Efficient removal of NH4+ accumulated during anaerobic treatment of distillery wastewater from Shochu making

Propionate and NH₄⁺ were accumulated in the effluent during anaerobic treatment of five-fold diluted distillery wastewater from shochu making. Propionate could be removed efficiently during biological denitrification by the addition of NO₃⁻ (4.2 g/l) to the anaerobically treated wastewater. At a hydraulic retention time of more than 2 h, a TOC removal efficiency of 90% could be achieved. The wastewater was then treated aerobically by biological nitrification. With a hydraulic retention time of more than 14 h the efficiency of reduction of NH₄⁺ could be maintained above 97%. In order to reduce the amount of NO₃⁻ addition necessary for the removal of propionate, simultaneous removal of propionate and NH₄⁺ was studied by recirculating the effluent from a nitrification process to a denitrification process using denitrification and nitrification reactors connected in series. At a recirculation ratio of 2, the amount of NO₃⁻ that had to be added was reduced to 0.3 g/l of anaerobically treated wastewater, which corresponds to 6.9% of the theoretical value. Under the same conditions except for the addition of NO₃⁻ at 1.0 g/l, TOC and BOD in the effluent from the nitrification were 23 and 5 mg/l respectively, which are sufficiently low to allow discharge into river water. Moreover, the NO₃⁻ concentration in the effluent decreased with increases in the recirculation ratio.     

Anaerobic degradation of aromatic compounds coupled to Fe(III) reduction by Ferroglobus placidus

Aromatic compounds are an important component of the organic matter in some of the anaerobic environments that hyperthermophilic microorganisms inhabit, but the potential for hyperthermophilic microorganisms to metabolize aromatic compounds has not been described previously. In this study, aromatic metabolism was investigated in the hyperthermophile Ferroglobus placidus . F. placidus grew at 85°C in anaerobic medium with a variety of aromatic compounds as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. Aromatic compounds supporting growth included benzoate, phenol, 4-hydroxybenzoate, benzaldehyde, p -hydroxybenzaldehyde and t -cinnamic acid (3-phenyl-2-propenoic acid). These aromatic compounds did not support growth when nitrate was provided as the electron acceptor, even though nitrate supports the growth of this organism with Fe(II) or H₂ as the electron donor. The stoichiometry of benzoate and phenol uptake and Fe(III) reduction indicated that F. placidus completely oxidized these aromatic compounds to carbon dioxide, with Fe(III) serving as the sole electron acceptor. This is the first example of an Archaea that can anaerobically oxidize an aromatic compound. These results also demonstrate for the first time that hyperthermophilic microorganisms can anaerobically oxidize aromatic compounds and suggest that hyperthermophiles may metabolize aromatic compounds in hot environments such as the deep hot subsurface and in marine and terrestrial hydrothermal zones in which Fe(III) is available as an electron acceptor.     

Carbohydrate oxidation coupled to Fe(III) reduction, a novel form of anaerobic metabolism

An isolate, designated GC-29, that could incompletely oxidize glucose to acetate and carbon dioxide with Fe(III) serving as the electron acceptor was recovered from freshwater sediments of the Potomac River, Maryland. This metabolism yielded energy to support cell growth. Strain GC-29 is a facultatively anaerobic, gram-negative motile rod which, in addition to glucose, also used sucrose, lactate, pyruvate, yeast extract, casamino acids or H2 as alternative electron donors for Fe(III) reduction. Stain GC-29 could reduce NO3(-), Mn(IV), U(VI), fumarate, malate, S2O3(2-), and colloidal S0 as well as the humics analog, 2,6-anthraquinone disulfonate. Analysis of the almost complete 16S rRNA sequence indicated that strain GC-29 belongs in the Shewanella genus in the epsilon subdivision of the Proteobacteria. The name Shewanella saccharophilia is proposed. Shewanella saccharophilia differs from previously described fermentative microorganisms that metabolize glucose with the reduction of Fe(III) because it transfers significantly more electron equivalents to Fe(III); acetate and carbon dioxide are the only products of glucose metabolism; energy is conserved from Fe(III) reduction; and glucose is not metabolized in the absence of Fe(III). The metabolism of organisms like S. saccharophilia may account for the fact that glucose is metabolized primarily to acetate and carbon dioxide in a variety of sediments in which Fe(III) reduction is the terminal electron accepting process.     

Perchlorate reduction by a mixed culture in an up-flow anaerobic fixed bed reactor

Wolinella succinogenes HAP-1 is a Gram-negative microaerophile which reduces perchlorate to chloride by the proposed pathway ClO₄ to ClO₃ to ClO₂ to Cl + O₂. A cost-effective perchlorate treatment process has been established using a consortium of facultative anaerobic organisms and W. succinogenes HAP-1. The mixed anaerobic bacterial culture containing W. succinogenes HAP-1 was examined for the ability to form a biofilm capable of perchlorate reduction. An up-flow anaerobic fixed bed reactor (UAFBR) was packed with diatomaceous earth pellets and operated at residence times of 1.17 and 0.46 h to insure a viable biofilm had attached to the diatomaceous earth pellets. Reduction rates of perchlorate to chloride in the UAFBR could be maintained at 1 g of perchlorate reduced h⁻¹ L⁻¹. Studies with the same bacterial consortium in continuously stirred tank reactors (CSTR) generally reduced 0.5–0.7 g of perchlorate h⁻¹. Viable cell counts were performed periodically on the diatomaceous earth pellets and demonstrated that the W. succinogenes HAP-1 population was maintained at 28–47% of the total microbial population. Scanning electron micrographs demonstrated that the external and internal surfaces of the diatomaceous pellets were densely colonized with microbial cells of multiple cell types. This is the first report of an anaerobic mixed culture forming a biofilm capable of perchlorate reduction. Received 22 May 1997/ Accepted in revised form 07 January 1998     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Studies of the reactivity of trolox with Mn3+/Fe3+ complexes by pulse radiolysis

The rates and mechanisms for the reactions between Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a synthetic analogue of α-tocopherol, Br₁⁻ and Mn³⁺-phosphate were determined under aerobic and anaerobic conditions by fast kinetic methods. In addition, the reaction between Fe(OH)₂⁺ and Trolox at pH 6.0 was shown not to occur under the conditions of the experiment, leading to a limiting rate for that reaction of k < 10³M⁻¹s⁻¹. These results are compared to studies of the reactivity of Trolox with HO₂/O₂⁻ and are discussed in light of the known antioxidant properties of vitamin E.     

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium

Late-exponential-phasePenicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 μmol/L (2.2±0.6 μmol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe³⁺ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified majorP. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the β-lactam production and intracellular iron level. Neither 150 nor 300 μmol/L extracellular Fe³⁺ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H₂O₂. Nevertheless, when iron was applied in the Fe^II oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.     

Anaerobic digestion of secondary residuals from an anaerobic bioreactor at a brewery to enhance bioenergy generation

Many beer breweries use high-rate anaerobic digestion (AD) systems to treat their soluble high-strength wastewater. Biogas from these AD systems is used to offset nonrenewable energy utilization in the brewery. With increasing nonrenewable energy costs, interest has mounted to also digest secondary residuals from the high-rate digester effluent, which consists of yeast cells, bacteria, methanogens, and small (hemi)cellulosic particles. Mesophilic (37 °C) and thermophilic (55 °C) lab-scale, low-rate continuously-stirred anaerobic digestion (CSAD) bioreactors were operated for 258 days by feeding secondary residuals at a volatile solids (VS) concentration of ∼40 g l⁻¹. At a hydraulic retention time (HRT) of 15 days and a VS loading rate of 2.7 g VS l⁻¹ day⁻¹, the mesophilic bioreactor showed an average specific volumetric biogas production rate of 0.88 l CH₄ l⁻¹ day⁻¹ and an effluent VS concentration of 22.2 g VS l⁻¹ (43.0% VS removal efficiency) while the thermophilic bioreactor displayed similar performances. The overall methane yield for both systems was 0.21 l CH₄ g⁻¹ VS fed and 0.47–0.48 l CH₄ g⁻¹ VS removed. A primary limitation of thermophilic digestion of this protein-rich waste is the inhibition of methanogens due to higher nondissociated (free) ammonia (NH₃) concentrations under similar total ammonium (NH₄ ⁺) concentrations at equilibrium. Since thermophilic AD did not result in advantageous methane production rates or yields, mesophilic AD was, therefore, superior in treating secondary residuals from high-rate AD effluent. An additional digester to convert secondary residuals to methane may increase the total biogas generation at the brewery by 8% compared to just conventional high-rate digestion of brewery wastewater alone. JIMB-2008: BioEnergy—Special issue.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Rhizosphere acidification and Fe3+ reduction in lupins and peas: Iron deficiency in lupins is not due to a poor ability to reduce Fe3+

While lupins suffer severely from Fe deficiency when grown on calcareous soils, field peas under the same conditions grow normally. This paper aimed to identify whether these differences were related to differences in either the pattern or capacity for rhizosphere acidification or Fe³⁺ reduction between these species. Two lupin species (Lupinus angustifolius, L. cosentinii) and field peas (Pisum sativum) were grown in solution culture for 5 weeks with both an adequate and a low supply of Fe. Plants were reliant on symbiotically fixed N. The extent of iron reduction was determined using the chelates TPTZ and BPDS. The pattern of reactions around roots was determined by placing roots in agar containing either bromocresol purple or TPTZ. The low supply of Fe decreased the growth of lupins by over 30% and induced severe chlorosis and necrosis. Growth of the peas was reduced by less than 15% and no symptoms appeared. All species acidified the solutions by about 1 pH unit regardless of the Fe treatment. The level of Fe³⁺ reduction was higher for all species grown with low Fe than with adequate Fe. Capacity for Fe³⁺ reduction was higher for all species grown with low Fe than with adequate Fe. Capacity for Fe³⁺ reduction was similar for all species. The pattern of acidification and reduction around roots was also similar between species. Thus it appears that the capacity of lupins to reduce Fe³⁺ in the rhizosphere is not the primary cause of Fe deficiency in lupins.     

In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area

Anaerobic oxidation of methane (AOM) and sulphate reduction were examined in sediment samples from a marine gas hydrate area (Hydrate Ridge, NE Pacific). The sediment contained high numbers of microbial consortia consisting of organisms that affiliate with methanogenic archaea and with sulphate-reducing bacteria. Sediment samples incubated under strictly anoxic conditions in defined mineral medium (salinity as in seawater) produced sulphide from sulphate if methane was added as the sole organic substrate. No sulphide production occurred in control experiments without methane. Methane-dependent sulphide production was fastest between 4 degree C and 16 degree C, the average rate with 0.1 MPa (approximately 1 atm) methane being 2.5 micro mol sulphide day(-1) and (g dry mass sediment)(-1). An increase of the methane pressure to 1.1 MPa (approximately 11 atm) resulted in a four to fivefold increase of the sulphide production rate. Quantitative measurements using a special anoxic incubation device without gas phase revealed continuous consumption of dissolved methane (from initially 3.2 to 0.7 mM) with simultaneous production of sulphide at a molar ratio of nearly 1:1. To test the response of the indigenous community to possible intermediates of AOM, molecular hydrogen, formate, acetate or methanol were added in the absence of methane; however, sulphide production from sulphate with any of these compounds was much slower than with methane. In the presence of methane, such additions neither stimulated nor inhibited sulphate reduction. Hence, the experiments did not provide evidence for one of these compounds acting as a free extracellular intermediate (intercellular shuttle) during AOM by the presently investigated consortia.     

Isolation of a multiheme protein with features of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizing enrichment culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V(max) and K(m) for hydrazine are 6.2 +/- 0.3 micromol/min.mg and 5.5 +/- 0.6 microM, respectively. Hydrazine (25 microM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 microM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of "Candidatus Kuenenia stuttgartiensis" (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.     

Fluorescent carbon nanoparticles obtained from charcoal via green methods and their application for sensing Fe3+ in an aqueous medium

Two green methods (microwave and hydrothermal) were employed for the preparation of water dispersible fluorescent carbon nanoparticles (CNPs) from activated charcoal. Microwave and hydrothermally synthesized carbon nanoparticles, (MW‐CNPs) and (HT‐CNPs), respectively were characterized by microscopic and spectroscopic techniques. A detailed study of their fluorescence characteristics was made. MW‐CNPs and HT‐CNPs were tested for metal ion selectivity in aqueous medium. MW‐CNPs showed selectivity for Fe³⁺ among the tested metal ions and important studies such as for interference, linear range and limit of detection were carried out. The application of MW‐CNPs for detection of Fe³⁺ in water was demonstrated.     

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na(+)-exchange reaction of the sodium pump unaltered and shifts its high-affinity ATP-binding site to a Na(+)-like form.

Inactivation of Na+/K(+)-ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb/Rb+ exchange, but leaves the 22Na+/Na(+)-exchange activity and the uncoupled ATP-supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K(+)-ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi-supported pathway (ouabain-enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP-supported pathway (ouabain-enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K(+)-ATPase. When eosin was used as a marker for the high-affinity ATP-binding site of the E1 conformation, formation of stable E'2.Co(NH3)4PO4 complex led to a shift in the high-affinity ATP-binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4-inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.     

Anaerobic energy-yielding reaction associated with transhydrogenation from glycerol 3-phosphate to fumarate by an Escherichia coli system.

A particulate subcellular fraction from Escherichia coli K-12 induced in anaerobic sn-glycerol 3-phosphate (G3P) dehydrogenase and fumarate reductase can catalyze under anaerobic conditions the transfer of hydrogens from G3P to fumarate, with attendant generation of high-energy phosphate. The phsophorylation process is more sensitive than the transhydrogenation process to inhibition by the detergent Triton X-100. The same is true with respect to sensitivity to sodium azide, carbonyl cyanide m-chlorophenylhydrazone and N,N'-dicyclohexylcarbodiimide. Such a preparation derived from cells with beta-galactoside permease can accumulate thiomethyl beta-D-galactoside anaerobically, and the accumulation can be stimulated twofold by adding G3P and fumarate. Mutants lacking the membrane-associated Mg2+-dependent adenosine triphosphatase cannot grow anaerobically on glycerol with fumarate as the hydrogen acceptor, although they can grow aerobically on glycerol alone.     

Biosorption of Fe3+ from aqueous solution by a bacterial dead Streptomyces rimosus biomass

The iron biosorption capacity of a Streptomyces rimosus biomass treated with NaOH was studied in batch mode. After pretreatment of biomass at the ambient temperature, optimum conditions of biosorption were found to be: a biomass particle size between 50 and 160 μm, an average saturation contact time of 4 h, a biomass concentration of 3 g/l and a stirring speed of 250 rpm. The equilibrium data could be fitted by Langmuir isotherm equation. Under these optimal conditions, 122 mg _Fe/g_biomass were fixed.     

A heterodimeric cytochrome c complex with a very low redox potential from an anaerobic ammonium-oxidizing enrichment culture

A dimeric cytochrome c with an apparent molecular mass of 25 kDa was isolated from an anammox bacterium, strain KSU-1, in a relatively large quantity. This protein was named the NaxLS complex. The spectrum of the oxidized form exhibited a peculiar Soret peak at 419 nm. The reduction of NaxLS was not complete even with the addition of excess dithionite, but was complete with titanium (III) citrate, indicating that the NaxLS complex has a very low redox potential. The genes encoding the two subunits, naxL and naxS, are adjacent on the genome. The deduced amino-acid sequences of the genes showed high identities with those of two genes encoding 'unknown proteins' in the genome of Candidatus Kuenenia stuttgartiensis, but had lower identities with other c-type heme proteins. The electron paramagnetic resonance spectra of NaxLS exhibited low-spin signals of two heme species in the range between g=2.6 and g=1.8, which strongly suggested an unusual His/Cys coordination. This unique coordination might account for the low redox potential of the hemes in NaxLS. NaxLS might participate in the transfer of low redox potential electrons in the intracellular anammoxosome compartment or the cytoplasm.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.