Inactivation of Salmonella typhi by high levels of volatile fatty acids during anaerobic digestion

Survival of Salmonella typhi was investigated in an anaerobic digester for cattle dung with volatile fatty acid (VFA) levels of 5000 mg l(-1) and pH 6.0. The organism was added to the digester only once in the first experiment and daily in the other. Survival was monitored on alternate days. In the single dose experiment, the counts of Salm. typhi declined rapidly and the pathogen was completely eliminated within 12 d in the experimental digester (VFA ca 5000 mg l(-1) and pH 6.0), whereas 26 d were required in the control digester (VFA ca 100 mg l(-1) and pH 6.8). T90 values for the experimental and control digesters were 2.44 d and 4.80 d, respectively. In the daily dose experiment, a four log reduction in the pathogen count was observed in the experimental digester, but only a two log reduction in the control digester at the end of the experimental period. The mean T90 values for the experimental and the control digester were 4.22 d and 18.63 d, respectively. In both the experiments, statistical analysis of the data showed significant differences in the survival pattern of Salm. typhi in the two digesters.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Effect of acute and chronic ammonia and nitrite exposure on oxygen consumption and growth of juvenile big bellied seahorse

Juvenile big bellied seahorse Hippocampus abdominalis were exposed acutely and chronically to elevated ammonia and nitrite {24 h exposure: 0·01, 5·0, 10·1, 14·8 and 19·9 mg l⁻¹ total ammonia-nitrogen [TA-N] and <0·001, 74·4, 99·2 and 123·6 mg l⁻¹ [NO₂-N] nitrite-nitrogen and 35 days exposure: 0·11, 0·55, 1·67 and 3·07 mg l⁻¹ TAN and <0·001, 0·92, 4·67 and 9·10 mg NO₂-N l⁻¹}. Significant ( P <0·001) increases in oxygen consumption rate and ventilation frequency occurred at 14·8, 19·9 mg l⁻¹ TA-N and 99·2, 123·6 mg l⁻¹ NO₂-N for acutely exposed fish. Oxygen consumption rate was significantly ( P <0·05) elevated at 1·67 and 3·07 mg l⁻¹ TA-N in chronically treated fish and ventilation frequency increased significantly ( P <0·05) at 0·55, 1·67, 3·07 mg l⁻¹ TA-N and 4·59, 9·10 mg l⁻¹ NO₂-N. There were no significant differences in growth between controls and ammonia exposed fish. Mortalities occurred at 14·8, 19·9 mg l⁻¹ TA-N.     

Optimization of D-ribose production with a transketolase-affected Bacillussubtilis mutant strain in glucose and gluconic acid-based media

When the transketolase-deficient and D -ribose-producing Bacillus subtilis strain ATCC 21951 was grown in a glucose (200 g l⁻¹)-based medium (Kintaka et al. 1986), only 11 g l⁻¹ D -ribose was synthesized, in addition to acetic acid (12 g l⁻¹) and acetoin plus 2,3-butanediol (24 g l⁻¹), within 1 week of fermentation. After optimizing the process conditions at 2 l fermentor scale (simplified medium composition, pH 5·0 or 6·0, highly oxidative (1000 rev min⁻¹, 3 vvm)), 40 g l⁻¹ D -ribose was obtained from 200 g l⁻¹ D -glucose, in addition to 14·5 g l⁻¹ acetoin, during 1 week of fermentation. By partially substituting D -glucose with D -gluconic acid (100 g l⁻¹ D -glucose plus 50 g l⁻¹ D -gluconic acid) under highly oxidative (1000 rev min⁻¹, 3 vvm) and pH-controlled (pH 6·5) conditions, D -ribose productivity increased (45 g l⁻¹) and acetoin formation (7·5 g l⁻¹) dropped, as did the fermentation time (3·5 d). The mixed carbon substrate procedure here developed provides an excellent alternative to the less efficient glucose-based processes described so far.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

The effects of environmental factors on rainbow smelt Osmerus mordax embryos and larvae

Experiments were conducted to identify environmental factors that influence the survival of rainbow smelt Osmerus mordax during their early life stages. Developing rainbow smelt embryos and yolk-sac larvae were cultured under controlled conditions with different dissolved oxygen (DO; 1·09, 2·18, 4·37 and 6·55 mg l⁻¹, pH (4·0, 4·5, 5·0, 5·5, 6·0 and 7·0), nitrate ( 0·7, 3·6, 7·3, 14·6 and 29·2 mg l⁻¹), phosphate (0·04, 0·21, 0·42, 2·08 and 4·17 mg l⁻¹) and salinity (0, 5, 10, 15, 20 and 30) levels. Rainbow smelt embryos were also incubated with simulated tidal salinity fluctuations (2–28), ultraviolet radiation (irradiances of 2·8, 6·2 and 5·1 W m⁻²) and under natural conditions in two rainbow smelt spawning rivers. In the laboratory, hatch was only impaired under the lowest DO and pH conditions (0 and 13% hatch, respectively) and at highest constant salinity levels (0% hatch). Larval survival was only affected by pH levels ≤5·0. The experiment that compared hatch under natural conditions was terminated when embryos became covered with silt and fungus. These results suggest that water acidification, sediment and fungal growth may affect rainbow smelt survival during their early life stages.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Histidine carboxylase of Leuconostoc œnos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc ' nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. ' nos 9204 HDC was synthesized as a precursor proHDC π₆ (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α (Mr 25 380) and β (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)₆. At the optimal pH of 4·8, the HDC activity exhibited a simple Michaelis-Menten kinetic ( K _m = 0·33 mmol l⁻¹, V _max = 17·8 μmol CO₂ min⁻¹ mg⁻¹), while at pH 7·6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor ( K _i = 32 mmol l⁻¹). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.     

Assessment of microbial involvement in the elevation of copper levels in drinking water

A study of bacterial populations in metropolitan Adelaide domestic reticulation pipes was conducted to investigate a possible link between copper in drinking water and biofilms. Biofilm densities from cold water copper pipes at 10 sample sites were measured by viable cell counts. The range detected was from <2 × 10¹ to 3·25 × 10⁷ cfu cm⁻². Five isolates were selected for further experiments as they represented a range of responses to solvated copper and relative tendency for adhesion on glass slides. Drinking water supplied to the Adelaide Hills is high in total organic carbon (TOC; 22·57 mg C l⁻¹) and has a negative Langelier Index (LI; −1·16), whereas Adelaide metropolitan water undergoes filtration and has both a lower TOC and LI (10·72 mg C l⁻¹, LI, −0·49). Copper coupons were exposed to biofilm isolates (24 h), washed and resuspended in Adelaide metropolitan and Adelaide Hills water. Copper coupons not exposed to biofilm isolates were suspended in respective waters as a control. After 5 d of incubation, the copper content of Adelaide Hills water (4·71 ± 0·87 mg Cu l⁻¹), in which the copper coupons were suspended, consistently exceeded values obtained in the metropolitan Adelaide water (1·17 ± 0·249 mg Cu l⁻¹). The concentration of copper in the Adelaide Hills water was influenced by the bacterial species forming the biofilm on the coupon, with Agrobacterium sp. producing significantly higher levels of soluble copper than the control. The experiments reported here indicate that the suspended organic carbon, the aggressivity of the water and the biofilm may independently or synergistically increase the dissolution of copper from pipes into drinking water.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.     

Purification and characterization of galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K _m values for o -nitrophenyl-β- d -galactopyranoside and lactose were 14·3 and 5·5 mmol l⁻¹, respectively, and the V _max values for these substrates were 33·4 and 94·5 μmol min⁻¹ mg of protein⁻¹, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml⁻¹ galacto-oligosaccharide was produced from 200 mg ml⁻¹ lactose.     

Characterization of the interaction of bovine plasmin with Streptococcus uberis

The binding of plasmin to Streptococcus uberis strain 0140 J was optimal in the pH range 5·0–5·5. Plasmin binding decreased exponentially with increasing NaCl concentration (0–0·8 mol l⁻¹), reaching a minimum at NaCl concentrations exceeding 0·55 mol l⁻¹. Neither K⁺, Mg²⁺ nor the metal chelator EDTA had any effect on the interaction. Plasmin binding was prevented, in a concentration-dependent manner, by the amino acids lysine, arginine and ε-aminocaproic acid. Bound plasmin was also eluted from the bacterial cell using the same amino acids. Bound plasmin was lost from the bacterium in a time- and temperature-dependent fashion, the rate of plasmin loss increased with increasing temperature over the range 4–55 °C, and the elution of plasmin from live and heat-killed bacteria was similar. Cell-bound plasmin was only partially inhibited by the physiological inhibitor α₂-antiplasmin whereas the serine protease inhibitor aprotinin, and the active site titrant p -nitrophenyl- p -guanidiniobenzoate, inhibited the activity of the cell-bound plasmin by more than 95%.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6·8, but not at pH 4·0, when incubated at 37°C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30°C at pH 4·0 was 0·03 mol/l and for Escherichia coli it was 0·09 mol/l. Fermented pig wastes in a digester, maintained at pH 5·9, were inoculated with Salm. typhimurium and then incubated at 37°C for 24 h. The pH was adjusted to either 4·0 or 5·0 and after a further 48 h at 30°C, Salm. typhimurium survived at pH 5·0 but not at pH 4·0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

The effect of chlorine on β- D- galactosidase activity of sewage bacteria and Escherichia coli was studied. β- D- galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0·05 mg Cl₂ l⁻¹) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0·1 mg Cl₂ l⁻¹, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu⁻¹ increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu⁻¹ decreased when the chlorine dosage was increased from 0·1 to 0·5 mg Cl₂ l⁻¹, but were not significantly different ( P > 0·05) for dosages of 0·2–0·7 mg Cl₂ l⁻¹. After chlorination, β- D- galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.     

Lipid formation and γ-linolenic acid production by Mucorales fungi grown on sunflower oil

Lipid formation and γ-linolenic acid (GLA) production by 48 species of Mucorales fungi grown on sunflower oil (which consists of 70% linoleic acid ; LA) were studied. The strains accumulated 42·7–65·8% lipid in biomass (7·66–13·39 g l⁻¹). Eight cultures produced more than 200 mg GLA l⁻¹. Highest GLA yields exhibited Mucor mucedo CCF-1384 and Cunninghamella echinulata CCF-103 (379 and 373 mg l⁻¹, respectively). Mortierella alpina CCF-185 synthesized 465 mg l⁻¹ arachidonic acid. While the decrease of LA utilization index (ratio of LA content of cell lipid/LA content of oil source) was accompanied with growth of delipidized biomass and with reduction of lipid accumulation within the cells, high lipid yield was as a consequence of the direct oil source incorporation into intracellular lipid.     

In vitro analysis of intestinal absorption of cadmium and calcium in rainbow trout fed with calcium- and cadmium-supplemented diets

The protective effects of dietary Ca²⁺ supplementation against Cd accumulation in rainbow trout Oncorhynchus mykiss fed with Cd-contaminated food were evaluated in relation to chronic changes in intestinal absorption rates. The changes were measured ' in vitro '. The control diet contained c. 20 mg Ca²⁺ g⁻¹ food and 0·25 μg Cd g⁻¹ food; the experimental diets were supplemented with CaCO₃ and Cd(NO₃)₂·4H₂O to levels of 50 mg Ca²⁺ g⁻¹ food and 300 μg Cd g⁻¹ food, alone and in combination. The Ca²⁺ and Cd absorption rates were measured using radiotracers (⁴⁵Ca, ¹⁰⁹Cd) at total Ca²⁺ and Cd concentrations of 3·0 and 0·12 mmol l⁻¹, respectively in the intestinal saline. Chronically elevated dietary Cd caused a significant increase in Cd absorption rate by up to 10-fold at 30 days in the mid-intestine. The high Ca²⁺ diet prevented this up-regulation of Cd transport rate. Conversely, intestinal Ca²⁺ absorption was significantly increased by two- to five-fold by the Ca²⁺-supplemented diet at 30 days in both the mid- and posterior intestine, and this effect was eliminated when Cd was simultaneously elevated in the diet. Ca²⁺ and Cd probably interact at common pathways and transport mechanisms in the intestine, though independent pathways may also exist.