Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O₂/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.     

Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations

AIMS: To obtain ammonia-oxidizing bacterial (AOB) strains inhabiting low dissolved oxygen (DO) environments and to characterize them to better understand their function and ecology. METHODS AND RESULTS: Using a serial dilution method, two AOB strains (ML1 and NL7) were isolated from chemostat reactors operated with low DO concentrations (0.12-0.24 mg l(-1)). Phylogenetically, strains ML1 and NL7 are affiliated to AOB within the Nitrosomonas europaea and Nitrosomonas oligotropha lineages, respectively. Kinetically, strain ML1 had high affinity for oxygen (0.24 +/- 0.13 mg l(-1)) and low affinity for ammonia (1.62 +/- 0.97 mg N l(-1)), while strain NL7 had high affinity for ammonia (0.48 +/- 0.35 mg l(-1)), but a surprisingly low affinity for oxygen (1.22 +/- 0.43 mg l(-1)). A co-culture experiment was used to iteratively estimate decay constants for both strains. CONCLUSIONS: The results indicated that AOB without high affinity for oxygen may have other mechanisms to persist in low DO environments, with high affinity for ammonia being important. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a method to determine AOB growth kinetic parameters without assuming or neglecting decay constant. And, this is the first report on oxygen affinity constant of a N. oligotropha strain.     

Partial nitrification under limited dissolved oxygen conditions

Partial nitrification to nitrite is technically feasible and economically favourable, especially when wastewaters contained high ammonium concentrations or low C/N ratios. Partial nitrification can be obtained by selectively inhibiting nitrite-oxidizing bacteria (NOB) through appropriate regulation of the pH, temperature and dissolved oxygen (DO) concentrations. The effect of pH, DO levels and temperature on ammonia oxidation rate and nitrite accumulation was investigated in order to determine the optimal conditions for partial nitrification of synthetic wastewater with high ammonia concentration. The experiments performed at low DO levels to lower the total oxygen needed in the nitrification step, which means great saving in aeration. During the start-up stage pH and DO were set at 7.0–7.4 and 0.5 mg/l, respectively. The reactor was operated until complete partial nitrification was achieved. The effect of pH, DO on partial nitrification was studied, as pH was kept at 6.5, 7.5, 8.5, 9.5 and DO at 0.5±0.2, 1.5±0.2 and 2.5±0.2 mg/l, and temperature at 30 °C. The influence of temperature on k_a value was studied by keeping pH=7.5, DO=1.5 mg/l and temperature was controlled at 12, 20 and 30 °C, respectively. The results showed that partial nitrification to nitrite was steadily obtained and the optimal operational parameters were pH=7.5, DO=1.5 mg/l, T=30 °C based on ammonia oxidation rate and nitrite accumulation rate. The maximum k_a was achieved and to be 115.1×10⁻³ mg NH₄⁺–N (mg VSS h)⁻¹ under this condition.     

Costs of egg ventilation for male common gobies breeding in conditions of low dissolved oxygen

This study examines the effects of harsh environmental conditions on life history trade-offs in parental care in a marine fish, the common goby, Pomatoschistus microps. We compared male parental care and hatching success over two sequential brood cycles in fish breeding in conditions of either low dissolved oxygen or normal levels of oxygen. Males compensated for a low oxygen environment by increasing the time they spent fanning water over their eggs, as well as their fanning tempo. They also increased the frequency of egg-directed activities and decreased nest-building activities. Males in the low oxygen treatment lost more weight than control fish during the first spawning, and were more likely to abandon care during the second spawning. Males that completed care under low oxygen conditions did not differ from control males in the hatching success of their offspring or the size of young at hatching, but hatching started on average 1 day later. Thus, greater parental allocations to offspring while breeding in a harsh environment led to reduced future allocations. Copyright 1999 The Association for the Study of Animal Behaviour.     

Enhanced production of heteropolysaccharide-7 by Beijerinckia indica HS-2001 in pilot-scaled bioreactor under optimized conditions involved in dissolved oxygen using sucrose-based medium

Heteropolysaccharide-7 (PS-7) is a possible alternative to xanthan or gellan used as food additives due to its properties and potential applications. Sucrose was developed as a carbon source for production of PS-7 by Beijerinckia indica HS-2001 to overcome catabolite repression against glucose. The inoculum size was established to be 5.0% (v/v) for the pilot-scaled production of PS-7 by B. indica HS-2001 based on productivity and economic aspect. The optimal agitation speed and aeration rate for cell growth of B. indica HS-2001 were found to be 495 rpm and 1.8 vvm using response surface methodology (RSM), whereas those for production of PS-7 were 440 rpm and 1.2 vvm. The optimal inner pressure for cell growth of B. indica HS-2001 in a 100 L bioreactor was 0.02 MPa, whereas that for production of PS-7 was 0.04 MPa. The production of PS-7 by B. indica HS-2001 from 30.0 g/L sucrose under an optimized inner pressure in a 100 L bioreactor was 10.20 g/L, which was 1.32 times higher than that without inner pressure. The maximal production of PS-7 under optimal conditions involved in the dissolved oxygen using the sucrose-based medium developed in this study was 1.55 times higher than that before optimization.     

Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus

Clavulanic acid (CA), a potent β-lactamase inhibitor, is produced by a filamentous bacterium. Here, the effect of DO and shear, expressed as impeller tip velocity, on CA production was examined. Cultivations were performed in a 4 L fermentor with speeds of 600, 800 and 1,000 rpm and a fixed air flow rate (0.5 vvm). Also, cultivation with automatic control of dissolved oxygen, at 50% air saturation, by varying stirrer speed and using a mixture of air and O₂ (10% v/v) in the inlet gas, and a cultivation with fixed stirrer speed of 800 rpm and air flow rate of 0.5 vvm, enriched with 10% v/v O₂, were performed. Significant variations in CA titer, CA production rate and O₂ uptake-rate were observed. It was also found that the DO level has no remarkable effect on CA production once a critical level is surpassed. The most significant improvement in CA production was related to high stirrer speeds.     

Partial nitrification to nitrite using low dissolved oxygen concentration as the main selection factor

Partial nitrification to nitrite (nitritation) can be achieved in a continuous process without sludge retention by wash out of nitrite oxidising bacteria (NOB) while retaining ammonia oxidising bacteria (AOB), at elevated temperatures (the SHARON process) and, as demonstrated in this paper, also at low dissolved oxygen (DO) concentrations. Enriched AOB was attained at a low DO concentration (0.4 mg l⁻¹) and a dilution rate of 0.42 day⁻¹ in a continuous process. A higher oxygen affinity of AOB compared to NOB seemed critical to achieving this. This was verified by determining the oxygen half saturation constant, K _o, with similar oxygen mass transfer resistances for enriched AOB and NOB as 0.033 ± 0.003 mg l⁻¹ and 0.43 ± 0.08 mg l⁻¹, respectively. However, the extent of nitritation attained was found to be highly sensitive to process upsets.     

Metabolic readjustment in juvenile South African abalone (Haliotis midae) acclimated to combinations of temperature and dissolved oxygen levels

When ectotherms are exposed to high temperatures, cellular oxygen demand increases. When oxygen demand exceeds oxygen availability, animals may experience functional hypoxia regardless of environmental oxygen levels.     

Cofacial heme binding is linked to dimerization by a bacterial heme transport protein

Campylobacter jejuni is a leading bacterial cause of food-borne illness in the developed world. Like most pathogens, C. jejuni requires iron that must be acquired from the host environment. Although the iron preference of the food-borne pathogen C. jejuni is not established, this organism possesses heme transport systems to acquire iron. ChaN is an iron-regulated lipoprotein from C. jejuni proposed to be associated with ChaR, an outer-membrane receptor. Mutation of PhuW, a ChaN orthologue in Pseudomonas aeruginosa, compromises growth on heme as a sole iron source. The crystal structure of ChaN, determined to 1.9 A resolution reveals that ChaN is comprised of a large parallel beta-sheet with flanking alpha-helices and a smaller domain consisting of alpha-helices. Unexpectedly, two cofacial heme groups ( approximately 3.5 A apart with an inter-iron distance of 4.4 A) bind in a pocket formed by a dimer of ChaN monomers. Each heme iron is coordinated by a single tyrosine from one monomer, and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Sequence analyses reveal that these residues are conserved among ChaN homologues from diverse bacterial origins. Electronic absorption and electron paramagnetic resonance (EPR) spectroscopy are consistent with heme binding through tyrosine coordination by ChaN in solution yielding a high-spin heme iron structure in a pH-dependent equilibrium with a low-spin species. Analytical ultracentrifugation demonstrates that apo-ChaN is predominantly monomeric and that dimerization occurs with heme binding such that the stability constant for dimer formation increases by 60-fold.     

Liposomal heme as oxygen carrier under semi-physiological conditions orientation study of heme embedded in a phospholipid bilayer by an electrooptical method

The meso-tetra(α,α,α,α(o-pivalamidophenyl))porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37°C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.     

Enhanced Production of carboxymethylcellulase by a marine bacterium,Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Bacillus velezensis A-68 at a flask scale have been previously reported. In this study, the parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase. The optimal agitation speed and aeration rate for cell growth of B. velezensis A-68 were 323 rpm and 1.46 vvm in a 7 L bioreactor, whereas those for the production of CMCase were 380 rpm and 0.54 vvm, respectively. The analysis of variance (ANOVA) implied that the highly significant factor for cell growth was the aeration rate, whereas that for the production of CMCase was the agitation speed. The optimal inner pressures for cell growth and the production of CMCase by B. velezensis A-68 in a 100 L bioreactor were 0.00 and 0.04 MPa, respectively. The maximal production of CMCase in a 100 L bioreactor under optimized conditions using rice hulls was 108.1 U/ml, which was 1.8 times higher than that at a flask scale under previously optimized conditions.     

Differential survival of juvenile sockeye and coho salmon exposed to low dissolved oxygen during winter

Juvenile sockeye salmon (43–78 mm) survived 100% for 24 h in cages in ice–covered Black Lake, Alaska at oxygen saturations >65% (9 mg l^–1), but only 45% at 24% saturation (3·0–3·3 mg l^–1) and none at <17% saturation (2·3 mg l^–1). All juvenile coho (50–120 mm) survived 100% for 24 h down to 21% oxygen saturation (3·1 mg l^–1), and all 50 coho survived 4–5 days at 23–24% saturation (3·2–3·3 mg l^–1).     

Invasion success of a freshwater fish corresponds to low dissolved oxygen and diminished riparian integrity

Biological Invasions - Invasions are a common occurrence in many ecosystems but predicting the establishment and impacts of the invader can be difficult. Understanding how and why invasion...     

Liposomal heme as oxygen carrier under semi-physiological conditions. Orientation study of heme embedded in a phospholipid bilayer by an electrooptical method

The meso-tetra(alpha,alpha,alpha,alpha(o-pivalamidophenyl]porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37 degrees C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.     

Phenotypic convergence in a natural Daphnia population acclimated to low temperature

Fluidity of a given membrane decreases at lower ambient temperatures, whereas it rises at increasing temperatures, which is achieved through changes in membrane lipid composition. In consistence with homeoviscous adaptation theory, lower temperatures result in increased tissue concentrations of polyunsaturated fatty acids (PUFAs) in Daphnia magna, suggesting a higher PUFA requirement at lower temperatures. However, so far homeoviscous adaptation has been suggested for single or geographically separated Daphnia genotypes only. Here, we investigated changes in relative fatty acid (FA) tissue concentrations in response to a lower temperature (15°C) within a D. magna population. We determined juvenile growth rates (JGR) and FA patterns of 14 genotypes that were grown on Chlamydomonas klinobasis at 15°C and 20°C. We report significant differences of JGR and the relative body content of various FAs between genotypes at either temperature and between temperatures. Based on slopes of reaction norms, we found genotype‐specific changes in FA profiles between temperatures suggesting that genotypes have different strategies to cope with changing temperatures. In a hierarchical clustering analysis, we grouped genotypes according to differences in direction and magnitude of changes in relative FA content, which resulted in three clusters of genotypes following different patterns of changes in FA composition. These patterns suggest a lower importance of the PUFA eicosapentaenoic acid (EPA, C20:5ω3) than previously assumed. We calculated an unsaturation index (UI) as a proxy for membrane fluidity at 15°C, and we neither found significant differences for this UI nor for fitness, measured as JGR, between the three genotype clusters. We conclude that these three genotype clusters represent different physiological solutions to temperature changes by altering the relative share of different FAs, but that their phenotypes converge with respect to membrane fluidity and JGR. These clusters will be subjected to different degrees of PUFA limitation when sharing the same diet.