Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.     

A kinetic model for suspended and attached growth of a defined mixed culture

Kinetic experiments were carried out in a semicontinuous wastewater treatment process called self-cycling fermentation (SCF) using a defined mixed culture and various concentrations of synthetic brewery wastewater. The same consortium, which had been previously identified as Acinetobacter sp., Enterobacter sp., and Candida sp., were used in these experiments. The overall rate of substrate removal was attributable to both suspended microbes and the biofilm that formed during the treatment process. A rate expression was developed for the SCF system for a range of synthetic wastewaters containing glucose and various initial concentrations of ethanol and maltose. The data indicated that substrate removal by the suspended cells was directly related to the biomass concentration. However, substrate removal by the biofilm was apparently not affected by the biofilm thickness and was a function of substrate concentration only.     

Production of metabolites in the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of Mycobacterium sp. and Sphingomonas sp

The effects of the mixed culture of Mycobacterium sp. strain A1-PYR and Sphingomonas sp. strain PheB4 on the degradation characteristics of single polycyclic aromatic hydrocarbon were investigated. In the mixed bacterial culture, phenanthrene, fluoranthene and pyrene were degraded by 100% at Day 3, 71.2% and 50% at Day 7, respectively. Compared to their respective pure cultures, the degradation of phenanthrene and fluoranthene decreased, but that of pyrene increased significantly. Based on GC-MS analysis, eight and six new metabolites were produced from the biodegradation of phenanthrene and fluoranthene, respectively, while only two new metabolites were formed from pyrene. To our knowledge, this is the first report that the mixed bacterial culture could increase the diversity of metabolites from PAH, but the diverse metabolite pattern was not necessarily beneficial to the degradation of the recalcitrant PAH. The enhancement on pyrene degradation was possibly attributed to the rapid growth of strain PheB4.     

亚硝化单胞菌（Nitrosomonas sp．）THD-1分离鉴定及高密度培养

从浙江舟山桃花岛近海海域分离筛选到一株高活性氨氧化细菌,命名为THD-1。菌株THD-1为革兰氏阴性,球状至椭球状,大小约为（0．5～0．8）μm×（0．7～1．3）μm;菌落呈无色透明状,针尖大小,边缘光滑;可在盐度为0～50％0的培养基中生长。16SrDNA序列比对表明,菌株THD—1与Nitrosomonas europaea ATCC25978^T的相似性为96．3％。培养液的pH值对菌体生长和氨氧化活性影响明显,当pH值降至6．0以下时,菌株的生长和氨氧化活性几乎完全被抑制,但这种抑制可通过回调pH值的方法解除;在所试的4种碱液中,碳酸氢铵调节pH值对菌体生长和氨活性改善效果最好。建立了菌株THD-1的分批补料式高密度培养方法,最终OD600可达0．214,最大活菌数可达7．5×10^8cells／mL。     

Effect of adhesion to particles on the survival and activity of Nitrosomonas sp. and Nitrobacter sp.

The adhesion of Nitrosomonas sp. and Nitrobacter sp. cells isolated from fishpond sediment to different solid particles was studied. Nitrosomonas and Nitrobacter cells rapidly attached to particles of bentonite, calcium carbonate, amberlite, and fishpond sediment, however they did not adhere to phenyl-sepharose beads. The nitrifying activity of attached bacteria was greater than the activity of freely suspended cells or the activity of cells which have been detached from CaCO₃ particles. The enhancement in the nitrifying activity was rapid and was already observed within the first hour after attachment (which equals only 1/24 to 1/50 of the generation time of Nitrosomonas sp. or Nitrobacter sp. In addition, the survival of the attached bacteria under both anaerobic and under aerobic incubation was extended to weeks, compared to only a few days for the free cells. The presence of substrate (ammonia or nitrite) during the anaerobic incubation period was found not to affect the survival time of the bacteria. Finally, it was found that the attachment of Nitrosomonas and Nitrobacter cells to CaCO₃ particles affected the dispersal and sinking rate of these particles.     

Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Ethanol production by a mixed culture of flocculent strains of Zymomonas mobilis and Saccharomyces sp.

 Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l^-1 h^-1. No levan was produced even if a sucrose-based medium was used. Received: 20 April 1995/Received revision: 26 July 1995/Accepted: 13 September 1995     

Kinetic analysis of the growth of Spirulina sp. in batch culture

Batch cultivation of Spirulina sp. was carried out under limited light at 30°C in the pH range of 9.2 to 9.7. The specific growth rate D was calculated from the tangent of the growth curve and the cell concentration at that time, and the amount of light energy absorbed per unit time per unit cell weight (E_x), namely, the specific absorption rate of light energy, was also calculated from the total amount of radiant flux of transmitted light at the surface of the culture vessel and cell concentration of the culture solution. A plot against E_x of D in the linear growth phase in batch culture and at various phases in continuous culture gave, for E_x of less than 1.0 kcal/g·h, points scattered near a straight line with slope m 0.037 g/kcal and an intercept on the ordinate, −b, of −0.0046 h⁻¹, and, for higher E_x values, points scattered near a curve of gradually decreasing slope which tended to approach a constant value.A Lineweaver-Burk plot of the reciprocal of D plus b against that of E_x yielded an equation for the growth rate which represented well the growth curve in batch culture. This equation also expressed the linear increase of D with increase of E_x at high cell concentration in the culture solution. The relation between cell growth rate and cell fluidity is discussed by use of a vector equation obtained by applying this relation to a culture solution contained in a given closed surface.     

Calorimetric measurement of free energy utilization by Nitrosomonas and Nitrobacter

Summary Calorimetric estimates of the utilization efficiency of the free-energy derived from substrate oxidation by cell suspensions of two nitrifying bacteria, Nitrosomonas and Nitrobacter, provided two ranges of values: 11 to 27% and 15 to 51%, respectively. About 15 to 30% of the utilized free-energy is used for driving endergonic reactions other than CO₂ fixation, probably the synthesis of polyphosphates.The molar heat of substrate oxidation does not seem to be influenced by the age of cells harvested during growth or by the length of the incubation period during which cells have been kept in a buffer suspension in a starved condition. The loss of respiratory activity measured either by oxygen uptake or heat evolution in the presence of the specific substrate, nitrite or ammonium, decreases according to kinetics which are influenced by the aerobiosis of the suspension. The viability of the starved cells decreases in a way which is similar to that of the respiratory activity. It seemed impossible to obtain cells which had lost their viability but kept the ability to oxidize their substrate.Two inhibitors of the respiratory chain, quinacrine and cyanide, are without effect on the molar heat of substrate oxidation and consequently on the free-energy utilization efficiency. 2.4 dinitrophenol did decrease the rate of heat evolution during substrate oxidation at concentrations at which the rate of oxygen uptake was not depressed, with the consequences that free-energy efficiency was apparently increased.     

Toxicity and kinetic parameters of the aerobic biodegradation of the phenol and alkylphenols by a mixed culture

A mixed culture aerobically metabolized phenol, cresol isomers (o-,m-,p-), 2-ethylphenol and xylenol isomers (2,5-DMP and 3,4-DMP) as the sole carbon and energy source. This culture had a high tolerance towards phenol with values of maximum degradation rate (V_\max) of 47 M phenol mg^–1 protein h^–1 and inhibition substrate constant (Ki) of 10 mM. These kinetic parameters were considerably diminished and the toxicity increased with the alkylphenols. For example with 2,5-xylenol, V_\max and Ki values of 0.8 M 2,5-xylenol mg^–1 protein h^–1 and 1.3 mM, respectively, were obtained. The cresols were 5-fold more toxic than phenol, whereas 2-ethylphenol and 3,4-xylenol were 11-fold more toxic, and 2,5-xylenol was 34-fold more toxic than phenol.     

The effect of glucose and ammonium sulfate on kinetic acidification by heterogeneous mixed culture

Summary The effect of glucose and ammonium sulfate concentration on the kinetics of lactic acid formation by a heterogeneous mixed culture was evaluated by the sole product formation using the Gompertz model, which can be used to define culture media composition taking into account product accumulation and acidification rate constant. A compromise between ionic inhibition and nitrogen limitation was found by using ammonium sulfate as nitrogen source. The sugar tolerance was similar to that in exenic cultures.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Growth of Nitrosomonas europaea in batch and continuous culture

Summary A clear medium has been used to grow pur cultures of Nitrosomonas europaea in flasks and in a continuous culture apparatus.Of several metallic ions examined in flask cultures of Nitrosomonas, Fe at 2 ppm and Co, Mn and Zn at 1 ppm were not toxic, Ni and Cr at concentrations greater than 0.25 ppm inhibited growth and Cu stopped growth completely at 0.5 ppm and inhibited at 0.1 ppm. Stainless steel of the specification EN58 B did not affect growth.In the continuous culture vessel, Nitrosomonas showed a growth response to Fe only when the population exceeded about 500×10⁶ organisms/ml. The minimum doubling time was about 8 hours in flasks and 11 hours in the culture vessel. With effective aeration and automatic P_H control, cultures of Nitrosomonas were grown successfully in continuous culture and gave a yield of 2.14 g dry weight of bacteria from 30 litres of culture in 5 days.     

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:307–315, 2015     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

Nitric oxide generation upon growth of Antarctic Chlorella sp. cells

The aim of this work was to characterize nitric oxide (NO) generation during the growth of Chlorella sp. cells. The profile of NO production was compared with that observed in Chlorella from temperate climate and Antarctic Chlamydomonas sp. A distinctive electron paramagnetic resonance (EPR) signal for the adduct MGD-Fe-NO was detected in the Antarctic Chlorella sp. cells on days 6 and 7 of growth. An assay based upon the time-dependent detection of NO by EPR was developed to assess both nitrate reductase (NR) and NO synthase (NOS)-like activities. A significant increase in both enzymatic activities was observed on day 6 of growth of Antarctic Chlorella sp. cells. However, NOS-like activity proved undetectable on day 10 of growth, while NR activity was observed until day 22 of growth. When the cultures were supplemented with Nω-nitro- l -arginine methyl ester ( l -NAME) from day 0 to day 13, no growth was observed. After administration of l -NAME from day 5 to day 12, growth rate at log phase was inhibited from 0.18 ± 0.02 (control) to 0.11 ± 0.01 per day. Removal of l -NAME on day 12 increased the growth rate to 0.15 ± 0.02 per day. Supplementation with S-nitroso- N -acetyl-penicillamine on day 5, in the presence of l -NAME from day 0, restored the growth rate to control values. The same profile of NO generation was observed during the growth of Antarctic Chlamydomonas sp.; however, Chlorella cells from temperate climate did not show any difference in NO generation over the growth period. The data reported here are the first observations, by employing EPR, of NO and the activity of the enzymes responsible for its generation during the growth of Antarctic photosynthetic alga.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

Stoichiometric analysis of dissolved organic carbon flux into storage and growth in aerobic granules culture

This study attempted to address a fundamental question of whether metabolic behaviors of aerobic granules are different from their counterparts, such as activated sludge and biofilms. A series of respirometric experiments were carried out using mature aerobic granules with mean sizes of 0.75–3.4 mm. Results suggested that metabolism of aerobic granules comprised three consecutive phases: (i) conversion of external dissolved organic carbon to a poly-β-hydroxybutyrate-like substance; (ii) growth of aerobic granules on the stored poly-β-hydroxybutyrate-like substance derived from phase I, and (iii) subsequent endogenous metabolism of aerobic granules. The stoichiometric analysis revealed that the conversion yields of external dissolved organic carbon to the poly-β-hydroxybutyrate-like substance, the growth yields of biomass on storage, and the overall growth yields of biomass on external dissolved organic carbon were not significantly correlated to the sizes of aerobic granules, i.e., the metabolism of aerobic granules would be size independent. The conversion coefficients and growth rates of aerobic granules were found to be comparable with those reported in the activated sludge and biofilms cultures, indicating that there would not be significant difference in the metabolisms of aerobic granules over activated sludge and biofilms. This information will be useful for modeling and designing aerobic granular sludge processes.     

Growth of Nitrosomonas europaea in batch and in continuous culture

Summary Techniques are described in this paper for growing Nitrosomonas europea in batch and in continuous culture with apparatus constructed from readily available laboratory materials.The methods employed in batch culture have enabled the collection of cells concentrated in small volumes. Nitrosomonas europaea has been grown successfully in continuous culture and yields of 23 g wet weight were obtained, an average of 0.16 g/l with a flow rate of 300 ml/h over twenty days. Results show that high levels of nitrite do not materially affect the growth of the bacterium. The continuous culture equipment has also been used to grow Azotobacter vinelandii and Thiobacillus concretivorus.