cultures and cell suspensions of both acetogens incorporated ¹⁴CO specifically into the carboxyl group of acetate. This CO fixation into C₁ of acetate was unaffected by cyanide (1 mM). The findings are taken to indicate that CO (in a bound form) is the physiological precursor of the C₁ of acetate in acetate synthesis from CO₂. The cyanide inhibition experiments support the hypothesis that the cyanide-sensitive carbon monoxide dehydrogenase may serve to reduce CO₂ to CO rather than to incorporate the carbonyl into C₁ of acetate.     

Aeration strategy: a need for very high ethanol performance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> fed-batch process

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions—oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)—on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l^–1), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l^–1 in 45 h) and average productivity (3.3 vs 2.6 g l^–1 h^–1) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l^–1. We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l^–1 in 45 h) in order to achieve a highly competitive dynamic process.