Presence, distribution, and diversity of iron-oxidizing bacteria at a landfill leachate-impacted groundwater surface water interface

We examined the presence of iron-oxidizing bacteria (IOB) at a groundwater surface water interface (GSI) impacted by reduced groundwater originating as leachate from an upgradient landfill. IOB enrichments and quantifications were obtained, at high vertical resolution, by an iron/oxygen opposing gradient cultivation method. The depth-resolved soil distribution profiles of water content, Fe²⁺, and total Fe indicated sharp gradients within the top 10 cm sediments of the GSI, where the IOB density was the highest. In addition, the vertical distribution of iron-reducing bacteria at the same sampling site mirrored the IOB distribution. Clone libraries from two separate IOB enrichments indicated a stratified IOB community with clear differences at short vertical distances. Alpha- and Betaproteobacteria were the dominant phylotypes. Clones from the near-surface sediment (1–2 cm below ground surface) were dominated by members of the Bradyrhizobiaceae and Comamonadaceae ; clones from the deeper sediments were phylogenetically more diverse, dominated by members of the Rhodocyclaceae . The iron deposition profiles indicated that active iron oxidation occurred only within the near-to-surface GSI sediments. The match between the iron deposition profiles and the IOB abundance profiles strongly hints at the contribution of the IOB community to Fe oxidation in this Fe-rich GSI ecosystem.     

Consortial N2 fixation: a strategy for meeting nitrogen requirements of marine and terrestrial cyanobacterial mats

Abstract: Four microbial mat-forming, non-axenic, strains of the non-heterocystous, filamentous, cyanobacterial genus Microcoleus were maintained in culture and examined for the ability to fix atmospheric nitrogen (N₂). Each was tested for nitrogenase activity using the acetylene reduction assay (ARA) and for the presence of the dinitrogenase reductase gene ( nifH ), an essential gene for N₂ fixation, using the polymerase chain reaction (PCR). The Microcoleus spp. cultures were incapable of growth without an exogenous nitrogen source and never exhibited nitrogenase activity. Attempts to amplify a 360-bp segment of the nifH gene using DNA purified from the cyanobacterial cultures did not produce any cyanobacteria-specific nifH sequences. However, several non-cyanobacterial homologous nifH sequences were obtained. Phylogenetic analysis showed these sequences to be most similar to sequences from heterotrophic bacteria isolated from a marine microbial mat in Tomales Bay (California, USA), and bulk DNA extracted from a cryptobiotic soil crust in Moab (Utah, USA). Microcoleus spp. dominated the biomass of both systems. Cyanobacteria-specific 16S rDNA sequences obtained from the cultured cyanobacterial strains demonstrate that the lack of cyanobacteria-specific nifH sequences was not due to inefficiency of extracting Microcoleus DNA. Hence, both the growth and genetic data indicate that, contrary to earlier reports, Microcoleus spp. appear incapable of fixing N₂ because they lack at least one of the requisite genes for this process. Furthermore, our study suggests epiphytic N₂-fixing bacteria form a diazotrophic consortium with these Microcoleus spp. and are likely key sources of fixed N₂ generated within soil crusts and marine microbial mats.     

The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4°C

The development of the microflora of smoked pork loin and frankfurter sausage was followed during storage in vacuum, N₂ and CO₂ atmospheres at 4°C. The total aerobic count on the smoked pork loin reached 10⁷ organisms/g after 37 d in vacuum, 43 d in N₂ and 49 d in CO₂. The corresponding value for the sausage was 77 d in vacuum, while the growth stopped at 6 times 10⁴ organisms/g after 98 d in N₂, and at 4 times 10² organisms/g after 48 d in CO².
The predominant organisms on the fresh products were Bacillus spp., coryneform bacteria, Flavobacterium spp. and Pseudomonas spp.
At the end of the storage time the microflora on both products in the three gas atmospheres, consisted mainly of Lactobacillus spp. and two large groups of organisms that could not be identified as any described genus. Some of the unidentified strains could be classified as a Lactobacillus sp. after subsequent subculturing on laboratory media.
The numbers of Lactobacillus spp. at the end of storage decreased in the order, CO₂ > N₂ > vacuum. Lactobacillus viridescens generally constituted a substantial part of the Lactobacillus flora (5–72%). On the sausages two large uniform groups of unidentifiable homofermentative Lactobacillus spp. were also found.     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Effect of oxygen tension, salinity, temperature and organic matter concentration on the growth and nitrifying activity of an estuarine strain of Nitrosomonas

Abstract The effects of O₂ tension, temperature, salt concentration and organic matter concentration on the growth and nitrifying activity of Nitrosomonas N₃ isolated from Tay Estuary sediments have been investigated. Chemostat-grown cultures were able to grow and nitrify at dissolved O₂ concentrations as low as 0.1 mg O₂· 1⁻¹ (cell population densities were 15% of those obtained in fully aerated cultures). This bacterium was sensitive to reduced temperatures as chemostat-grown cultures washed out at growth temperatures below 15°C, at dilution rates > 0.025 · h⁻¹. Batch-grown cultures of Nitrosomonas N₃ were used to study the effects of NaCl and complex organic matter concentration on nitrifying activity. Maximum rates of NH⁺₄ oxidation were recorded at NaCl concentrations of 1% w/v, whilst tryptone soya broth (TSB), nutrient broth (NB), yeast extract broth (YEB) and peptone were inhibitory at concentrations > 10 mg · 1⁻¹.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Inhibition of nitrous oxide reduction by a component of Hamilton Harbour sediment

Abstract: A component of Hamilton Harbour sediment prevented nitrous oxide (N₂O) reduction in denitrification assays with a mixed population of endogenous bacteria and a pure culture (HH1) isolated from the sediment. A 5% (v/v) concentration of sediment in nutrient broth caused near maximum inhibition of N₂O reduction. Sediment taken from a site closer to pollution sources (Site 906) was twice as inhibitory (as measured by N₂O accumulation) as sediment from Site 910, further from pollution sources. N₂O persistence was associated with the particulate sediment fraction only. Several heavy metals were tested at in situ concentrations, and ionic cadmium (Cd) and chromium (Cr) caused N₂O accumulation. Ashed sediment did not cause N₂O accumulation, but did decrease initial nitrate reduction rates with HH1.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species

Cells of Geobacter metallireducens , Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N₂-dependent growth, the first anaerobically with Fe(III) as the electron acceptor, and the latter three species microaerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N₂ under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5 ± 5.9 nmol C₂H₄ produced min⁻¹ mg⁻¹ cell protein) while growing with Fe(III) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum , indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum , fix atmospheric dinitrogen.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.     

Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.     

Ecophysiology and the energetic benefit of mixotrophic Fe(II) oxidation by various strains of nitrate-reducing bacteria

In order to assess the importance of nitrate-dependent Fe(II) oxidation and its impact on the growth physiology of dominant Fe oxidizers, we counted these bacteria in freshwater lake sediments and studied their growth physiology. Most probable number counts of nitrate-reducing Fe(II)-oxidizing bacteria in the sediment of Lake Constance, a freshwater lake in Southern Germany, yielded about 10⁵ cells mL⁻¹ of the total heterotrophic nitrate-reducing bacteria, with about 1% (10³ cells mL⁻¹) of nitrate-reducing Fe(II) oxidizers. We investigated the growth physiology of Acidovorax sp. strain BoFeN1, a dominant nitrate-reducing mixotrophic Fe(II) oxidizer isolated from this sediment. Strain BoFeN1 uses several organic compounds (but no sugars) as substrates for nitrate reduction. It also reduces nitrite, dinitrogen monoxide, and O₂, but cannot reduce Fe(III). Growth experiments with cultures amended either with acetate plus Fe(II) or with acetate alone demonstrated that the simultaneous oxidation of Fe(II) and acetate enhanced growth yields with acetate alone (12.5 g dry mass mol⁻¹ acetate) by about 1.4 g dry mass mol⁻¹ Fe(II). Also, pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans strains can oxidize Fe(II) with nitrate, whereas Pseudomonas fluorescens and Thiobacillus denitrificans strains did not. Our study demonstrates that nitrate-dependent Fe(II) oxidation contributes to the energy metabolism of these bacteria, and that nitrate-dependent Fe(II) oxidation can essentially contribute to anaerobic iron cycling.     

Protective mechanisms of nitrogenase against oxygen excess and partially-reduced oxygen intermediates

Diazotrophic systems have developed a number of strategies to protect nitrogenase (N₂ase; EC 1.18.6.1) from O₂ excess and active-oxygen species (AOS). Protection against O₂ excess is given by biochemical modifications of N₂ase, increased rates of low-efficiency respiration, temporal segregation of N₂ fixation and photosynthesis, physical barriers to O₂ diffusion, and hemoglobins. On the other hand, AOS may originate from oxidation of N₂ase components, ferredoxins, flavodoxins and hemoglobins; interaction among the AOS themselves, or between H₂O₂ and hemoglobins; and during reactions catalyzed by hydrogenase (EC 1.18.99.1), xanthine oxidase (EC 1.1.3.22) and uricase (EC 1.7.3.3). Active-oxygen species are scavenged enzymatically [superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6). peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11)] or through non-enzymic reaction with low-molecular-weight compounds (ascorbate, α-tocopherol, glutathione).     

The distribution of denitrifying bacteria in soils monitored by DNA-probing

Abstract DNA probes of dissimilatory NO₂⁻- and N₂O-reductases have been used to screen for the distribution of denitrifying bacteria in soils. In control experiments with known organisms, denitrifiers gave positive DNA-DNA hybridization signals in the dot-blot experiments whereas non-denitrifiers did not hybridize in almost all cases. Bacteria from soil of our institute garden were isolated, grown up and assayed for denitrification activity and for DNA-hybridizations with both gene probes. A correlation of about 75% was obtained between activities and signals. The remainder of the isolates gave a weak signal in the dot blots and only half of them expressed denitrification activities in the cultures. The confidence of about 75% was good enough to assay for the distribution of denitrifying bacteria in five different soil-types of the Düsseldorf area. In all cases, the percentage of bacteria which gave no hybridization signal was high in the plant-free, bulk soil, whereas bacteria with strong or very strong signals favourably associated with the roots of plants isolated from the different soils. The result that denitrifying bacteria predominantly occur at the surface of roots was obtained when the samples were taken both in June and November. The probes for either NO₂⁻- or N₂O-reductase gave essentially the same results. For comparison, a gene probe for nitrogenase was included in this investigation. N₂-fixing bacteria showed a tendency to associate with the roots of plants in some but not in all soils. The overall number of bacteria was much higher at the roots of the plants than in the bulk soil in any of the samples assayed in the present investigation.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Associative cellulolysis and N2 fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum: the effects of ammonium-N on these processes

Mixed cultures of the cellulolytic fungus Trichoderma harzianum with the anaerobic diazotroph Clostridium butyricum were shown to co-operatively degrade cellulose and utilize the degradation products for N₂ fixation. Cellulose degradation and N₂ fixation were stimulated by small (0.1 mg/ml) additions of (NH₄)₂SO₄. The (NH₄₂SO₄ stimulates cellulolysis thereby increasing the supply of cellulose degradation products to the diazotroph. In aerobic environments the anaerobe depends on the respiration of the aerobe to create anaerobic microsites. The N source increased O₂ uptake by the fungus increasing the number of sites suitable for the development of the anaerobe. Stimulation in the growth of T. harzianum by (NH₄₂SO₄ resulted in increased growth and N₂ fixation by Cl. butyricum.     

Reduction of nitrous oxide by a soil Cytophaga in the presence of acetylene and sulfide

Abstract A denitrifying Cytophaga was isolated from soil enriched by anaerobic incubation with glucose, sulfide (S²⁻), nitrous oxide (N₂O), and acetylene (C₂H₂). Such soil enrichments and pure cultures of the isolated Cytophaga reduced N₂O rapidly even in the presence of a normally inhibitory concentration of C₂H₂ (4 kPa) providing S²⁻ was present (8 μmol/g soil or 0.4 μmol/ml culture). Since C₂H₂ inhibition of the reduction of N₂O is used as a tool in the assay of denitrification, the presence in large numbers of such a Cytophaga may influence the effectiveness of this assay especially in sulfidic environments.     

自然微生物挂膜处理水产养殖废水的效果及微生物群落分析

以弹性填料和流化床填料为硝化反应的生物挂膜材料, 聚羟基丁酸/戊酸共聚酯(PHBV)为反硝化反应的碳源和生物膜载体, 通过微生物自然挂膜处理低C/N比水产养殖废水, 去除水体中的氨氮、亚硝酸盐氮及总氮。应用Miseq高通量测序技术对生物膜的微生物群落组成和结构进行分析。结果表明: 温度25—30℃, 该处理系统首次挂膜成功需要4周, 启动后运行稳定, 对2种不同来源和氮污染程度的养殖废水均有较好的脱氮效果, 氨氮、亚硝酸盐氮及总氮的去除率均在90%以上。硝化生物膜(a)的优势菌分别归属变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)。反硝化生物膜(b)微生物群落的多样性指数和丰度指数均远大于前者, 主要为变形菌门、厚壁菌门、拟杆菌门、螺旋体门(Spirochaetae)及绿菌门(Chlorobi)。其中, 归属于变形菌门β-变形菌纲(Betaproteobacteria)的丛毛单胞菌科(Comamonadaceae)和红环菌科(Rhodocyclaceae)在2种生物膜中占比均较高。由于所处环境(载体, 碳源、溶氧等)不同, 在属分类水平上, 2种生物膜的细菌群落结构表现出明显差异。生物膜a中属的种类仅为b的三分之二, 相对丰度>0.5%的优势菌属, a为8个, b为18个。其中, 隶属丛毛单胞菌科和红环菌科未知属的优势种群分别占到a、b总序列数的56.67%和45.51%。磁螺菌属(Magnetospirillum)和硝化螺菌属(Nitrospira)是a中特有的优势功能菌群, 梭菌属(Clostridium)、动胶菌属(Zoogloea)、管道杆菌属(Cloacibacterium)、脱硫弧菌属(Desulfovibrio)等具有反硝化功能的菌群为b的优势菌属。     

Nitrogen-fixing legumes and silvigenesis in a rain forest in French Guiana: a taxonomic and ecological approach

The nodulation status and nodule morphology of 62 taxa of Leguminosae in a rain forest in French Guiana are reported according to the taxonomy of the family. The N₂-fixing species are then fitted into 'functional groups' according to their behaviour towards illumination, in order to evaluate their importance in the global dynamics of the stand. The results showed that 67% of the observed species were nodulated (50, 71 and 77% of the Caesalpiniaceae, Mimosaceae and Papilionaceae, respectively). In the Caesalpiniaceae, nodule-like structures were reported in the genus Crudia and in the species Senna quinquangulata , although this needs to be confirmed. All the nodules studied in this subfamily were astragaloid and mucunoid. In the Mimosaceae, the ability of a new genus ( Balizia ) to form nodules was reported, as well as nodulation on aerial roots in Inga stipularis . The nodules studied were mainly mucunoid. In the Papilionaceae, nodulation on aerial roots in Poecilanthe hostmannii and on conventional roots of the genus Paramachaerium were reported for the first time. All types of nodular structures were found in this subfamily but the structures were quite uniform at the tribal level. These are consistent with suggestions that nodule morphology has a taxonomic value. Eight functional groups of N₂-fixing species are proposed, ranging from light dependance to shade tolerance. These results indicate the important role played by N₂-fixing species in the global dynamics of the stand and that N inputs by N₂ fixation were continuous along the gradient of energetic resources that characterizes the silvigenetic process. The interactions between the photosynthetic capacities of the species and the ability to fix N₂ in low light conditions are discussed.