Interactions between respiration and denitrification during growth of Thiosphaera pantotropha in continuous culture

Abstract The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h⁻¹. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h⁻¹. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.     

Continuous deacidification of wine by immobilized Schizosaccharomyces pombe cells: evaluation of malic acid degradation rate and analytical profiles

Schizosaccharomyces pombe cells entrapped in Ca alginate gel were used under continuous fermentation conditions to evaluate the extent of malic acid degradation at different dilution rates ( D , h⁻¹) and the analytical profiles of wines obtained. Gel entrapped cells caused the deacidification of wine without affecting the analytical profiles. Maximum deacidification rate was obtained at 0.076 h⁻¹ D while higher dilution rates (up to 0.100 h⁻¹ D ) resulted in a clear reduction of activity. At D -value of 0.055 h⁻¹, the deacidification activity remained constant during the observation period of 360 h.     

Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates

Abstract From chemostat enrichments conducted at dilution rates of 0.025, 0.12 and 0.25 h⁻¹ glutamate- and aspartate-fermenting bacteria were isolated. The dominant aspartate-fermenting strains in all these enrichments belonged to the genus Campylobacter , whereas 3 dissimilar types of glutamate-fermenting bacteria predominated at the different dilution rates. One of these strains was identified as Clostridium cochlearium . The remaining two were designated as strain DKglu16 (glutamate → acetate + propionate + ammonium + carbon dioxide) and DKglu21 (glutamate → acetate + formate + ammonium + carbon dioxide). Grown in continuous culture under glutamate limitation, strain DKglu16 (μ_max= 0.13 h⁻¹; K _s= 1.9 μM) outcompeted C. cochlearium (μ_max= 0.36 h⁻¹; K _s= 7 μM) at low dilution rates, but was outgrown at higher rates of dilution (0.044 h⁻¹). In glutamate-limited continuous culture the competitiveness of strain DKglu16 increased considerably when lactate was added to the feed in addition to glutamate.     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Oxygen and nitrate respiration in a reed swamp sediment from a eutrophic lake

Seasonal measurements of the oxygen and nitrate uptake by a reed swamp sediment were carried out in a shallow, eutrophic Danish lake, Arreskov Sø. The oxidation of organic carbon in the sediment by aerobic and nitrate respiration was 290 and 188 g C m⁻² yr⁻¹ respectively. During winter, nitrate respiration amounted to 94% of the total carbon oxidation, whereas it was zero during summer. On an annual basis nitrate respiration constituted 39% of total respiration. Sediment nitrate uptake was correlated to nitrate concentration. In consequence of this the nitrate uptake rates varied during the year from zero in summer to 55 mg N m⁻² d⁻¹ in spring.
Oxygen uptake rates varied from 30 to 250 mg O₂ m⁻² h⁻¹ during the year, with a maximum uptake in August. The oxygen uptake per year was calculated to 860 g O₂ m⁻². The oxygen uptake rate was correlated to lake temperature and Kjeldahl nitrogen content of the sediment. The oxygen uptake rate, however, showed no correlation with loss on ignition of the sediment. A Q₁₀-value of 2.2 was found for lake measurements in the temperature interval of 5–15°C. The corresponding O₁₀-value in the laboratory was 2.6. A high microbial biomass indicated by the maximum content of Kjeldahl nitrogen and the lowest ratio of loss on ignition on Kjeldahl nitrogen appeared in late August, when the maximum oxygen uptake occurred. The oxygen uptake rate increased during the time interval from sampling to the start of the experiments.     

Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection

Biofilm formation and function was studied in mixed culture using 20 bacterial strains isolated from a karst aquifer. When co-cultured in a glucose-limited chemostat, Vogesella indigofera and Pseudomonas putida were the dominant planktonic and biofilm organisms respectively. Biofilm formation and resistance to the iodine disinfectant betadine were then studied with monoculture and binary cultures of V. indigofera and P. putida and a 20-strain community. Biofilm population size [measured as colony-forming units (CFU) cm⁻²] increased with increasing species diversity. Significantly larger populations formed at dilution rates (DRs) of 0.0083 h⁻¹ than at 0.033 h⁻¹. P. putida populations were higher and V. indigofera lower in binary than in monoculture biofilms, suggesting that P. putida outcompeted V. indigofera . In binary biofilms, V. indigofera , a betadine-resistant organism, enhanced the survival of P. putida , a betadine-susceptible organism. In the 20-strain biofilms, this protective effect was not observed because of low concentrations of V. indigofera (< 1% of the total population), suggesting that resistant organisms contribute to overall biofilm disinfectant resistance. Growth at 0.033 h⁻¹ enhanced survival of V. indigofera biofilms against betadine. Although DR did influence survival of the other communities, its effects were neither consistent nor significant. All told, biofilm formation and betadine resistance are complex phenomena, influenced by community composition, growth rate and betadine concentration.     

The application of compound bi-directional flow diffusion chemostats to the study of microbial interactions

Abstract A multi-stage bi-directional chemostat system has been developed in which solutes but not cells are allowed to diffuse between the individual growth chambers which are separated by 0.2 micron pore size polyvinyledene difluoride membranes. The experimental system enables the generation of physico-chemical gradients which, together with the spatial separation of the individual microbial processes, provides a useful laboratory model to study microbial interactions. This paper describes the construction of a multi-stage diffusion chemostat and its application in studying carbon flow in anaerobic estuarine sediments. Populations of Clostridium butyricum, Desulfovibrio desulfuricans and Chromatium vinosum were grown in the compound diffusion chemostat at a dilution rate of 0.03 h⁻¹ at 25°C, and the effects of inorganic nitrogen source and availability on carbon flow and individual cell populations were determined. C. butyricum and D. desulfuricans both used NO⁻₃ as an e⁻ acceptor with an increase in cell numbers. Under these growth conditions, free S²⁻ concentrations were lower, resulting in more stable cell populations than in comparable cultures grown on NH⁺₄ as nitrogen source.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments

Abstract: Different reduced sulfur compounds (H₂S, FeS, S₂O₃²⁻) were tested as electron donors for dissimilatory nitrate reduction in nitrate-amended sediment slurries. Only in the free sulfide-enriched slurries was nitrate appreciably reduced to ammonia (     ), with concomitant oxidation of sulfide to S⁰ (     ). The initial concentration of free sulfide appears as a factor determining the type of nitrate reduction. At extremely low concentrations of free S²⁻ (metal sulfides) nitrate was reduced via denitrification whereas at higher S²⁻ concentrations, dissimilatory nitrate reduction to ammonia (DNRA) and incomplete denitrification to gaseous nitrogen oxides took place. Sulfide inhibition of NO- and N₂O- reductases is proposed as being responsible for the driving part of the electron flow from S²⁻ to NH₄⁺.     

Oxygen uptake in developing eggs and larvae of the cod, Gadus morhua L.

Unfertilised cod eggs showed a mean oxygen uptake rate at 5°C of 0.089 μl O₂, dry wt.⁻¹ h⁻¹; this gradually rose to 0.768 μl O₂ mg dry wt.⁻¹ h⁻¹ in eggs about to hatch. From hatching to complete yolk absorption larvae respired at 1.6 μl O₂, mg dry wt.⁻¹ h⁻¹. During starvation following yolk absorption, uptake fell significantly to 1.1 μl O₂, mg dry ⁻¹ h⁻¹. Much of this decrease in oxygen consumption was shown to be caused by reduction in activity. Loss of weight during the embryo and larval phases could not easily be reconciled with total oxygen consumption; it is suggested that cod embryos and larvae may not rely solely upon endogenous energy reserves during development.     

Simultaneous phototrophic and chemotrophic growth in the purple sulfur bacterium Thiocapsa roseopersicina M1

Abstract The anoxygenic phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in illuminated continuous cultures with thiosulfate as growth limiting substrate. Aeration resulted in completely colorless cells growing chemotrophically, whereafter the conditions were changed to a 23 h oxic/1 h anoxic regime. After 11 volume changes at a dilution rate of 0.031 h⁻¹ (35% of μ_max) a time dependent equilibrium was established. During the 23 h oxic periods bacteriochlorophyll a synthesis (BChl a ) was not observed, whereas during the 1 h anoxic periods synthesis was maximal (i.e. 1.1 μg (mg protein)⁻¹ h⁻¹). As a result the BChl a concentration gradually increased from zero to an average value over 24 h of 1.9 μg (mg protein)⁻¹. Concomitantly, the protein concentration increased from 13.9 mg 1⁻¹ during continuous oxic conditions to 28.8 mg 1⁻¹. For comparison, the protein concentration during fully phototrophic growth at an identical thiosulfate concentration in the inflowing medium was 53.7 mg 1⁻¹. The specific respiration rate was 8 μmol O₂ (mg protein)⁻¹ h⁻¹ during full chemotrophic growth and gradually decreased to 3.5 μmol O₂ (mg protein)⁻¹ h⁻¹ after 11 volume changes at the regime employed. These data show that T. rosepersicina is able to simultaneously utilize light and aerobic respiration of thiosulfate as sources of energy. The ecological relevance of the data is discussed.     

Oxygen consumption in Oreochromis niloticus (L.) in relation to development, salinity, temperature and time of day

Oxygen consumption of Oreochromis niloticus at different stages of development was studied in relation to salinity, temperature and time of day, using a Warburg apparatus. The oxygen consumption of newly hatched (0–14 h) larvae was 3.40 μl O₂ larva⁻¹ h⁻¹, of older yolk sac larvae 10.09 μl O₂ larva⁻¹ h⁻¹, and of one-month-old fry 32.99 μl O₂ larva⁻¹ h⁻¹. The QO₂ values showed a decrease with development and growth, ranging from 21.2–26.0 μl O₂ mg⁻¹ h⁻¹ in newly hatched larvae to 2.97 μl mg⁻¹ h⁻¹ in one-month-old fry. Changes in oxygen consumption occurred with salinity, the highest being at 17%o. Active larvae (12-24 mm T.L.) showed a doubling of consumption with a 10° C rise in temperature, and their Q₁₀ factor increased from 2.25 to 3.43 with increasing size. Day-old yolk-sac larvae, late yolk-sac larvae (5 days old) and fry of 12 14 mm length all showed a depression in oxygen consumption at midnight followed by a dawn rise.     

Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention

Abstract: The retentostat was developed for long-term continuous, axenic cultivation of microorganisms at those low growth rates which prevail in most natural habitats and which cannot be established properly in chemostats. How a microbial population approaches 'zero-growth' was studied in axenic cultures of Nitrosomonas europaea with complete biomass retention at 25°C and constant input of a nutrient solution containing ammonium (0.57 mM) as energy source. Since only cell-free filtrate left the reactor, biomass accumulated until a stable maximum of 2.7 × 10⁹ cells ml⁻¹ (398 mg l⁻¹ dry matter) was reached after about 5 weeks. In this state, growth rate approached zero, and the ammonium input just met the substrate demand required for maintenance energy (1.43 μmol NH₃–N mg dm⁻¹ h⁻¹). The potential of the retentostat for studying interactions between different microorganisms was demonstrated with a cascade of cultures of Nitrosomonas, Nitrobacter , and a denitrifying Pseudomonas . Thereby the ammonia was completely eliminated from artificial wastewater.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Reduction of nitrate in the perennial tissues of aerial parts of Alnus glutinosa

In vivo nitrate reductase (NR, EC 1.6.6.1.) activity was measured in leaves, branches and trunk of field-grown Alnus glutinosa (L.) Gaertn. All of the assayed tissues enzymatically reduced nitrate with a decreasing activity [μmol NO₂⁻ (g dry weight)⁻¹ h⁻¹] in the order: leaves > branch bark > inner branch tissues > trunk xylem. The NR activity of the various tissues of excised branches was inhibited by tungstate added to the transpiration stream. Part of the nitrate added to the feeding solution (0.2, 0.5 or 1 m M KNO₃) of excised branches disappeared during its transport via the transpiration stream in the perennial tissues. This disappearance was enzymatic since it was decreased by tungstate.
No evidence was obtained for the presence of nitrate in natural xylem sap nor for a significant correlation between nitrate content of soil and leaf NR activity. These results indicate that in the field-grown black alder, the nitrate not reduced in the roots could be reduced in the perennial tissues of aerial parts. Since the leaf NR activity does not reflect the actual in situ nitrate reduction, the existence of a constitutive NR activity in Alnus leaves is suggested.     

Competition between purple and brown phototrophic bacteria in stratified lakes: sulfide, acetate, and light as limiting factors

Abstract The affinities for sulfide and acetate under mixotrophic conditions have been determined for the brown Chlorobium phaeobacteroides and the purple Thiocapsa roseopersicina isolated from a bloom in Lake Kinneret (Israel) at a depth of about 18 m. C. phaeobacteroides exhibited a far higher affinity for sulfide than T. roseopersicina . For acetate, the opposite was observed.
In light-limited continuous cultures, C. phaeobacteroides preferentially used sulfide, whereas in mixotrophic cultures of T. roseopersicina sulfide could be detected without detectable acetate. Competition experiments under increasingly severe light limitation resulted in co-existence of the two strains. Relatively high light intensities resulted in a dominance of T. roseopersicina over C. phaeobacteroides , whereas at lower intensities C. phaeobacteroides became dominant. However, at light intensities below 2 μEin · m⁻²· s⁻¹, T. roseopersicina was completely excluded.
At low light intensities, C. phaeobacteroides is able to grow at a much higher rate than T. roseopersicina . The maintenance rate constant μ_e of C. phaeobacteroides is −0.001 h⁻¹, whereas that of T. roseopersicina is −0.011 h⁻¹. However, high light intensities inhibit the growth rate of C. phaeobacteroides , but not that of T. roseopersicina .
The explanation of the high numbers of C. phaeobacteroides in Lake Kinneret appears to be the combination of low light intensities and low sulfide concentrations. As a result, the incorporation of acetate is enhanced. The low numbers of T. roseopersicina can be explained by the high maintenance energy requirements of this organism, which exceed the available light at the depth of the bloom.     

Ammonium ion affecting tylosin production by Streptomyces fradiae NRRL 2702 in continuous culture

Nitrogen regulation in tylosin production by Streptomyces fradiae NRRL 2702 was studied in chemostat culture using a soluble synthetic medium. The maximum value of specific tylosin formation rate ( q _TYL) was 1·13 mg g⁻¹ h⁻¹ at the specific growth rate (μ) of 0·05 h⁻¹, and q _TYL decreased with increasing levels of the specific growth rate after reaching a rate of 0·1 h⁻¹. The optimum conditions for tylosin formation were that the specific ammonium ion uptake rate ( q _N) and μ were 0·13 mmol g⁻¹ h⁻¹ and 0·05 h⁻¹, respectively. The specific formation rates of threonine dehydratase (TDT) and tylosin were repressed by high levels of specific ammonium ion uptake rate. This study showed the adaptation to chemostat cultures of the nitrogen regulation of tylosin fermentations.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.