Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potential availability of anaerobic treatment with digester slurry of animal waste for the reclamation of acid mine water containing sulfate and heavy metals

The use of an anaerobic digester slurry of cattle waste for the reclamation of acid mine water was examined. When the digester slurry was mixed with acid mine water, anaerobic digestion, including sulfate reduction and methanogenesis, was enhanced. In the mixture of acid mine water and the digester slurry, sulfate reduction proceeded without diminishing methanogenesis. The digester slurry and its supernatant (SDF-sup) showed a significant capacity to act as a strong alkaline reagent, and the pH of the acid mine water was markedly elevated by the addition of the digester slurry of SDF-sup even at the low ratio of 1% (v/v). Precipitation of heavy metals in the acid mine water occurred as the pH was elevated by the addition of SDF-sup. When the digester slurry was added at the ratio of 5% (v/v) to acid mine water which had been pretreated with SDF-sup, the rate of sulfate reduction increased with increasing the concentration of sulfate in the mixture up to about 1,400 mg·l⁻¹. In acid mine water pretreated with SDF-sup and supplemented with the digester slurry at the ratio of 5% (v/v), the maximum amount of sulfate reduced within 20 d of incubation was about 1,000 mg·l⁻¹, and the maximum rate of sulfate reduction was about 120 mg SO₄²⁻·l⁻¹·d⁻¹.     

The gills as an important uptake route for the essential nutrient iron in freshwater rainbow trout Oncorhynchus mykiss

Short-term (3h) acquisition of iron (16 nmol ⁵⁹FeCl₃ l⁻¹) from oxic, alkaline fresh water was assessed in rainbow trout Oncorhynchus mykiss in the presence or absence of a range of iron chelators, all of which had differing binding affinities for ferric iron [100 μmol l⁻¹ of desferrioxamine (DFO), Log₁₀K₁ 32·5; citric acid Log₁₀K₁ 11·9; nitrilotriacetic acid (NTA) Log₁₀K₁ 15·9, CP20 and CP94 (Log₁₀K₁ > 30), as well as humic acid (HA), Log₁₀K₁ 5·04, 5 mg l⁻¹]. In the absence of chelators (control conditions) O. mykiss acquired iron from the water under laboratory lights (wavelength range of the lights 440–650 nm, peak intensity 548–626 nm) via the gill. In these conditions iron uptake onto the gill had a maximum transport capacity (J_max) of 11·2 pmol Fe g⁻¹ h⁻¹ (gill organ mass) and a K_m of 21·3 nmol Fe l⁻¹ h⁻¹. Furthermore, there were two components to iron accumulation into the carcass of these fish, a slow rate of aqueous iron uptake at low concentrations (6–24 nmol Fe l⁻¹), followed by a faster rate of uptake at higher iron concentrations (48–96 nmol Fe l⁻¹), suggesting that the rate-limiting step of iron uptake at low iron concentrations is the apical entry step. O. mykiss also acquired iron in the presence of HA, although the majority of the other chelators prevented iron uptake. Ultraviolet light (354 nm) treatment of Fe-DFO increased iron bioavailability. Results suggest that rainbow trout are able to access either the predicted very low concentrations (picomolar) of ferrous iron present in fresh water or the ferric oxide complexes present in oxic environments. The iron uptake rate measured (0·75 pmol g⁻¹ h⁻¹) would be sufficient to provide a substantial proportion (c. 85%) of the daily iron requirements of growing salmonid fry.     

The effect of Fe-EDDHA and of ascorbic acid on in vitro rooting of the peach rootstock GF-677 explants

The objective of this research was to study the effect of the chelated form of the iron salt of ethylenediamine di-o-hydroxyphenylacetic acid (Fe-EDDHA) (6% Fe) on in vitro rooting of the rootstock GF-677. The iron salt of ethylenediamine tetraacetic acid (Fe-EDTA) (12% Fe) of the MS basic medium was replaced by Fe-EDDHA, which was applied in three concentrations: 93.5, 187.0 and 280 mg l⁻¹ (5.6, 11.2 and 16.8 mg l⁻¹ Fe, respectively). For each treatment of Fe-EDDHA, the effect of ascorbic acid added in four concentrations (0, 0.1, 1.0 and 10 mg l⁻¹) was studied. After 4 weeks of culture, the explants growing on the medium with 280 mg l⁻¹ Fe-EDDHA gave the best rooting results. Regarding ascorbic acid, no clear stimulating effect on rooting was found.     

A comparative study of the effects of natural and synthetic ligands on iron uptake by plants

Summary The uptake of iron by wheat seedlings was investigated using half-strength Hoagland's nutrient solution containing 2.0 M ferric chloride labelled with⁵⁹Fe. The iron content of root tissue, which includes adsorbed iron, was depressed by the presence in the solution of the synthetic ligands EDTA and polymaleic acid (PMA) and by the natural ligands, humate, fulvate and a water-extractable soil polycarboxylate. The patterns of change in iron content of the shoots were in all cases different from those of the roots and were of two types. EDTA and humate increased the iron content of the shoots to maximum values, at ligand concentrations of 5.0 M and 2.5 mg l^–1 respectively, and decreased it at higher concentrations. Fulvate, water-extractable soil polycarboxylate and PMA increased the iron content of the shoots up to the maximum ligand concentrations tested (25 mg l^–1). These results are discussed in the light of the likely solution chemistry of iron and the various ligands.     

The lipophilic iron compound TMH-ferrocene [(3,5,5-trimethylhexanoyl)ferrocene] increases iron concentrations,neuronal L-ferritin,and heme oxygenase in brains of BALB/c mice

Mismanagement of intracellular iron is a key pathological feature of many neurodegenerative diseases. Our long-term goal is to use animal models to investigate the mechanisms of iron neurotoxicity and its relationship to neurodegenerative pathologies. The immediate aim of this experiment was to determine regional distribution of iron and cellular distribution of iron storage proteins (l- and h-ferritin) and an oxidative stress marker (heme oxygenase-1) in brains of mice fed the lipophilic iron compound (3,5,5-trimethylhexanoyl) (TMH)-ferrocene. We fed male and female weanling BALB/cj mice diets either deficient in iron (0 mg Fe/kg diet), adequate in iron (35 mg Fe/kg diet; control mice), or adequate in iron and supplemented with 0.1 or 1.0 g TMH-ferrocene/kg diet for 8 wk. Iron concentrations in cerebrum were higher in mice fed 1.0 g TMH-ferrocene/kg diet than in control mice (p<0.05). Liver iron concentrations were eightfold higher in mice fed 1.0 g TMH-ferrocene/kg diet than in control mice (p<0.0001). l-Ferritin and heme oxygenase-1 expression were elevated in striatum in mice fed 1.0 g TMH-ferrocene/kg diet. We conculde that administration of the lipophilic iron compound TMH-ferrocene leads to subtle perturbations of cellular iron within the brain, potentially representing a model of iron accumulation similar to that seen in various neuropathological conditions.     

Characterizing and modeling hydrogen sulfide production in anaerobic digestion of livestock manure,agro-industrial wastes,and wastewater sludge

Hydrogen sulfide (H₂S) is the most undesirable inorganic gas in biogas from anaerobic digestion (AD). However, H₂S production in AD is complex and understanding of its processes is still limited. This study performed six controlled batch anaerobic co-digestion experiments to investigate H₂S production. Materials were obtained from four field anaerobic digester systems and co-digestion feedstocks from agroindustry. An additional precipitation experiment was conducted to further examine H₂S production dynamics. Digesters containing highly soluble, carbohydrate-based wastes had a high H₂S final specific production (FSP) value. Additionally, the FSP values were negatively correlated with the initial Fe(II):S ratios in the digester liquid of the batch tests. The precipitation experiment indicated that iron sulfide precipitation was preferred in the presence of an anaerobic community. The H₂S production as a time series was successfully modeled using a generalized additive model (R² > 0.82). This study revealed that sulfate, phosphorus, and iron concentrations are important predictors and potential inhibitors of H₂S production in AD. Further examination of real-time H₂S modeling in AD is warranted.     

Isolation and study of two strains ofLeptospirillum-like bacteria from a natural mixed population cultured on a cobaltiferous pyrite substrate

Two strains ofLeptospirillum-like bacteria, L6 and L8, have been isolated from a mixed inoculum, also containingThiobacillus ferrooxidans andT. thiooxidans, cultured for one year with a colbaltiferous pyrite as energy substrate in a 100 I continuous bioleaching laboratory unit. Several physiological properties of the strains are described. The vibrio-shaped microorganisms grew at pH values lower than 1.3. Their growth rate was maximum between 2.5 and 8.0 g l¹ ferrous iron. The optimal growth temperature was 37.5° C. Ferric iron had a stimulative effect on bacterial development up to 8 g l^–1, and growth was as rapid at 14 g l^–1 ferric iron as at 8 g l^–1. The negative influence of cobalt on the final cell concentration was observed at 0.5 g l^–1, but the growth rate was not affected up to 2 g l^–1. The G + C content of strains L8 is 55.6 mol%.     

Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge

The phylogenetic and functional diversity of syntrophic propionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR–FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52–62% of the total active POB under all the propionate concentrations tested (0.5–15 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobacter accounted for 16–31% of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration.     

Air-side ammonia stripping coupled to anaerobic digestion indirectly impacts anaerobic microbiome

Air-side stripping without a prior solid–liquid phase separation step is a feasible and promising process to control ammonia concentration in thermophilic digesters. During the process, part of the anaerobic biomass is exposed to high temperature, high pH and aerobic conditions. However, there are no studies assessing the effects of those harsh conditions on the microbial communities of thermophilic digesters. To fill this knowledge gap, the microbiomes of two thermophilic digesters (55°C), fed with a mixture of pig manure and nitrogen-rich co-substrates, were investigated under different organic loading rates (OLR: 1.1–5.2 g COD l⁻¹ day⁻¹), ammonia concentrations (0.2–1.5 g free ammonia nitrogen l⁻¹) and stripping frequencies (3–5 times per week). The bacterial communities were dominated by Firmicutes and Bacteroidetes phyla, while the predominant methanogens were Methanosarcina sp archaea. Increasing co-substrate fraction, OLR and free ammonia nitrogen (FAN) favoured the presence of genera Ruminiclostridium, Clostridium and Tepidimicrobium and of hydrogenotrophic methanogens, mainly Methanoculleus archaea. The data indicated that the use of air-side stripping did not adversely affect thermophilic microbial communities, but indirectly modulated them by controlling FAN concentrations in the digester. These results demonstrate the viability at microbial community level of air side-stream stripping process as an adequate technology for the ammonia control during anaerobic co-digestion of nitrogen-rich substrates.     

Water for small‐scale biogas digesters in sub‐Saharan Africa

Biogas could provide a more sustainable energy source than wood fuels for rural households in sub‐Saharan African. However, functioning of biogas digesters can be limited in areas of low water availability. The water required is approximately 50 dm³ day^?1 for each cow and 10 dm³ day^?1 for each pig providing manure to the digester, or 25 (±6) dm³ day^?1 for each person in the household, using a digester volume of 1.3 (±0.3) m³ capita^?1. Here, we consider the potential of domestic water recycling, rainwater harvesting, and aquaculture to supply the water needed for digestion in different countries of sub‐Saharan Africa. Domestic water recycling was found to be important in every country but was usually insufficient to meet the requirements of the digester, with households in 72% of countries need to collect additional water. Rooftop rainwater harvesting also has an important role, iron roofs being more effective than thatched roofs at collecting water. However, even with an iron roof, the size of roof commonly found in sub‐Saharan Africa (15 to 40 m²) is too small to collect sufficient water, requiring an extra area (in m²) for each person of (R/100) (where R is the rainfall in mm). If there is a local market for fish, stocking a pond with tilapia, fed on plankton growing on bioslurry from the digester, could provide an important source of additional income and hold the water required by the digester. In areas where rainfall is low and seasonal, the fishpond might be stocked only in the rainy season, allowing the pond to be covered during the dry period to reduce evaporation. If evaporative losses (E in mm) exceed rainfall, an extra catchment area is needed to maintain the water level in the pond, equivalent to approximately (1.5 × ((E?R)/R)) m² for each person in the household.     

Decolorization kinetics of the azo dye Reactive Red 2 under methanogenic conditions: effect of long-term culture acclimation

The biological decolorization of the textile azo dye Reactive Red 2 was investigated using a mixed, mesophilic methanogenic culture, which was developed with mixed liquor obtained from a mesophilic, municipal anaerobic digester and enriched by feeding a mixture of dextrin/peptone as well as media containing salts, trace metals and vitamins. Batch decolorization assays were conducted with the unacclimated methanogenic culture and dye decolorization kinetics were determined as a function of initial dye, biomass, and carbon source concentrations. Dye decolorization was inhibited at initial dye concentrations higher than 100 mg l^-1 and decolorization kinetics were described based on the Haldane model. The effect of long-term culture exposure to the reactive dye on decolorization kinetics, culture acclimation, as well as possible dye mineralization was tested using two reactors fed weekly for two years with an initial dye concentration of 300 mg l^-1 and a mixture of dextrin/peptone. The maximum dye decolorization rate after a 2-year acclimation at an initial dye concentration of 300 mg l^-1 was more than 10-fold higher as compared to that obtained with the unacclimated culture. Aniline and the o-aminohydroxynaphthalene derivative resulting from the reductive azo bond cleavage of the dye were detected, but further transformation(s) leading to dye mineralization were not observed. Reactive Red 2 did not serve as the carbon and energy source for the mixed culture, and dye decolorization was sustained by the continuous addition of dextrin and peptone. Thus, biological decolorization of reactive azo dyes is feasible under conditions of low redox potential created and maintained in overall methanogenic systems, but supply of a biodegradable carbon source is necessary.     

Promotion of ε-poly-l-lysine production by iron in Kitasatospora kifunense

Kitasatospora kifunense, belonging to the Streptomycetaceae family, produces a basic homopolymer, ε-poly-l-lysine, which is used as a food preservative. We showed that ε-poly-l-lysine production in this bacterium on agar plates with iron started two or three days earlier than that on plates without iron. We also showed that iron added to a liquid culture medium increased ε-poly-l-lysine production by K. kifunense. Similarly, manganese and cobalt also promoted ε-poly-l-lysine production on agar plates. Moreover, cobalt promoted ε-poly-l-lysine production in liquid culture media. These results indicate that iron, manganese and cobalt are involved in regulating the ε-poly-l-lysine biosynthesis system in K. kifunense.     

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Factors in acid treated bagasse inhibiting ethanol production from d-xylose by Pachysolen tannophilus

The fermentation of d-xylose, the major sugar-cane bagasse hemicellulose component, to ethanol by Pachysolen tannophilus is inhibited by various factors produced or released during the acid hydrolysis of the bagasse or during the fermentation process. These include ethanol, iron, chromium, copper, nickel, acetic acid and furfural. Ethanol production by P. tannophilus is inhibited by ethanol fconcentrations >24 g l^?1. Furfural and acetic acid concentrations as low as 0.3 and 7 g l^?1, respectively, and iron, chromium, nickel and copper at concentrations of 0.07, 0.01, 0.01 and 0.004 g l^?1, respectively. Similar concentrations may be found in acid-hydrolysed bagasse. The removal of these factors by treatment with ion-exchange resin resulted in the fermentation of the sugars to ethanol. The d-glucose was used rapidly and completely whereas d-xylose utilization was slow and incomplete. An ethanol concentration of 4.1 g l^?1 was produced and an ethanol yield of 0.32 was obtained. Xylitol in significant amounts was produced.     

Oxidation of high ferrous iron concentrations by chemolithotrophic Thiobacillus ferrooxidans in packed bed bioreactors

The bacterial oxidation of high ferrous iron concentrations in batch culture has been studied in a packed bed bioreactor. It has been found that aeration rates from 0.49 to 1.2 VVM did not influence the biofilm oxidation activity during the period of biofilm formation up to 30 g l⁻¹ initial ferrous iron concentration. The contribution of swimming, attached and fixed bacteria to the Fe²⁺ oxidation process has been evaluated. Kinetics data showed that the oxidation rate depends on the aeration rate, when the initial Fe²⁺ concentration exceeded 30 g l⁻¹. The maximum overall Fe²⁺ oxidation rate was 1.8 g l⁻¹ h⁻¹, when the initial ferrous iron concentration was in the range 30 to 45 g l⁻¹.     

The effect of volatile fatty acids on the inactivation of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> in anaerobic digestion

The application of sludge digestion systems to remove pathogens has been employed to generate biosolids suitable for reuse in agriculture. Traditionally, temperature is considered the principal agent responsible for pathogen reduction in anaerobic digestion. However, other substances such as volatile fatty acids may also have an antimicrobial effect. The objective of this study was to assess the impact of fatty acid mixtures on the inactivation of C. perfringens over a range of digestion temperatures. An equimolar mixture of acetic acid, propionic acid and butyric acid was applied to digester effluent for a period of 24 h at temperatures of 35 °C, 42 °C, 49 °C and 55 °C. C. perfringens inactivation in digester effluents, when dosed with volatile organic acids, was found to depend on pH, acid concentration and temperature. Temperatures above 55 °C appeared to increase the inhibitory effects of the organic acids at higher concentrations. An interaction between temperature and pH on survival of C. perfringens was observed. The results suggest that high concentrations of organic acids at a pH value of 4.5–5.5 during thermophilic digestion substantially reduce concentrations of C. perfringens in municipal sludge.     

The limnology of a thermal lake: Lake Rotowhero,New Zealand: II. General biology with emphasis on the benthic fauna of Chironomids

Nitella hookeri A. BR., a native charophyte of the Rotorua lakes in the North Island of New Zealand, was investigated in terms of its nutrient requirements under laboratory culture in non-arenic conditions. Optimum growth in phosphate, nitrate, ammonia, iron and magnesium occurred at 20 mg l⁻¹ or lower. Higher concentrations of these ions were markedly inhibitory to growth when compared with the optimum. Calcium and potassium gave growth at higher levels and over a wider range of concentrations. Sulphate and sulphur-amino acids do not promote growth above the levels of the basal medium (1.2 mg l⁻¹ sulphur as SO²⁻ ₄). Amino acids did not appear to provide a nitrogen source for the plant. The optimum phosphate concentration was one thousand times higher than previously reported values. This may be due to the presence of arsenic in the vegetative propagules used from Lake Rotoiti. Iron toxicity effects may have resulted from the presence of EDTA. The results are considered in relation to the known levels of nutrients present in lake waters. Only phosphorus appears directly correlated with the natural distribution of the plant but it is suggested that anaerobic lake substrates may contribute iron and ammonia to the plants and further explain their distribution.     

Influence of trace elements on biogas production from mango processing waste in 1.5 m3 KVIC digesters

Summary The influence of trace elements (Co²⁺, Ni²⁺ and Fe³⁺) in varying concentrations and combination, was studied in 1.5 m³ Khadi & Village Industries Commission (KVIC) digesters for biogas generation from mangopeel. Addition of these trace metals enhanced the biogas yield and methane content moderately, the maximum being with the iron fed digester. The digesters were always found to be stable without much variation in total volatile fatty acids (VFA), pH, total alkalinity and other parameters. A methane content of 62% and biogas yield of 0.49 m³/kg VS added was obtained with 4000 mg/L FeCl₃ supplemented mangopeel fed digester as compared to control having biogas yield of 0.22 m³/kg VS added with a methane content of about 48–50%.