The mode of toxic action of the pesticide gliftor: the metabolism of 1,3-difluoroacetone to (-)-erythro-fluorocitrate

The biochemical toxicology of 1,3-difluoroacetone, a known metabolite of the major ingredient of the pesticide Gliftor (1,3-difluoro-2-propanol), was investigated in vivo and in vitro. Rat kidney homogenates supplemented with coenzyme A, ATP, oxaloacetate, and Mg2+ converted 1,3-difluoroacetone to (-)-erythro-fluorocitrate in vitro. Administration of 1,3-difluoroacetone (100 mg kg(-1) body weight) to rats in vivo resulted in (-)-erythro-fluorocitrate synthesis in the kidney, which was preceded by an elevation in fluoride levels and followed by citrate accumulation. Animals dosed with 1,3-difluoroacetone did not display the 2-3 hour lag phase in either (-)-erythro-fluorocitrate synthesis or in citrate and fluoride accumulation characteristic of animals dosed with 1,3-difluoro-2-propanol. We demonstrate that the conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone by an NAD+-dependent oxidation is the rate-limiting step in the synthesis of the toxic product, (-)-erythro-fluorocitrate from 1,3-difluoro-2-propanol. Prior administration of 4-methylpyrazole (90 mg kg(-1) body weight) was shown to prevent the conversion of 1,3-difluoro-2-propanol (100 mg kg(-1) body weight) to (-)-erythro-fluorocitrate in vivo and to eliminate the fluoride and citrate elevations seen in 1,3-difluoro-2-propanol-intoxicated animals. However, administration of 4-methylpyrazole (90 mg kg(-1) body weight) to rats 2 hours prior to 1,3-difluoroacetone (100 mg kg(-1) body weight) was ineffective in preventing (-)-erythro-fluorocitrate synthesis and did not diminish fluoride or citrate accumulation in vivo. We conclude that the prophylactic and antidotal properties of 4-methylpyrazole seen in animals treated with 1,3-difluoro-2-propanol derive from its capacity to inhibit the NAD+-dependent oxidation responsible for converting 1,3-difluoro-2-propanol to 1,3-difluoroacetone in the committed step of the toxic pathway.     

Metabolism of fluoroacetate in the skink (Tiliqua rugosa) and the rat (Rattus norvegicus)

Administration of 100 mg sodium fluoroacetate (compound 1080) per kilogram body weight to T. rugosa resulted in a 3.4-fold increase in plasma citrate levels 48 h after dosing while administration of 3 mg sodium fluoroacetate per kilogram body weight to R. norvegicus produced a fivefold increase in plasma citrate levels within 4 h. Administration of 300 mg sodium fluoroacetate per kilogram body weight reduced the oxygen consumption of the skink by between 2.5 and 11% while in the rat, 2 mg sodium fluoroacetate per kilogram body weight reduced oxygen consumption by between 28 and 57%. Aconitate hydratase activity in extracts of liver acetone powders from T. rugosa was less inhibited by (-)erythrofluorocitrate (Ki: 0.065 mM) than that in extracts derived from R. norvegicus (Ki: 0.026 mM). The rate of defluorination of fluoroacetate in erythrocytes and in extracts of liver acetone powders of T. rugosa was 8- and 4.5-fold greater, respectively, than that found in similar preparations from R. norvegicus. A rapid rate of defluorination together with a low reliance on aerobic respiration favoured detoxification of fluoroacetate in T. rugosa rather than its conversion into fluorocitrate. Though defluorination in this species helped to minimize the immediate effects of fluoroacetate on aerobic respiration, it resulted in rapid depletion of liver glutathione levels.     

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.     

Insulin-like effects of fluoroacetate on lipolysis and lipogenesis in adipose tissue.

Hormone-stimulated lipolysis in adipose tissue was inhibited by fluoroacetate and there was a concomitant decrease in both the basal and hormone-stimulated cyclic AMP levels. Adenylate cyclase (EC 4.6.1.1) activity in membrane preparations was inhibited by fluoroacetate. There was no influence of fluoroacetate on the low Km cyclic AMP phosphodiesterase (EC 3.1.4.17) activity. The rate of glucose conversion to fatty acids was increased when adipose tissue was incubated in the presence of fluoroacetate. The outputs of pyruvate and lactate into the incubation medium were decreased at this time, suggesting decreased tissue pyruvate levels and a site of activation of lipogenesis distal to pyruvate formation. Pyruvate dehydrogenase (EC 1.2.4.1) activity was increased twofold in adipose tissue incubated in the presence of fluoroacetate. This was attributed to a fluoroacetate-induced inhibition of pyruvate dehydrogenase kinase, the enzyme responsible for inactivating the pyruvate dehydrogenase complex. Glucose transport was increased to a small but significant degree by fluoroacetate. In addition, both the tissue content of citrate and its release into the incubation medium were increased, suggesting that fluoroacetate resulted in an inhibition of aconitase (EC 4.2.1.3). The tissue ATP content was unchanged. Because the antilipolytic and lipogenic effects of fluoroacetate parallel those of insulin, they may share a common mechanism.     

Studies on the biochemical basis of susceptibility and resistance of the housefly to fluoroacetate

1. Administration of fluoroacetate to sensitive houseflies in amounts close to the L.D.₅₀ range (0.25–0.3 μg/fly) brought about a prompt elevation of their citrate content. With about 10-fold higher doses of fluoroacetate a concurrent increase of both citrate and pyruvate levels took place in the fly tissues.

2. Incubation of sarcosomes of the sensitive housefly strain in the presence of oxidizable substrates and fluoroacetate resulted in accumulation of citrate, inhibition of respiration and uncoupling of oxidative phosphorylation. The magnitude of the effects varied considerably with the different substrates used, being particularly pronounced with pyruvate and malate and inappreciable with succinate and -glycerophosphate.

3. The respiratory inhibition induced by a brief exposure in the cold of housefly sarcosomes to fluoroacetate, persisted after the sarcosomes had been washed free from fluoroacetate. The toxic effect of fluoroacetate on the respiratory chain could be prevented by an excess of simultaneously added acetate.

4. The susceptibility of the respiratory function of the sarcosomes to fluoroacetate inhibition was abolished by sonication. The unresponsiveness of the sonicated sarcosomes to fluoroacetate was attended by a loss of their respiratory chain phosphorylation activity.

5. Sarcosomes derived from a partially resistant housefly strain, when incubated in the presence of fluoroacetate, failed to accumulate citrate, but displayed the characteristic respiratory-inhibition response. Sarcosomes from a highly resistant strain showed no impairment of their functional capacity by fluoroacetate. However, all the different housefly strains tested proved to be equally sensitive to the deleterious effect of fluorocitrate on sarcosomal respiration.

6. The possible biochemical mechanisms underlying the toxicity of fluoroacetate in the housefly are considered with particular reference to the altered response of the target systems exhibited by the fluoroacetate-resistant strains.     

The effect of sodium monofluoroacetate on plasma testosterone concentration in Tiliqua rugosa (gray)

• 1.1. Administration of multiple or single doses of sodium fluoroacetate (1080) to male Tiliqua rugosa caused a decrease in plasma testosterone concentration.
• 2.2. A single dose of 100 or 250 mg 1080 kg⁻¹ body weight decreased plasma testosterone by 52%. However, 25 mg kg⁻¹ had little apparent effect on testosterone levels. When lizards were given the multiple dose equivalent of these doses over 12 days at 3 day intervals, the effect was much less dramatic with plasma testosterone concentration steadily declining over 15 days for the two higher doses.
• 3.3. There was a suggestion of degeneration of seminiferous tubules in some individuals.

     

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP⁺) [threo-d_s-isocitrate:NADP⁺ oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH₂COO^?→acetylCoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml^?1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP⁺) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.     

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l⁻¹ of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (μ) and the 1,3-PDO yield from glucose (Y_P/S) in the disruptant were higher than those in the non-disruptant (ΔglpF, μ = 0·08 ± 0·00 h⁻¹, Y_P/S = 0·06 mol mol-glucose⁻¹; BW25113, μ = 0·06 ± 0·00 h⁻¹, Y_P/S = 0·02 mol mol-glucose⁻¹). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.     

The effects of arsenic and induced-phytoextraction methods on photosynthesis in Pteris species with different arsenic-accumulating abilities

Arsenic (As) accumulation and photosynthesis occur simultaneously in plants under As exposure. We investigated the effects of As and induced-phytoextraction methods on photosynthesis in two As hyperaccumulators (Pteris vittata and Pteris cretica var. nervosa) and two non-hyperaccumulators (Pteris semipinnata and Pteris ensiformis) under soil culture conditions. Chlorophyll fluorescence parameters (the maximum [F_v/F_m] and actual quantum efficiency [F_PSII]) and the activities of three photosynthetic enzymes (ATPase, ribulose-1, 5-bisphosphate carboxylase [RuBPC] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were measured. Arsenic accumulation and photosynthetic behaviours in response to enhanced-phytoextraction methods (trans-1, 2-cyclobexylenedinitrilotetraacetic acid [CDTA] and phosphorous [P] addition and soil pH adjustment) of P. cretica and P. semipinnata were monitored and compared under conditions of 100 mg As kg⁻¹. Significant decreases in the F_v/F_m (19.9%) and F_PSII (36.1%) were observed in P. vittata when exposed to 100 mg As kg⁻¹ in comparison to the control (0 mg As kg⁻¹). Compared to the control (0 mg As kg⁻¹), the activities of GAPDH increased by 0.5% in P. cretica var. nervosa and decreased by only 8.3% in P. vittata even when both of them were treated with 200 mg As kg⁻¹, whereas a significant decrease, 56.1% and 51.7%, of this enzyme was observed in P. semipinnata and P. ensiformis, respectively, when exposed to 50 mg As kg⁻¹. Compared to the control (0 mg CDTA kg⁻¹ or 0 mg P kg⁻¹), the activities of ATPase increased by 53.7% and 82.7% in P. cretica when exposed to 0.5 g CDTA kg⁻¹ and 50 mg P kg⁻¹, respectively, and an increase of up to 175% was also observed in P. semipinnata when exposed to 600 mg P kg⁻¹. The activities of GAPDH increased by 68.9% and 90.7% in P. cretica when exposed to 2 g CDTA kg⁻¹ and 600 mg P kg⁻¹, respectively, but a decrease of up to 60% was observed in P. semipinnata when exposed to 2 g CDTA kg⁻¹. The uptake of As in P. semipinnata increased by 80.9% and 73.3% when 1 g CDTA kg⁻¹ and 600 mg P kg⁻¹ were added, respectively, compared to the control (0 g CDTA kg⁻¹ or 0 mg P kg⁻¹). It was concluded that GAPDH played an important role in the photosynthesis of As hyperaccumulators under As treatments.     

The effect of dietary inclusion of starch,sucrose and xylose on the utilization of dietary energy in sheep

A calorimetric experiment of 4 × 4 Latin square design was undertaken to study the effect of sugar-beet pulp (SBP), maize starch, sucrose and xylose on energy metabolism in sheep. The four diets comprised a diet (A) of dried grass, soya-bean meal and SBP (450, 50 and 500 g kg⁻¹ on dry matter (DM) basis) and corresponding diets in which 400 g kg⁻¹ of SBP was replaced by maize starch (B), sucrose (C) or xylose (D); all diets were offered at a level of 600 g DM day⁻¹. After the Latin square was completed, energy value of the basal diet of dried grass and soya-bean meal (900 and 100 g kg⁻¹ DM; 600 g day⁻¹) was determined in the same four sheep.Treatment differences in organic matter, gross energy, nitrogen (N) and neutral detergent fibre (NDF) digestibility were non-significant. Differences in N retention were not significant.Digestible energy (DE) contents (MJ kg⁻¹ DM) were 13.27, 13.22, 13.21 and 13.21 MJ kg⁻¹ for diets A, B, C and D, respectively. Energy loss in methane was higher (P < 0.05) for Diet A than for other diets. Metabolizable energy (ME) contents for the diets A-D were 11.25, 11.22, 11.32 and 11.40 MJ kg⁻¹ DM, respectively. Metabolizability (q) of the diets averaged 0.642 and was not significantly affected by the diet given. There was a trend for heat production to be higher in sheep given the sucrose-containing diet (C) than in those given other diets (6.34 versus 6.04 MJ day⁻¹) and as a result, energy retention was lower (0.38 versus 0.64 MJ day⁻¹), but the difference did not reach statistical difference. Efficiencies of utilization of ME for maintenance and fattening (k_mf) averaged 0.67 and were in good agreement with those predicted from equations of the Agricultural Research Council (1980) excepting the lower k_mf (0.63) for Diet C.The mean ME content of SBP calculated by difference was 13.05 MJ kg⁻¹ DM and the corresponding values for mixtures of SBP + starch, SBP + sucrose and SBP + xylose (600 and 400 g kg⁻¹ DM) were 12.98, 13.16 and 13.36 MJ kg⁻¹ DM, respectively.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

The role of the tricarboxylic acid cycle in citric acid accumulation by Aspergillus niger

Summary Determinations of the momentary levels of various intermediates related to the activity of the tricarboxylic acid cycle have been made during citric acid production in high-accumulating (manganese deficient) and lowaccumulating (manganese supplemented) mycelia of Aspergillus niger. During the growth period the levels of almost all TCA cycle acids, with the exception of 2-oxo-acids, were unusually high; during the induction phase of citrate accumulation malate, fumarate, and isocitrate decreased, whereas pyruvate, oxalacetate, and citrate increased. The presence of succinate could not be demonstrated. The interrelations of the momentary concentrations of the intermediates mainly demonstrate a lack in activity of 2-oxoglutarate dehydrogenase, representing a block in the TCA cycle concomitant with a strongly operating glycolysis as a prerequisite for citrate accumulation. Inhibition studies with crude enzyme preparations suggest that an inhibition of malate dehydrogenase by citrate and also inhibition of isocitrate dehydrogenase by citrate and 2-oxoglutarate occur during the production phase as additional factors.     

Studies on the Metabolism of Fluoroacetate in the Liver of a Rat

In a rat poisoned with sodium fluoroacetate no accumulation of citrate was found in the liver, while in that of a frog fluoroacetate adtninistration was found to cause a marked increase of citrate. In this paper, it was shown that even in a rat when it was treated with insulin before fluoroacetate administration, citrate accuillulation could be found in the liver. In the experiments in vitro using slices of a rat liver it was also shown that fluoroacetate could cause an accumulation of citrate and inhibited the oxidation of malate in the slices of a normal as well as an insulinized liver.     

Carbohydrate and lignin contents of plant materials used in animal feeding

A total of 115 samples representing 38 different feedstuffs was analysed for carbohydrates and lignin. The samples were analysed for low-molecular weight (LMW) sugars by high-performance liquid chromatography, starch, fructan and mixed linked β(1 → 3;1 → 4)-D-glucan by colorimetry, total, soluble and insoluble non-starch polysaccharides (NSP) by gas-liquid chromatography and lignin by gravimetry. For all but alfalfa meal, almost quantitative recovery of carbohydrates and lignin was obtained with a deviation between calculated and analysed values of less than 2 g kg⁻¹ dry matter. The correlation between calculated and analysed values was 0.985 (P < 0.0001).The concentration (g kg⁻¹ dry matter) of LMW-sugars varied from 5 g kg⁻¹ and up to 137 g kg⁻¹ with the lowest values found in cereal substitutes, whole grain cereals and by-products while the protein concentrates in general had the highest content of LMW-sugars (57–137 g kg⁻¹). Starch was the main polysaccharide in whole grain cereals where it varied from 468 g kg⁻¹ in oats to 690 g kg⁻¹ in maize, in cereal by-products (93–902 g kg⁻¹) and in tapioca (768 g kg⁻¹). In contrast, the concentration of starch was low in all protein concentrates but peas and faba beans. The lowest levels of NSP and lignin were found in maize flour (NSP, 21 g kg⁻¹; lignin, 4 g kg⁻¹) and the highest levels in oat hull meal (NSP, 503 g kg⁻¹; lignin, 148 g kg⁻¹). There was also a significant variation in NSP and lignin in protein concentrates with the NSP value varying from 189 g kg⁻¹ in faba beans to 451 g kg⁻¹ in white lupins and with lignin varying from 12 g kg⁻¹ in white lupins to 133 g kg⁻¹ in sunflower cake. Grass meal, alfalfa meal and sugar beet fibre had in general high concentrations of NSP and lignin with values in grass and alfalfa meals of NSP: 329–426 g kg⁻¹ and lignin: 128–169 g kg⁻¹ and in sugar beet fibre 779 g kg⁻¹ and 35 g kg⁻¹, respectively.     

Examination of stability,and its effect on the nutritive value,of fish silage in diets for growing pigs

Fish silage was manufactured by the addition of formic acid (85% solution) to whole mackerel at a rate of 35 g kg⁻¹ [wet weight (ww)]. During 112 days of storage, the peroxide value of the silage declined from 164.3 meq O₂ kg⁻¹ oil on Day 1 to 55.0 meq O₂ kg⁻¹ oil by Day 42 and thereafter remained stable; microbial activity persisted at 10 colonies g⁻¹ silage ww. Four diets of similar crude protein, digestible energy and mineral concentrations were formulated with 0, 50, 100 or 150 g fish silage kg⁻¹ diet dry matter (DM). The diets were given to 72 Landrace × (Landrace × Large White) pigs (boars, gilts and castrated males) from 25 kg to slaughter at 55 kg.Animals on fish silage diets grew faster than those given no fish silage owing to an improved food conversion ratio (FCR); 100 g fish silage kg⁻¹ diet DM effected best performance (daily liveweight gain, 725 g; FCR, 1.96). Carcass measurements did not vary between dietary treatments. Soft, yellow fat was observed in carcasses from pigs given 150 g silage kg⁻¹ diet DM. Growth rates were similar between sexes; boars and gilts had less backfat than castrated males.     

Significance of sulfhydryl compounds in the manifestation of fluoroacetate toxicity to the rat, brush-tailed possum, woylie and western grey kangaroo

Levels of citrate in kidneys and livers of rats with normal glutathione levels increased 6.8 and 1.7-fold respectively 2 h after dosing with 1.5 mg of compound 1080 (= 95% sodium fluoroacetate) per kilogram body weight. In animals with liver glutathione levels 15% of normal, increases in plasma and liver citrate levels after dosing with fluoroacetate were significantly greater than those of control animals. Cysteamine and N-acetylcysteine, like glutathione, partially protected aconitate hydratase from fluorocitrate inhibition in rat liver preparations but were unable to replace glutathione as a substrate for the defluorination of fluoroacetate in vitro. N-Acetylcysteine did not diminish plasma citrate levels of glutathione-deficient rats dosed with fluoroacetate, while cysteamine inhibited the rate of in vivo defluorination in glutathione-deficient brush-tailed possums. It is suggested that non-physiological sulfhydryl compounds are ineffective antidotes to fluoroacetate intoxication in vivo. The in vivo defluorination patterns of four mammal species with differing sensitivities to fluoroacetate did not indicate a direct relationship between tolerance and rate of defluorination and it is also suggested that a high level of activity of the glutathione-S-transferase responsible for the defluorination of fluoroacetate is not the major mechanism for circumventing fluoroacetate toxicity in resistant mammals.     

Nutritive value of deep stacked and ensiled broiler litter for sheep

Broiler litter was deep stacked and ensiled with water to achieved 40% moisture before being added, with or without 5% sugarcane molasses or with rumen contents, to a basal diet. The influence of stacking and ensiling of broiler litter on the numbers of Salmonella, Shigella, Proteus and total number of colony forming units (CFU) was investigated. Nutritive value of broiler litter processed by deep stacking and ensiling was evaluated in a digestibility trial. The experiment was conducted with 30 wethers allotted to five diets. A basal diet (20% corn grain, 23% wheat bran, 37% cotton seed cake, 18% wheat straw and 2% dicalcium phosphate) was given alone (1) or with broiler litter processed by deep stacking (2), ensiling (3), ensiling with 5% added molasses (4), or ensiling with rumen contents (1:1, wet basis) (5). For Diets 2–5, the ratio of basal diet to silage was 1:1, dry basis. For the digestion trial, diets were given at 20 g dry matter (DM) kg⁻¹ body weight per day. Initial samples of broiler litter showed 2.5 × 10⁸ CFU and Salmonella, Shigella and Proteus were present. The processes of deep stacking and ensiling were equally effective in achieving a complete elimination of all the pathogens. Apparent digestibilities of organic matter (OM) and crude protein (CP) (559.1 g kg⁻¹ and 608.7 g kg⁻¹ DM) were lower (P < 0.05) for diet 2 (deep stacked litter) than for the other waste-containing diets (OM: 578.7 g kg⁻¹, 582.9 g kg⁻¹, 594.1 g kg⁻¹; CP: 688.6 g kg⁻¹, 675.8 g kg⁻¹, 709.0 g kg⁻¹ DM, for Diets 3, 4 and 5, respectively). Among the waste-containing diets, cellulose digestibility (398.7 g kg⁻¹ DM) was higher (P < 0.05) for Diet 5 (ensiled litter-rumen contents). The results indicate that deep stacking and ensiling are equally feasible and effective for eliminating the pathogens and processed broiler litter can be incorporated in the diet of ruminants at levels of up to 50% without any adverse effect on the health of the animals.     

A Medium for the Isolation of Capsular Bacteria from Kefir Grains

Secondary alcohols (C₃ to C₁₀) were oxidized to the corresponding methylketones by resting mycelia of Scedosporium sp. A-4 grown on propane, but 3-pentanol and 3-hexanol were not oxidized. The oxidation of 2-propanol to acetone was inhibited by pyrazole, potassium cyanide, sodium azide and Hg^{2 +}. Alcohol dehydrogenase activity was found in the cell-free soluble fraction and this activity requires a cofactor, specifically NAD⁺. The oxidation of both 1-propanol and 2-propanol may be catalyzed by the same alcohol dehydrogenase.     

Ecotoxicity of soils contaminated with industrial and domestic wastewater in western shenyang,China

Soil samples were collected from 7 sites in the up-, mid-and down-reach along and nearby the wastewater irrigation channel, western Shenyang of China. The concentrations of selected pollutants (mineral oil, PAHs - polycycle aromatic hydrocarbons and Cd) were determined by UV spectrometer, HPLC and AAS (atomic adsorption spectrometer) spectrometer, respectively. Toxicity effects of soils were evaluated by seedling emergence test with root length of wheat as the end-point and by earthworms test with the mortality rate and inhibition rates of body weight as endpoints. Results showed accumulation of pollutants for most soils with concentration of 200.2 mg.kg⁻¹∼1600 mg.kg⁻¹ for mineral oil, 0.33 mg.kg⁻¹∼1.81 mg.kg⁻¹ for Cd and 900.16 mg.kg⁻¹ ∼ 2737.91 mg.kg⁻¹ for PAHs. The inhibition rates of root elongation were from −20% up to 40 %, and mortality rates of earthworms ranged from 0%∼40% from the exposure period of two weeks to eight weeks by sampling interval of two weeks, the inhibition rates of earthworm growth were from −19.36% to 34.53%, showing effects of stimulation at 2 weeks to an increasing effects of inhibition at 4, 6 and 8 weeks, respectively. Mortality rates correlated with the loss of body weight of earthworms.

This study indicated the potential risk of pollutants of environmental low content in soil by the determination of selected chemicals combined with toxicity indexes.

     

Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)