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.     

Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions.

The effciency of denitrification, or anaerobic respiration, in Pseudomonas denitrificans was investigated, using growth yield as an index. Glutamate was mainly used as the sole source of energy and carbon. In batch culture, the growth yield per mole of electrons transported through the respiratory system under denitrifying conditions was about half that under aerobic conditions. Similar figures were also obtained in chemostat cultures under glutamate-limited conditions. The decrease in growth yield under denitrifying conditions could be due to the restriction of phosphorylation associated with nitrate reduction to nitrogen gas.     

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.     

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.     

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.     

Bacterial oxidation of sulphide under denitrifying conditions

Anoxic H2S oxidation under denitrifying conditions produced sulphur and sulphate in almost equal proportions by an isolated Thiobacillus denitrificans. Under nitrate reducing conditions the rate of sulphide oxidation was approximately 0.9 g sulphide/g biomass h. Nitrate was reduced to nitrite and accumulated during sulphide oxidation. Above 100 mg nitrite/l, the sulphide oxidation rate declined and at 500 mg/l it was totally arrested. The optimum pH for the anoxic sulphide oxidation was around 7.5. Concentrations of sulphate 1500 mg/l and acetate 400 mg/l had no effect on anoxic sulphide oxidation.     

The genome sequence of the obligately chemolithoautotrophic, facultatively anaerobic bacterium Thiobacillus denitrificans

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, beta-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including 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. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by beta-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.     

In vivo 13C NMR analysis of acetate metabolism in Thiobacillus versutus under denitrifying conditions

The disappearance of 2-¹³C-acetate and the subsequent incorporation of label into cellular metabolites were followed in denitrifying cells of Thiobacillus versutus by ¹³C NMR spectroscopy. In cells grown under acetate-limitation, the specific rate of consumption was idependent of the density of the cell suspension. An isotopic steady state was reached within 30 min if sufficient substrate was added to the cell suspension. In cells grown under nitrate-limitation, the consumption of 2-¹³C-acetate proceeded at a significantly lower rate. The decrease and final disappearance of 2-¹³C-acetate were accompanied by incorporation of ¹³C into glutamate, glutamine, and by the release of labeled HCO ₃ ^– and CO₂. The appearance of a broad resonance being the methyl endgroup of poly-3-hydroxybutyrate (PHB) was indicative for PHB mobilization during the incubation. The sequence of label incorporation and the distribution among the various carbon nuclei were consistent with the operation of the tricarboxylic acid cycle.     

Differential isotopic fractionation during Cr(VI) reduction by an aquifer-derived bacterium under aerobic versus denitrifying conditions

We studied Cr isotopic fractionation during Cr(VI) reduction by Pseudomonas stutzeri strain RCH2. Despite the fact that strain RCH2 reduces Cr(VI) cometabolically under both aerobic and denitrifying conditions and at similar specific rates, fractionation was markedly different under these two conditions (ε was ～2‰ aerobically and ～0.4‰ under denitrifying conditions).     

Production and consumption of nitric oxide by denitrifying bacteria under anaerobic and aerobic conditions

Abstract: Pseudomonas aeruginosa, P. stutzeri and Azospirillum brasilense showed highest NO production rates and NO consumption rate constants when anaerobically grown cells were tested under anaerobic conditions. Aerobic assay conditions resulted in 20–75-fold lower NO production rates. NO consumption rate constants, however, decreased by less than a factor of four. NO consumption activity was observed even in aerobically grown P. aeruginosa , provided the assay was done under anaerobic conditions. Obviously, NO consumption was less O₂-sensitive than NO production so that compensation between production and consumption occurred at lower NO mixing ratios under aerobic than under anaerobic conditions.     

Phosphate removal under denitrifying conditions by Brachymonas sp. strain P12 and Paracoccus denitrificans PP15

In this study, we used the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12 to investigate the enhanced biologic phosphorus-removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation can enhance PHB formation efficiency and that PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. strain P12 for PHB, phosphorus, and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth is divided into two main phases: the early log phase, in which acetate and glycogen are consumed to supply enough energy and reducing power for PHB formation and cell growth (phosphorus assimilation), and the late log phase, which ends the simultaneous degradation of PHB and remaining acetate for polyphosphate accumulation. Glycogenolysis plays a significant role in the alternate responses between PHB formation and phosphorus uptake under anoxic or aerobic conditions. After the application of the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12, aerobic cultivation increases the level of PHB production, and anoxic cultivation further increases phosphorus uptake.     

Biodegradation of free phytol by bacterial communities isolated from marine sediments under aerobic and denitrifying conditions

Biodegradation of (E)-phytol [3,7,11, 15-tetramethylhexadec-2(E)-en-1-ol] by two bacterial communities isolated from recent marine sediments under aerobic and denitrifying conditions was studied at 20 degrees C. This isoprenoid alcohol is metabolized efficiently by these two bacterial communities via 6,10, 14-trimethylpentadecan-2-one and (E)-phytenic acid. The first step in both aerobic and anaerobic bacterial degradation of (E)-phytol involves the transient production of (E)-phytenal, which in turn can be abiotically converted to 6,10,14-trimethylpentadecan-2-one. Most of the isoprenoid metabolites identified in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations. Since (E)-phytenal is less sensitive to abiotic degradation at the temperature of the sediments (15 degrees C), the major part of (E)-phytol appeared to be biodegraded in situ via (E)-phytenic acid. (Z)- and (E)-phytenic acids are present in particularly large quantities in the upper section of the core, and their concentrations quickly decrease with depth in the core. This degradation (which takes place without significant production of phytanic acid) is attributed to the involvement of alternating beta-decarboxymethylation and beta-oxidation reaction sequences induced by denitrifiers. Despite the low nitrate concentration of marine sediments, denitrifying bacteria seem to play a significant role in the mineralization of (E)-phytol.     

Inhibition of bacteriochlorophyll synthesis in Rhodobacter sphaeroides subsp. denitrificans grown in light under denitrifying conditions.

The inclusion of nitrate or nitrite in cultures of Rhodobacter spaeroides subsp. denitrificans grown heterotrophically in light depressed the formation of bacteriochlorophyll a. The pigment biosynthesis was inhibited at the stage of the reduction of chlorophyllide (chlorin) to bacteriochlorophyllide (tetrahydroporphyrin) since 3-hydroxyethylchlorophyllide a accumulated in the culture medium. The addition of exogenous 5-aminolevulinic acid to these cultures resulted in a complete restoration of bacteriochlorophyll synthesis accompanied by the accumulation of 3-vinylbacteriopheophorbide. This indicates that under these conditions bacteriochlorophyll was formed via an alternative route, in which the reduction of chlorins to tetrahydroporphyrins precedes modifications of the C-3 side chain. The multiple forms of 5-aminolevulinic acid synthase were purified from cells grown with and without nitrate. Antibodies against these proteins were raised in rabbits and used in enzyme-linked immunosorbent assays for various forms of 5-aminolevulinic acid synthase. In denitrifying cells, the amount and activity of fraction I of the enzyme was reduced by approximately 40 and 30%, respectively. Partly active enzymes from both types of cells were activated by cystine trisulfide.     

Biodegradation of atrazine under denitrifying conditions

Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by ¹⁴CO₂ evolution. Denitrification was confirmed by detection of ¹⁵N₂ in headspace samples of K¹⁵NO₃-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [¹⁴C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media. Received: 3 September 1997 / Received revision: 4 December 1997 / Accepted: 2 January 1998     

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min^-1·mg^-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min^-1·mg^-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a R_f value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg²⁺ and cysteine.Abbreviations TCA Tricarboxylic acid - ICL isocitrate lyase - MS malate synthase - FPLC fast protein liquid chromatography - maximum specific oxygen consumption rate