Permeability of Nitrobacter agilis to Organic Compounds.

Ida, S. (Cornell University, Ithaca, N.Y.), and M. Alexander. Permeability of Nitrobacter agilis to organic compounds. J. Bacteriol. 90:151-156. 1965.-None of a variety of inorganic ions or organic compounds served as a sole energy source for the growth of Nitrobacter agilis, and the test substrates were not oxidized by either intact cells or extracts of the obligate chemoautotroph. The organic substances did not serve as sole carbon sources for the bacterium in a synthetic medium, and they failed to enhance the rate of nitrite oxidation. The organism was permeable to acetate and a number of other simple carbon compounds, however, and exogenously supplied acetate was converted to a number of products. On the basis of these findings, possible reasons are examined for the inability of the chemoautotroph to use exogenous organic compounds as energy or carbon sources.     

Chemolithoorganotrophic growth of Nitrosomonas europaea on fructose

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from CO(2) and all its energy and reductant for growth from the oxidation of NH(3) and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea. The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of CO(2). In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two- to threefold increase in optical density and protein content in 3 to 4 days. Studies with [(14)C]fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitol, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in CO(2)-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.     

Growth of Nitrobacter in the presence of organic matter

1. Culture filtrates of heterotrophic bacteria were tested for their stimulatory effect on nitrification of three strains of Nitrobacter.
2. Yeast extract-peptone solution, in which Pseudomonas fluorescens had grown, after removal of the cells was added to autotrophically growing cultures of Nitrobacter agilis; it caused a stimulated nitrite oxidation and growth of Nitrobacter agilis.
3. The degree of stimulation depended on: a) the proportion of the culture filtrate to the autotrophic medium; b) the composition of the complex medium in which Pseudomonas fluorescens had been grown; c) the time the heterotrophic bacterium had been grown in the complex medium.
4. The stimulatory effect was highest with Nitrobacter agilis, less with Nitrobacter winogradskyi and negligible with Nitrobacter K ₄.
5. It was possible to adapt nitrifying cells of Nitrobacter agilis to higher concentrations of yeast extract and peptone. After the nitrite had been completely oxidized the cell-N still increased up to 30% before growth stopped.

     

A PCR-MPN BASED QUANTITATIVE APPROACH TO ENUMERATE NITRIFYING BACTERIA IN ZEOPONIC SUBSTRATES

Self-sustaining, regenerative life-support systems are required for long duration missions to the Moon and Mars. Improved activity of nitrifying bacteria to convert NH₄⁺ to NO₃^- has been shown to promote plant growth in zeoponic substrates. Due to physiological characteristics, such as slow growth and low yield, nitrifying bacteria are not easily enumerated by traditional microbiological techniques. A method for rapid detection and enumeration of a commercial inoculum of nitrifying bacteria in a zeoponic substrate was developed using a polymerase chain reaction (PCR)-most probable number (MPN) approach. Samples from four-week laboratory incubation studies were processed to extract their total microbial community DNA and the sequences specific to 16s rRNA of Nitrobacter spp. were PCR amplified. The detection limit of the methodology was 2,000 Nitrobacter cells per assay. The quantitative assay demonstrated that the zeoponic substrate was capable of supporting 10⁵ to 10⁶ MPN Nitrobacter cells per gram of substrate. The PCR-MPN method can be an effective and rapid approach to enumerate nitrifying bacteria in zeoponic substrates.     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h^-1. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h^-1, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10⁵ and 12 × 10⁵ flagellates ml^-1 were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h^-1, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.     

The Nitrifying Bacteria in Soils from Rothamsted Classical Fields and Elsewhere

S ummary . Ammonia oxidizing autotrophic nitrifying bacteria in soil samples from 3 classical Rothamsted fields and elsewhere were counted by a modified 2-layered silica gel plating method. Counts ranged from 0-18000/g of dry soil, the maximum number being found in the Broadbalk farmyard manure plot. Nitrosomonas europaea was isolated in pure culture and detected only in soils treated with dung or other organic fertilizer. Nitrosocystis coccoides and Nitrosospira spp. were found in other soils. Nitrobacter spp. were present in many soils and of the 18 pure cultures isolated there seemed to be 3, or possibly 4, different colony types.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3)d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L(-1). Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosomonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662.     

Effects of Organic Acids on Heterotrophic Nitrification by Alcaligenes faecalis OKK17

Factors affecting heterotrophic nitrification by Alcaligenes faecalis OKK17, which was isolated from sewage sludge, were examined. Specific nitrifying activity increased as the pH increased up to 8.5. Most of the nitrogenous compounds (88%) in the culture supernatant were converted to hydroxylamine or nitrite at pH 9 but 87% of them remained as ammonium at pH 7. These results imply that the substrate for heterotrophic nitrification is ammonium and that the organism oxidizes ammonium to lower its toxic effect. Although the addition of acetate to a defined medium increased growth of the bacterium up to C/N = ca. 6, the accumulation of nitrification products almost paralleled the growth and the specific nitrifying activity decreased. Pyruvate and oxaloacetate increased the specific nitrifying activity six- to eightfold compared with the other organic acids examined, but the key enzyme activities in the glyoxylate cycle were not increased. Acetate, glyoxylate, and malonate did not increase the specific nitrifying activity, but they increased the enzyme activities. These results imply that the involvement of acetate metabolism in the heterotrophic nitrification is unlikely.     

新异养硝化菌Colloides sp.JZ1-1的鉴定及其硝化性能

从驯化后的活性污泥中筛分、诱变出一株性能较好的异养硝化菌JZ1-1.经形态及生理生化特性分析,鉴定菌株JZ1-1为胶样菌属(Colloides sp.).分别考察了碳源、C/N、pH、溶解氧、温度和铵态氮初始浓度对JZ1-1硝化性能的影响.结果表明:菌株对柠檬酸钠的利用较好;C/N为10 ～14、30℃、pH 6-9和转速150 r·min-1以上有利于铵态氮的降解;菌株对中高浓度铵态氮废水(100 mg·L-1≤铵态氮浓度≤500 mg·L-1)的降解效果显著.经5次继代培养,菌株的稳定性较好.     

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.     

Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems

The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. (15)N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [(15)N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered.     

Stimulation of Nitrobacter agilis by Biotin.

Krulwich, Terry A. (Goucher College, Baltimore, Md.), and Helen B. Funk. Stimulation of Nitrobacter agilis by biotin. J. Bacteriol. 90:729-733. 1965.-Addition of biotin to nitrite-mineral medium greatly stimulated the autotrophic growth of four strains of Nitrobacter agilis. Comparisons of cultures of the organisms grown in parallel at 30 C in nitrite medium and in the medium supplemented with 150 mmug of biotin per ml showed that the vitamin promoted: (i) 2- to 4-fold greater rates of utilization of nitrite, and (ii) 100- to 1,000-fold greater populations of cells per milliliter. Avidin specifically inhibited the biotin stimulation of nitrite utilization at an avidin-biotin ratio of 133:1. Incubation of the four strains of N. agilis at 37 C imposed a requirement for biotin that could be met by daily addition of 150 mmug of the vitamin per ml of medium. The stimulatory effects of the vitamin at 30 C suggest that in N. agilis the synthesis of biotin is rate-limiting for growth.     

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.     

Steady state and transient growth of autotrophic nitrifying bacteria

Growth of the autotrophic nitrifying bacteria Nitrosomonas europaea and Nitrobacter sp. was studied in continuous culture. Steady state growth kinetics of both organisms conformed with that predicted by chemostat theory, modified to account for maintenance energy requirement. Steady state data were used to calculate the maximum specific growth rate, the saturation constant for growth, the true growth yield and the maintenance coefficient. Transient growth was studied by imposing step changes in dilution rate. Step increases resulted in overshoots and oscillations in substrate concentration before establishment of a new steady state while step decreases in dilution rate were followed by monotonic changes in substrate concentration. The size of overshoots in substrate concentration following step increases in dilution rate was dependent on both the magnitude of the increase and of the dilution rate prior to the change.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR

Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results, the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at concentrations of a few micrograms per liter.

Five Pseudomonas aeruginosa strains were tested for the utilization of 47 low-molecular-weight compounds as their sole sources of carbon and energy for growth at a concentration of 2.5 g/liter. Of these compounds, 31 to 35 were consumed. Growth experiments in tap water at 15 degrees C were carried out with one particular strain (P1525) isolated from drinking water. This strain was tested for the utilization of 30 compounds supplied at a concentration of 25 microgram of C per liter. The growth rate (number of generations per hour) of strain P1525 in this tap water was approximately 0.005 h-1, and with 10 compounds it was larger than 0.03 h-1. An average yield of 6.2 x 10(9) colony-forming units per mg of C was obtained from the maximum colony counts (colony-forming units per milliliter). The average yield and maximum colony count of strain P1525 grown in tap water supplied with a mixture of 45 compounds, each at a concentration of 1 microgram of C per liter, enabled us to calculate that 28 compounds were utilized. Growth rates of two P. aeruginosa strains (including P1525) in various types of water at 15 degrees C were half of those of a fluorescent pseudomonad. The concentrations of assimilable organic carbon calculated from maximum colony counts and average yield values amounted to 0.1 to 0.7% of the total organic carbon concentrations in five types of tap water. The assimilable organic carbon percentages were about 10 times larger in river water and in water after ozonation.     

Chemolithotrophic haloalkaliphiles from soda lakes

This paper summarizes recent data on the occurrence and properties of lithotrophic prokaryotes found in extremely alkaline, saline (soda) lakes. Among the chemolithotrophs found in these lakes the obligately autotrophic sulfur-oxidizing bacteria were the dominant, most diverse group, best adapted to haloalkaline conditions. The culturable forms are represented by three new genera, Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira in the Gammaproteobacteria. Among them, the genus Thioalkalivibrio was most metabolically diverse, including denitrifying, thiocyanate-oxidizing and facultatively alkaliphilic species. Culturable methane-oxidizing populations in the soda lakes belong to the type I methanotroph group in the Gammaproteobacteria, mostly in the genus Methylomicrobium. The nitrifying bacteria in hyposaline soda lakes were represented by a new species Nitrobacter alkalicus (Alphaproteobacteria), and by an alkaliphilic subspecies of Nitrosomonas halophila (Betaproteobacteria). Both belonged to the low salt-tolerant alkaliphiles. The facultatively autotrophic haloalkaliphilic isolates able to grow with hydrogen as electron donor were identified as representatives of the alpha-3 subclass of the Proteobacteria (aerobic) and of the Natronolimnicola - Alkalispirillum group in the gammaproteobacteria (nitrate-reducing). While all chemolithotrophic isolates from soda lakes belong to the alkaliphiles with a pH optimum for growth around 10, only the sulfur-oxidizing group included species able to grow under hypersaline conditions. This indicates that carbon and nitrogen cycles in the hypersaline alkaline lakes might not be closed.