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Invertase from a strain of Rhodotorula glutinis.

An invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from Rhodotorula glutinis was purified by ammonium sulfate fractionation, gel filtration and anion exchange chromatography. Invertase molecular weight was estimated to be 100 kDa by analytical gel filtration and 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular mass determinations indicated that the native enzyme exists as a homodimer. It is a glycoprotein that contains 19% carbohydrate. The enzyme attacks beta-D-fructofuranoside (raffinose, stachyose and sucrose) from the fructose end. It has a K(m) of 0.227 M and a V(max) of 0.096 micromol/min with sucrose as a substrate. Invertase activity is stable between pH 2.6 and 5.5 for 30 min, maximum activity being observed at pH 4.5. The activation energy was 6520 cal/mol. The enzyme is stable between 20 and 60 degrees C. Mg(2+) and Ca(2+) ions stimulated invertase activity 3-fold, while Fe(2+), K(+), Co(2+), Na(+) and Cu(2+) increased activity about 2-fold. The transfructosylation reaction could not be observed. This enzyme is of particular interest since it appears to have a high hydrolytic activity in 1 M sucrose solution. This fact would make the enzymatic hydrolysis process economically efficient for syrup production using by-products with high salt and sugar contents such as sugar cane molasses.

Chromatography, Gel↗

New process for simultaneous removal of nitrogen and sulphur under anaerobic conditions.

A granular activated carbon (GAC) anaerobic fluidised-bed reactor treating vinasse from an ethanol distillery of sugar beet molasses was operated for 90 days, the first 40 days of start-up followed by 50 days of operation at constant organic loading rate of 1.7g COD/Ld. The reactor showed good performance in terms of organic matter removal but an anomalous behaviour in terms of unusual high concentrations of molecular nitrogen in the biogas. The analysis of the different nitrogenous and sulphur compounds and the mass balances of these compounds in the liquid and gas phases clearly indicated an uncommon evolution of nitrogen and sulphur in the reactor. About 50% of the nitrogen entering the reactor as total Kjeldahl nitrogen (TKN) was removed from the liquid phase appearing as N2 in the gas phase. Simultaneously, only 20% of the S-SO4(2-) initially present in the influent appears as S-S2- in the effluent or S-H2S in the biogas, indicating that 80% of the sulphur is removed. This behaviour has not been reported previously in the literature. These observations may suggest a new anaerobic removal process of ammonia and sulphate according to an uncommon mechanism involving simultaneous anaerobic ammonium oxidation and sulphate reduction.

Anaerobiosis↗

Utilization of agricultural wastes of Aspergillus awamori for the production of glucoamylase.

The principal industrial use of glucoamylase is in the production of crystalline D-glucose. Aspergillus awamori was used. Variations in the specific type of carbohydrate source affected the yield of enzyme markedly. Of all the substrates tried, maltose supported satisfactory enzyme yields. Sucrose, lactose, fructose, and galactose gave abundant growth which seemed to be necessary but no sufficient condition for maximum yields. The specificity of the carbohydrate structure for gluco-amylase production was tried. The synthesis of enzyme was significantly stimulated when two-third of the starch in the medium was replaced by maltose. The adaptation seemed to be specific to maltose structure, since the substitution of maltose by structurally related compounds, a.o. lactose, in the medium did not give a comparable glucoamylase yield. Maltose or compounds composed of maltose gave maximum yields of enzyme. These results seemed to favour the theory that glucoamylase production is adaptive. Agricultural wastes produced locally in large quantities from plants manufacturing starch and other products were discussed. Rice bran, followed by glucose syrup, corn bran, wheat bran, and can sugar molasses supported good yields of glucoamylase.

Agriculture↗

Anaerobic incorporation of the radiolabeled explosive TNT and metabolites into the organic soil matrix of contaminated soil after different treatment procedures.

Four bioreactor designs were performed to evaluate the level of incorporation of 14C-labeled 2,4,6-trinitrotoluene (TNT) and metabolites into the organic soil matrix of different anaerobically treated contaminated soils. The contaminated soils were amended with molasses slivers (80:20% per weight) as auxiliary substrate to enhance microbial activity. After 5 weeks (bioreactors 1 and 2), 8 weeks (bioreactor 3) and 12 weeks (bioreactor 4) of anaerobic incubation, we determined 41%, 58%, 72%, and 54%, respectively, of the initially applied radioactivity immobilized in various soil fractions. After alkaline hydrolyses of the solvent-extracted soils, low quantities of radiolabel were found in the humic and fulvic acid fractions, whereas the bulk of 14C activity was found to be strongly bound to the humin fraction (solid soil residues). The amounts of solvent extractable radioactivity were 53%, 40%, 16%, and 29% for bioreactors 1, 2, 3, and 4, respectively. The level of TNT transformation at the end of the experiments was within 90-94%. Regarding the results presented in this study, we can assume that there is the possibility of high incorporation levels of TNT metabolites into the soil organic matrix mediated by microbial cometabolism under strictly anoxic conditions.

Bacteria, Anaerobic↗

Assessment of arsenic level in the hair of the nonoccupational Egyptian population: pilot study.

Arsenic level of hair samples of apparently healthy Egyptian was measured by means of hydride atomic absorption spectrophotometery. It ranged between 0.04 and 1.04 mg As/kg hair, about 55% of the analysed hair samples were within the range of allowable values (0.08-0.25 mg As/kg hair), but 45% were not. There were no considerable sex-related differences (0.303 and 0.292 mg As/kg hair for males and females, respectively). Different educational levels did not influence it either, when the effect of the age had been excluded. Children and adolescents proved to be more susceptible to arsenic as their mean levels (0.353 microg/g), and were significantly higher than those in the adults (0.233 microg/g). Smoking and some dietary habits had an important role in the elevation of arsenic levels among the nonoccupational Egyptian population: 60% of smokers and 66.7% of indoor passive smokers had arsenic levels >0.25 mg As/kg hair. Arsenic levels were also dependent on the kind of smoking, as hair arsenic of the subject smoking molasses tobacco was found to be significantly higher than that of cigarette smokers (0.459 and 0.209 mg As/kg hair, respectively). The frequency of meat and fish consumption per week was also found to be positively, significantly correlated with arsenic levels. On the other hand, the frequency of consumption of fruits, fresh and cooked vegetables, milk and milk products per week beneficially influenced the arsenic level of the hair samples examined. Arsenic content of the consumed water in Egypt was 0.001 mg/l, which is below the maximum drinking water level allowed by World Health Organisation (WHO). Therefore, the arsenic content of domestic tap water hardly contributed to the arsenic exposure of the Egyptian population in the regions of the study. It is likely that exposure routes by smoking, fish and animal protein consumption are the principal cause of arsenic accumulation in the general Egyptian population.

Adolescent↗

The seroprevalence of antibodies to Sarcocystis neurona in Michigan equids.

A cross-sectional study of serum antibodies to Sarcocystis neurona (the etiologic agent of equine protozoal myeloencephalitis, EPM) was performed on Michigan equids. Our objectives were to determine the seroprevalence of antibodies to S. neurona in Michigan equids and to identify specific risk factors for seropositivity. A random, weighted sample of Michigan horse farms (stratified by the state's opossum (Didelphis virginiana) population and the number of equids on each operation) was selected. Ninety-eight equine-operation owners agreed to participate, and blood collection occurred from late March through October of 1997. Data regarding the 98 farms' feeding and management practices were collected, as well as descriptive data for each of the 1121 individual horses. Serum samples were tested for antibodies to S. neurona using a Western blot test. The true seroprevalence of antibodies specific to S. neurona was estimated to be 60%. Chi-square analysis showed that seroprevalence was lowest in the colder parts of the state that had the fewest opossums (P<0.0001). In two multivariable logistic-regression analyses with random effects grouped by herd, age and exposure to pasture were associated with increased odds of seropositivity, and feeding of sweet feed (grains mixed with molasses) was associated with decreased odds of testing positive. No association was found between farm size, animal gender, hay types, horse-housing types or exposure to natural surface water and seropositivity.

Animals↗

Risk factors associated with faecal shedding of verocytotoxin-producing Escherichia coli O157 in eight known-infected Danish dairy herds.

A risk-factor study was performed in eight dairy herds found to excrete verocytotoxin-producing Escherichia coli (VTEC) O157 in a former prevalence study. Associations between excretion of VTEC O157 and management factors such as housing and feeding were analysed in a generalised linear mixed model. The animals were stratified in three age groups and sampled four times during 1 year. The risk of excreting VTEC O157 was higher among weaned calves than non-weaned calves. Among the calves aged 1-4 months, the risk was reduced if the calf had suckled colostrum from the mother or if the calf had stayed >2 days with the mother after calving. Calves aged 5-24 months that had been moved within the last 2 weeks had a higher risk, but risk was reduced if fed barley silage. Cows fed grain or molasses had a higher risk of excreting VTEC O157.

Aging↗

Principal component ANN for modelling and control of baker's yeast production.

Modelling of baker's yeast production by the principal component based artificial neural networks (ANN) is presented. The models are derived for their application in adaptive control of fermentation by the internal model control (IMC) method. Modelling data are from industrial production in 40 m3 deep jet bioreactor and from computer simulations. The modelling effort is focused on selection of ANN structure and model verification. Principal component analysis of process variables results in projection of patterns to a space of low dimension, which enables determination of ANN structure, removes data colinearity and random components of measurement signals, and model degradation by over-training is eliminated. In view of IMC application, the models for prediction of the controlled variable (ethanol partial pressure) and the inverse model for manipulative variable (molasses feed rate) are determined. The models are tested for their predictability in the time horizon from 1 to 20 min. ANN models are derived with average relative errors for untrained patterns in the range from 1 to 10%.

Bioreactors↗

Utilization of waste products of dehydrated onion industry for production of fodder yeast by Saccharomyces cerevisiae.

One strain of Saccharomyces cerevisiae was selected from different yeasts, isolated from black strap molasses. This microorganism was cultivated on seven fermentation media for the production of protein. Medium I exhibited the highest potentiality for formation of protein. Therefore strain 1 of S. cerevisiae and medium I were used for further studies in the formation of protein. Factors controlling production of protein were explored. The required incubation period for the fermentation process was 72 hrs, while the initial pH value of the medium was 6.0. Sucrose supported the microorganism for higher production of protein (40.96%), while the best concentration of sucrose was shown to be 10.0 g/l. The best inorganic and organic nitrogen sources for protein formation were (NH4)2HPO4, (NH4)3PO4 and yeast extract, respectively. The best concentrations of (NH4)2HPO4 and yeast extract, supporting protein formation, were 5.0 g/l and 10.0 g/l, respectively. Addition of MgSO4, ZnSO4, ferrous ammonium sulphate, copper sulphate, biotin, Ca-pantothenate, thiamine, pyridoxine, and inositol to the synthetic medium did not markedly influence high level of protein formation. Glutamic acid was the best amino acid, supporting protein formation by S. cerevisiae. Onion juice was found to be a good medium, after deletion of inhibitory volatile sulphur organic compounds, for the production of protein by S. cerevisiae. Addition of (NH4)2HPO4 to the best concentration of onion juice assisted the onion medium in production of fodder yeast, containing high level of protein. Addition of MgSO4 to onion juice and (NH4)2HPO4 did not increase the total nitrogen of the biomass. Fodder yeast, produced by onion juice medium, contained more valuable ingredients than fodder yeast, produced by synthetic medium.

Amino Acids↗

Bull exposure and an increased within-day milking to suckling interval reduced postpartum anoestrus in dual purpose cows.

It is hypothesized that the combined effects of suckling and milking in the dual purpose cows is one of the main suppressors of reproductive efficiency in this production system. The experiment described here examined whether managing the interval between milking and suckling could reduce the postpartum anoestrous period and whether the presence of a teaser bull could enhance the effects of these managements. The experiment involved 39 Bos taurus x Bos indicus cows which had an average weight of 523.0 +/- 12.8 kg (mean +/- S.E.M.) and body condition score of 5-7 (scale 1-9) at calving. The cows and calves grazed separate pastures and the cows were supplemented with 2 kg 17% CP concentrates and 1 kg molasses per cow per day. The experiment was conducted over the first 100 days postpartum. Cows were hand-milked once per day in the presence of the calf to stimulate milk release. The factors in the 2 x 2 design were the milking to suckling interval (0 h, control suckling; CS versus 8 h prolonged-delay suckling; PDS) and no exposure versus exposure to a teaser bull (B). Cows were assigned at random within calving date to the four treatments: CS (n = 10), PDS (n = 10), CS-B (n = 9) and PDS-B (n = 10). Cows on treatments CS and CS-B had three-quarters of the udder milked and one-quarter was not milked. The entire udder was milked on those treatments where there was an interval between milking and suckling. The bull was introduced 7 days after calving in treatments where the cows were exposed to a teaser bull. Body weights of cows and calves and cow milk yield were recorded. Weekly blood samples were collected for plasma progesterone assay. Data were analyzed by ANOVA in a 2 x 2 factorial design and by chi(2)-test. There were no statistically significant differences between treatments in cow body weight at calving and at 100 days postpartum, nor in milk yield (overall mean 6.0 +/- 1.1 kg per day). Calf daily gain was 598 +/- 25 g for treatments CS and CS-B in which suckling immediately followed milking and 833 +/- 24 g for treatments PDS and PDS-B (P < 0.001). The proportion of cows ovulating was CS 80.0%, PDS 100.0%, CS-B 88.8% and PDS-B 100.0%. The difference between CS and PDS or PDS-B was significant (P < 0.05). CS-B did not differ significantly from the other three treatments. The calving to first ovulation interval was not improved when the interval between milking and suckling was increased from 0 h (56.5 +/- 5.0 days) to 8 h (64.8 +/- 4.5 days). Exposure to a teaser bull, however, significantly (P < 0.001) reduced this interval from 77.0 +/- 2.7 days (no bull exposure) to 44.4 +/- 2.7 days (exposure to teaser bull). Exposure to a teaser bull significantly reduced the calving to first anoestrus interval. Delaying the start of suckling after milking did not improve the reproductive performance of the cows or alter the effect of bull exposure, however, delaying suckling after milking improved the performance of the calves without affecting milk yield.

Anestrus↗

Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors.

In the first of two experiments investigating the effect of dietary urea on the survival and metabolism of ovine embryos, 30 Border Leicester x Scottish Blackface ewes received a maintenance diet (milled hay, molasses, minerals, vitamins) with no urea (control, C; n = 10) or with added urea at 15 g (low urea, LU; n = 10) or 30 g (high urea, HU; n = 10) kg-1 feed for a 12 week period. The degraded nitrogen (N) status relative to estimated rumen microbial N requirements was -2, +9 and +20 g per day, respectively. One week after allocation to diets, progesterone priming (12 days) commenced. Ewes received 800 IU of equine chorionic gonadotrophin at progesterone withdrawal, were inseminated 52 h later (Day 0) and embryos were collected from five ewes per group at Day 4 and from five ewes at Day 11. If available, one embryo was returned to each ewe; the rest were cultured in vitro. There was no effect of treatment on progesterone, luteinizing hormone (LH), or time of oestrus onset C, LU and HU plasma urea (P < 0.001) and ammonia levels (C vs. HU, P < 0.01; LU vs. HU, P < 0.05) differed. Day 4 HU embryos were retarded relative to C and LU embryos. After 3 days of culture, 70%, 66% and 0% of C, LU and HU embryos, respectively, were viable. Mid-term pregnancy rates following transfer were 63%, 43% and 33%. Only one HU lamb (male) was born following embryo transfer, its birthweight (10.1 kg) exceeded that of its C (n = 3; 7.0, 7.0, 7.5 kg) and LU (n = 2; 7.3, 8.2 kg) counterparts (P < 0.025). In the second experiment, C2 (2.5 g urea kg-1; n = 5) and HU2 (30 g kg-1; n = 7) diets which provided similar intakes of degraded N relative to microbial requirements as those for C and HU ewes in Experiment 1 were fed to Border Leicester x Scottish Blackface ewes superovulated with 16 mg of porcine follicle-stimulating hormone. Urea and ammonia levels in utero-oviductal samples were elevated in HU2 ewes (P < 0.05). At collection (Day 3), HU2 embryos used more glucose (P < 0.01) and, following culture, some exhibited up to a 2.8-fold increase in metabolism. In conclusion, excess rumen degradable N in ewe diets elevates urea and ammonia in plasma and in utero, with an associated increase in embryo mortality. Nevertheless, metabolism appears to be up-regulated in some embryos and, among those that survive, fetal growth appears to be enhanced.

Ammonia↗

Citrus pulp for cattle.

Citrus pulp is classified as an energy concentrate by-product feed. Citrus by-products fed to beef cattle include citrus molasses, citrus meal, wet citrus pulp, dried citrus pulp, and pelleted citrus pulp; however, in current production systems, pulp (wet, dry, and pelleted) is the only by-product commonly used. Citrus pulp production in the United States is limited to specific subtropical regions, of which south central Florida remains the largest with additional production in California and Texas.

Animal Feed↗

The effect of dietary crude protein level on intake, growth, protein retention and utilization of growing male Saanen kids.

The effects of increasing dietary levels of crude protein on growth, feed intake, feed efficiency and composition of gain in male Saanen kids were studied. Four groups of four kids each initially weighing 12.1+/-0.18kg and having a weight gain of 195+/-16g/d were penned individually and fed for 73 to 124 days up to 25kg of BW. They were fed chaffed wheat straw (45g CP/kg DM) which had been molasses sprayed (10%) and pelleted concentrates containing 8.7, 11.7, 14.4 and 17.6% crude protein (CP) on DM basis, the ratio of straw to concentrate being 1:5. Kids were fed controlled to satiation in which case small amounts of the feeds were offered about five times a day as long as the kids wanted to eat. Retention of protein, fat and energy were calculated from their initial and final concentrations in the empty body homogenates of the slaughtered kids. With increasing CP level in the diet, feed intake increased from 448 to 608g DM/d, weight gain from 94 to 181g/d, retention of protein from 9.7 to 27.8g/d, retention of fat from 9.6 to 19.1g/d and feed efficiency improved from 4.79 to 3.39kg DM/kg weight gain. Protein composition of gain increased from 103 to 154g/kg BWG while fat (103-105g/kg BWG) remained constant. Regression analyses showed that BWG can be optimized at 136g CP/kg DM and protein retention at 180g CP/kg DM, whereas, dietary nitrogen was utilized most efficiently at 120g CP/kg DM. Extrapolated from the regression equations, maintenance N requirement of the kids at zero N-retention and at zero BWG were 0.38 and 0.16g N/kg W(0.75), respectively. Recommended dietary CP concentrations and maintenance N requirements depend on the traits desired.

Journal Article↗

Nutritional and cultural parameters influencing antidipteran delta-endotoxin production.

In this study, various nutritional and cultural parameters influencing diptera-specific delta-endotoxin synthesis by Bacillus thuringiensis subsp. israelensis HD500 were investigated. Of various inorganic nitrogen sources, the highest yields of Cry11Aa and Cry4Ba proteins were obtained on (NH(4))(2)HPO(4). Among carbon sources, inulin, dextrin, maltose, lactose, sucrose, whey and glycerol were all stimulatory, while glucose, starch and molasses were suppressive. High concentrations of inorganic phosphate (50 to 100 mM K(2)HPO(4)) were required for an effective synthesis of Cry4Ba. Mn was the most critical element for the biosynthesis of both toxins at 10(-6) M concentration. Mg and Ca favored production when provided at 8 x 10(-3) M and 5.5 x 10(-4) M concentrations, respectively, while Fe, Zn and Cu negatively influenced biosynthesis. Cry4-toxin synthesis was best at neutral pH and also when the organism was grown at 25 degrees C. Throughout the study, the extent of growth and sporulation of the producer organism was also monitored.

Bacillus thuringiensis↗

Regulation of crystal protein biosynthesis by Bacillus thuringiensis: II. Effects of carbon and nitrogen sources.

The regulation of crystal protein production in Bacillus thuringiensis 81 by sources of carbon and nitrogen was investigated. The highest titers of toxin were obtained on sucrose, lactose and inulin which also supported sporulation. Whey and molasses were also potential carbon substrates for toxin production. Other carbohydrates including glucose, glycerol, maltose, starch and dextrin yielded lower amounts of toxin. Nitrogen sources were found to exert the most profound controls. Peptone was the best organic nitrogen source, supporting optimum production and sporulation as well as high cell density. The formation of CryI and CryII toxin proteins was found to be differentially regulated by the inorganic nitrogenous compounds incorporated into the medium.

Bacillus thuringiensis↗

Yeast production from virgin grape marc.

An alternative utilization of virgin grape marc (VGM) to produce SCP from S. ceretisiae is reported. A simple extraction method of fresh grape marc produces a sugar-rich solution: through fed-batch fermentation, a high-value yeast biomass instead of a low-value product like ethanol can be produced. Productivity and quality of yeast are similar to these obtainable from molasses. The convenience of yeast production from VGM is briefly discussed; it appears of great interest in south Italy and generally in grape-producing countries, specially if these lack relevant sources of fermentable sugars.

Alcoholic Beverages↗

Batch co-digestion of multi-component agro-wastes.

In certain parts of the developing world conventional energy supplies such as electricity, gas, coal and petroleum by-products are either unavailable, too capital intensive to install, are unjustifiable due to low population densities in some semi-arid regions, or are simply unaffordable to the target population. In Zimbabwe, it has been assessed that only biomass energy can conveniently provide both lighting and space heating. Therefore, means of generating biogas from agricultural and other organic wastes, and to encourage their use is a policy which has been adopted by Zimbabwe's Department of Energy. In this study cattle slurry was mixed with a range of solid wastes and allowed to digest in 11 batch digesters. The mixtures which were used were selected on the basis of centroid design with the objective of determining whether there was either synergism or antagonism. Two trials were carried out, one based on cattle slurry, chicken manure (CM) and molasses (Mol), the other based on sheep and goat manure, chicken manure and surplus activated sludge. The criteria for judging the success of a co-digestion were volatile solids (VS) reduction, total methane production and methane yield. In the first trial, the analysis based on the methane yield showed that there was no antagonism and that the mixture of 30% cattle slurry/30% CM/40% Mol gave a synergistic effect. The analysis based on the VS destruction, however, did show that there was some very slight antagonism. In the second trial, the analysis based on the methane yield showed that there was both antagonism and synergism and that the synergism produced an extra 6.7% methane. The analysis based on the VS destruction also showed that there was both antagonism and synergism but that the effects were small.

Agriculture↗

Comparison of laboratory-scale thermophilic biofilm and activated sludge processes integrated with a mesophilic activated sludge process.

A combined thermophilic-mesophilic wastewater treatment was studied using a laboratory-scale thermophilic activated sludge process (ASP) followed by mesophilic ASP or a thermophilic suspended carrier biofilm process (SCBP) followed by mesophilic ASP, both systems treating diluted molasses (dilution factor 1:500 corresponding GF/A-filtered COD (COD(filt)) of 1900+/-190 mgl(-1)). With hydraulic retention times (HRTs) of 12-18 h the thermophilic ASP and thermophilic SCBP removed 60+/-13% and 62+/-7% of COD(filt), respectively, with HRT of 8 h the removals were 48+/-1% and 69+/-4%. The sludge volume index (SVI) was notably lower in the thermophilic SCBP (measured from suspended sludge) than in the thermophilic ASP. Under the lowest HRT the mesophilic ASP gave better performance (as SVI, COD(filt), and COD(tot) removals) after the thermophilic SCBP than after the thermophilic ASP. Measured sludge yields were low (less than 0.1 kg suspended solids (SS) kg COD(filt removed)(-1)) in all processes. Both thermophilic treatments removed 80-85% of soluble COD (COD(sol)) whereas suspended COD (COD(susp)) and colloidal COD (COD(col)) were increased. Both mesophilic post-treatments removed all COD(col) and most of the COD(susp) from the thermophilic effluents. In conclusion, combined thermophilic-mesophilic treatment appeared to be easily operable and produced high effluent quality.

Aerobiosis↗