Anaerobic, Nitrate-Dependent Oxidation of U(IV) Oxide Minerals by the Chemolithoautotrophic Bacterium Thiobacillus denitrificans

Under anaerobic conditions and at circumneutral pH, cells of the widely distributed, obligate chemolithoautotrophic bacterium Thiobacillus denitrificans oxidatively dissolved synthetic and biogenic U(IV) oxides (uraninite) in nitrate-dependent fashion: U(IV) oxidation required the presence of nitrate and was strongly correlated with nitrate consumption. This is the first report of anaerobic U(IV) oxidation by an autotrophic bacterium.     

Development of a genetic system for the chemolithoautotrophic bacterium Thiobacillus denitrificans

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.     

Development of a Genetic Transformation System for an Alga-Lysing Bacterium

Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in Escherichia coli and Alteromonas sp. strain A28 was constructed by fusing pAS28 and E. coli vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10⁶ transformants per μg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28. This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.     

Anaerobic Production of Thiobacillus denitrificans for the Enzyme Rhodanese

A method for the anaerobic growth of Thiobacillus denitrificans in a 140-liter (total capacity) stainless-steel culture vessel is described. As a result of controlling the pH value of cultures, and of ensuring that certain essential nutrients were in excess, cell yields approaching 700 mg (dry weight) per liter were obtained. These were over threefold higher than the best yields hitherto reported. The average rhodanese content of the cells from four cultures was 176,000 units per gram (dry weight). Adenosine-5′-phosphosulfate reductase (average content, 238 units per gram dry weight) and adenylate kinase (average content, 15,300 units per gram, dry weight) were also present.     

Control of a Thiobacillus denitrificans bioreactor using machine vision

A PC-based machine vision system has been used to continuously monitor changes in biomass concentration and to control the undesirable production of colloidal elemental sulfer (a reactor upset condition due to an excessive concentration of inhibitory sulfide substrate) in a bioreactor containing Thiobacillus denitrificans. A field of view of a video camera was established which contained regions of different background lighting. Mean values of the distribution of red, green, and blue intensity components within corresponding regions of a digital image image captured from the camera were used to monitr color changes associated with changes in biomass concentration, and to determine if the reactor was in an upset condition. The ration of red to blue intensity components was an important parameter in detecting the formatin of an elemental sulfur precipitant. Using a stepper motor-driven pressure regulator, intelligent process control was performed by altering the hydrogen sulfide feed flow rate setpoint on the vision system measurements.     

Intermediates of denitrification in the chemoautotroph Thiobacillus denitrificans

Summary Intact cells obtained from Thiobacillus denitrificans grown autotrophically with thiosulfate as the oxidizable substrate and nitrate as the final electron acceptor catalyzed the reduction of nitrate, nitrite and nitric oxide stoichiometrically to nitrogen gas with the concomitant oxidation of thiosulfate. In addition, nitrous oxide was also capable of acting as the terminal oxidant of the respiratory chain with thiosulfate as the reductant. The anaerobic oxidation of thiosulfate by NO₃ ^-, NO, and N₂O was sensitive to the flavoprotein inhibitors, antimycin A or NHQNO, and cyanide or azide thus, implicating the participation of flavins, and cytochromes of b-, c-, and a-types in the denitrification process. The nitrite reductase system, however, was not markedly affected by the electron transport chain inhibitors. The experimental observations suggest that the dissimilatory nitrate reduction in the chemoautotroph T. denitrificans involves nitrite, nitric oxide, and nitrous oxide as theintermediates with nitrogen gas as the final reduction product.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - NHQNO 2-n-nonyl-4-hydroxyquinoline N-oxide     

Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans

From cell yields of Thiomicrospira denitrificans grown in the chemostat at different growth rates under anaerobic conditions a value of 1.4mm S₂O _inf3 ^sup= per g dry wt and per h could be calculated for maintenance energy requirements, and of 5.65 g dry wt per mole S₂O _inf3 ^sup= for the true growth yield.Cell yields of Thiomicrospira denitrificans appeared to be almost half of those of Thiobacillus denitrificans. Though in Thiobacillus denitrificans at D=0.03 h^-1 under anaerobic conditions a value was found of 11.60 g dry wt per mole of thiosulphate used for energetic purposes, a value of 5.72 g dry wt per mole of thiosulphate was found under comparable conditions in Thiomicrospira denitrificans. Under aerobic conditions at D=0.03 h^-1 values of 18.54 g dry wt per mole of thiosulphate were found in Thiobacillus denitrificans whereas Thiomicrospira denitrificans yielded only 9.38 g dry wt per mole of thiosulphate.As in Thiobacillus denitrificans anaerobic cell yields on sulphide were comparable to those on thiosulphate.Calculations have been made which indicate that the biosynthetic efficiency of Thiomicrospira denitrificans is lower than that of Thiobacillus denitrificans. This can only partly be explained by the absence of adenosine-phosphosulphate (APS) reductase.     

脱氮硫杆菌对硫酸盐还原菌生长的抑制作用

为了研究脱氮硫杆菌(Thiobacillus denitrificans,TDN)对硫酸盐还原菌(sulfate-reducing bacteria,SRB)生长的影响,将从污水中分离到的硫酸盐还原菌和从pH为2~3的酸性土壤中分离到的脱氮硫杆菌(TDN)用于含适当浓度硫酸盐和硝酸盐的模拟污水的处理,并测定单菌或混菌培养后系统中硫酸盐、硝酸盐浓度的变化以及硫化氢的产量。结果表明,在仅接种硫酸盐还原菌的培养系统中,硫酸盐和硝酸盐的含量分别降低4.8%和1.0%;而在同时接种脱氮硫杆菌和硫酸盐还原菌的系统中硫酸盐的含量升高了4.7%,但硝酸盐氮含量降低了25%,这一作用随着培养基中硝酸盐起始浓度的提高而得到加强。另外,混菌培养系统的硫化氢浓度比单一硫酸盐还原菌培养系统降低了65.93%。由此推断,在混菌培养系统中,脱氮硫杆菌通过其反硝化过程产生的代谢产物使硫酸盐还原菌的生长环境条件发生改变,从而抑制其生长并减少了硫化氢的产生。这对预防硫酸盐还原菌带来的不利影响提供了有效的措施。     

Some properties of the rhodanese system of Thiobacillus denitrificans

1. Rhodanese has been extracted from Thiobacillus denitrificans by ultrasonic disintegration of the cells. 2. Studies with Sephadex columns have shown that the enzyme aggregates, forming a tetramer. 3. The molecular weights of the monomer and of an enzymically active sub-unit one-quarter this size have been determined by gel filtration. 4. Higher-molecular-weight forms of rhodanese are broken down by mercaptoethanol to enzymically active fragments of mol.wt. 7000 and 2000 respectively. 5. It is suggested that these fragments are linked in vivo via disulphide bridges to form the monomer, which can then aggregate via further disulphide links. 6. The fragment of mol.wt. 7000 has been obtained in a substantially pure state. 7. Both disulphide and thiol groups are necessary for enzyme activity. 8. Similarities and differences existing between bacterial rhodanese, mammalian rhodanese and beta-mercaptopyruvate sulphurtransferase are discussed.     

Thermotolerance of adenylylsulfate reductase from Thiobacillus denitrificans

Abstract Adenylylsulfate (APS) and APS reductase are important in the energy-generating processes of sulfate-reducing bacteria and sulfur lithotrophs (phototrophs and nonphototrophs). APS reductase from an extremely thermophilic archaebacterial sulfate-reducer was recently shown to be thermophilic with optimal activity at 85°C (Speich and Truper (1988) J. Gen. Microbiol. 134, 1419–1425). APS reductase of Thiobacillus denitrificans , a mesophilic eubacterium, has biochemical and physical properties in common with the thermophilic enzyme and is also thermotolerant (up to 75°C). APS reductase and other enzymes of dissimilative inorganic sulfur metabolism may commonly be thermotolerant is mesophilic eubacteria; perhaps a vestige of their primordial significance.     

Assessment of the addition of Thiobacillus denitrificans and Thiomicrospira denitrificans to chemolithoautotrophic denitrifying bioreactors

The aim of the present study was to assess the impact of adding cultures of Thiobacillus denitrificans and Thiomicrospira denitrificans to two upflow anaerobic sludge bed (UASB) reactors: one inoculated with granular sludge and the other filled only with activated carbon (AC). The performances of the bioreactors and the changes in biomass were compared with a non-bioaugmented control UASB reactor inoculated with granular sludge. The reactors inoculated with granular sludge achieved efficiencies close to 90% in nitrate and thiosulfate removal for loading rates as high as 107 mmol-NO3 -/l per day and 68 mmol-S2O3 2-/l per day. Bioaugmentation with Tb. denitrificans and Tm. denitrificans did not enhance the efficiency compared to that achieved with non-bioaugmented granular sludge. The loading rates and efficiencies were 30-40% lower in the AC reactor. In all the reactors tested, Tb. denitrificans became the predominant species. The results strongly suggest that this bacterium was responsible for denitrification and sulfoxidation within the reactors. We additionally observed that granules partially lost their integrity during operation under chemolithoautotrophic conditions, suggesting limitations for long-term operation if bioaugmentation is applied in practice.     

Tetrathionate-Forming Thiosulfate Dehydrogenase from the Acidophilic,Chemolithoautotrophic Bacterium Acidithiobacillus ferrooxidans

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg⁻¹) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg⁻¹) observed at pH 2.5 and 50°C.     

Autotrophic denitrification by Thiobacillus denitrificans in a packed bed reactor

Summary An upflow packed bed reactor with lava stones as support for the microbial growth proved to be very useful for the denitrification of industrial waste water by Thiobacillus denitrificans. The application of the plug flow principle allowed higher concentrations of nitrate to be employed than in a stirred tank reactor because inhibitory concentrations of sulfate from thiosulfate oxidation built up only in the upper part of the column — if at all. In experiments with synthetic media nitrate solutions of different strength (NO ₃ ^– g/l: 1.8; 3.0; 4.3; 6.1) were tested, each at 5 different residence times (5; 3.3; 2.5; 2.0; 1.7 h). The combination of the two parameters which still allowed 95% denitrification was 3 g NO ₃ ^- /l and 2.5 h residence time; this corresponded to a volumetric nitrate loading of about 25 kg/m³·d. Higher nitrate loadings led to incomplete denitrification coupled with the occurence of nitrite in the outflow. Below the critical loading rate nitrite accumulated only in the lower part of the column and was then gradually reduced. Experiments with simulated middle active waste from processing nuclear fuel which contained numerous heavy metals yielded similar results. — Although pure inorganic media were fed into the reactor the microflora developing as a dense layer covering the lava stones consisted not only of T. denitrificans but also of heterotrophic denitrifiers, mainly Pseudomonas aeruginosa.     

Denitrification with Thiobacillus denitrificans in the ANANOX® process

Summary Thiobacillus denitrificans is a chemoautotrophic microorganism which is able to denitrify utilizing sulphides as electron donors. It has been found in the anoxic chamber of a ANANOX pilot plant, where the effluent from an anaerobic step is mixed with a clarified nitrified stream. Its presence resulted in a high denitrification rate, oxidizing sulphide to sulphate, and reducing nitrate to nitrogen gas.     

Genome Sequence of the Chemolithoautotrophic Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Nb-255

The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C₂ to C₄ metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C₆ molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.     

Aerobic oxidation of hydrogen sulfide by Thiobacillus denitrificans

It has been demonstrated that Thiobacillus denitrificans may be readily cultured aerobically in batch and continuous flow reactors on H(2)S(g) under sulfide limiting conditions. Under these conditions sulfide concentrations in the culture medium were less than 1muM resulting in very low concentrations of H(2)S in the reactor outlet gas. Biomass yield under aerobic conditions was much lower than previously reported for anaerobic conditions, presumably because of oxygen inhibition of growth. However, biomass yield was not affected by steady state oxygen concentration in the range of 45muM-150muM. Biomass yield was also observed to be essentially independent of specific growth rate in the range of 0.030-0.053 h(-1). Indicators of reactor upset were determined and recovery from upset conditions demonstrated. Maximum loading of the biomass for H(2)S oxidation under aerobic conditions was observed to be 15.1-20.9 mmol/h/g biomass which is much higher than previously reported for aerobic conditions. Other aspects of the stoichiometry of aerobic H(2)S oxidation are also reported.     

Proton translocation in intact cells of Thiobacillus denitrificans

Proton translocation assessed by the quinacrine fluorescence technique was compared with oxygen uptake during thiosulphate oxidation by cells of Thiobacillus denitrificans. The addition of thiosulphate to cell suspensions resulted in an outwardly directed proton translocation as reflected by an increased quinacrine fluorescence. Compared to the O₂ uptake activity, the proton translocating system was much more sensitive to proton conductors, other ionophores and inhibitors of electron transport. The results indicate that (a) the proton-translocation activity (membrane energization) is enhanced in aged cell suspensions, (b) intactness of the cytoplasmic membrane is essential for establishing a protonmotive force in cells, (c) the fluorescence increase and proton translocation are reversible processes, (d) inhibitors of electron transport may also act as proton conductors by altering the integrity of the cytoplasmic membrane.Abbreviations CCCP carbonyl cyanide m-chlorophenyl-hydrazone - DBP 2,4-dibromophenol - DNP 2,4-dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - PCP pentachlorophenol - TPB tetraphenyl boron - TTFA 1-[thenoyl-(2)]-3,3,3-trifluoracetone     

Sulfur metabolism in Thiobacillus denitrificans evidence for the presence of a sulfite reducatase activity.

The relatively high specific sulfite reductase activity of 25 mU/mg protein was found in extracts from Thiobacillus dentrificans. The absorption spectrum of the partially pruified enzyme was similar to the siroheme containing sulfite reductases from other sources. It is suggested that the T. denitrificans sulfite reductase may function during the oxidation of reduced sulfur compounds.     

脱氮硫杆菌特异引物/探针的设计和评价

自脱氮硫杆菌(Thiobacillus denitrificans)16S rRNA基因V3可变区中发现一条27 bp的特异序列, 以该序列为反向引物, 对高效同步脱硫反硝化系统污泥DNA进行了温度梯度PCR扩增和基因文库构建, 结果证实了该引物的高度专一性。应用该探针在去离子甲酰胺和NaCl的浓度分别为35%和100 mmol/L, 杂交/洗脱温度为48°C条件下对污泥样品杂交得到较好的阳性结果, 软件分析表明脱氮硫杆菌在污泥中约占15%。脱氮硫杆菌专一性引物/探针的提出, 将为不同生态环境中该种微生物的时空分布、结构动态以及实时定量等研究提供分子生物学工具。