Production of alcohol from raw wheat flour by Amyloglucosidase and Saccharomyces cerevisiae

Ethanol production, by a simultaneous saccharification and fermentation process from raw wheat flour, has been performed by Saccharomyces cerevisiae and a low level of amyloglucosidase enzyme. The fermentation time was about 60 h after a 6 h pre-saccharification, with an amyloglucosidase (AMG) level of 270 AGU. kg(-1) starch, but only 31 h with a simultaneous saccharification fermentation process (SSF). When an AMG level of 540 AGU. kg(-1) starch was used, the time decreased to 21 h, giving an ethanol concentration of 67 g. l(-1). Sugar composition of the wort after the liquefaction may be responsible of the difference between these two process. Maltose, a fermentable sugar, was produced in high concentration during the liquefaction, allowing a shorter process period, counteracting the effect of the slow starch hydrolysis at 35 degrees C (SSF temperature).     

Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica

Ethanol was produced using the simultaneous saccharification and fermentation (SSF) method with macroalgae polysaccharide from the seaweed Saccharina japonica (Sea tangle, Dasima) as biomass. The seaweed was dried by hot air, ground with a hammer mill and filtered with a 200-mesh sieve prior to pretreatment. Saccharification was carried out by thermal acid hydrolysis with H(2)SO(4) and the industrial enzyme, Termamyl 120 L. To increase the yield of saccharification, isolated marine bacteria were used; the optimal saccharification conditions were 10% (w/v) seaweed slurry, 40 mM H(2)SO(4) and 1 g dcw/L isolated Bacillus sp. JS-1. Using this saccharification procedure, the reducing sugar concentration and viscosity were 45.6 ± 5.0 g/L and 24.9 cp, respectively, and the total yield of the saccharification with optimal conditions and S. japonica was 69.1%. Simultaneous saccharification and fermentation was carried out for ethanol production. The highest ethanol concentration, 7.7 g/L (9.8 ml/L) with a theoretical yield of 33.3%, was obtained by SSF with 0.39 g dcw/L Bacillus sp. JS-1 and 0.45 g dcw/L of the yeast, Pichia angophorae KCTC 17574.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Optimization of laccase production by Botryosphaeria sp. in the presence of veratryl alcohol by the response-surface method

Botryosphaeria sp. produced two laccases (PPO-I and PPO-II) constitutively, whose titers were enhanced by veratryl alcohol. The effect of veratryl alcohol and yeast extract concentration, time of cultivation and agitation speed were evaluated by factorial analysis to select variables for optimizing the production of laccases. Maximal laccase production was determined using a second-order central-composite design and analyzed by the response-surface method. Veratryl alcohol concentration and time of cultivation were the main factors increasing laccase production, while yeast extract had no influence within the range 0.2–2.0% w/v. Response-surface analysis showed that 30.4 mM veratryl alcohol, for 4.5 days at 28°C and 180 rpm, were the optimal conditions to maximize PPO-I production, while conditions for maximal PPO-II production occurred within a range of 28–35 mM veratryl alcohol over a growth period of 4–5.5 days. The model predicted 5.6 U ml⁻¹ for PPO-I, and 0.6–1.0 U ml⁻¹ for PPO-II, which agreed with the experimentally observed results.     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Efficient plant regeneration from seedling explants of two commercially important temperate eucalypt species–Eucalyptus nitens and E. globulus

A reliable and reproducible method for plant regeneration in vitro of two important temperate eucalypts, Eucalyptus nitens and E. globulus, has been developed which utilises seedling explants. Highly regenerative callus was obtained from individual cotyledon and hypocotyledon explants of both species following cultivation on Murashige and Skoog’s (MS) basal nutrient medium supplemented with 30 g l⁻¹ sucrose, 5–10% (v/v) coconut water, 0.8% agar, 1 mg l⁻¹ -naphthalene-acetic acid (NAA) and 0.5 mg l⁻¹ N⁶ benzylaminopurine (BAP). Shoot differentiation was observed 7–8 weeks after transfer of callus onto regeneration medium containing 0.5 mg l⁻¹ NAA and 1 mg l⁻¹ BAP. In a few instances, direct shoot regeneration occurred without an intervening callus phase in both species. The frequency of plant regeneration was higher for callus derived from hypocotyl segments (30–35%) compared to cotyledonary explants (20–25%) though the average number of shoots per cotyledonary explant was generally higher than for hypocotyl explants. Somatic embryos were observed occasionally in E. nitens, arising from the surface of organogenic callus. Organised structures closely resembling somatic embryos were also observed in E. globulus. Regenerated shoots (30–40%) of both species could be rooted in modified MS media containing indole-3-butyric acid (IBA) and plantlets were successfully transferred to soil.     

Nutrient removal by thermophilic Fischerella (Mastigocladus laminosus) in a simulated algaculture process

A novel nutrient removal/waste heat utilization process was simulated using semicontinuous cultures of the thermophilic cyanobacterium Fischerella. Dissolved inorganic carbon (DIC)-enriched cultures, maintained with 10 mg l⁻¹ daily productivity, diurnally varying temperature (from 55°C to 26–28°C), a 12:12 light cycle (200 μE sec⁻¹ m⁻²) and 50% biomass recycling into heated effluent at the beginning of each light period, removed > 95% of NO₃⁻ + NO₂⁻−N, 71% of NH₃-N, 82% of PO₄³⁻ −P, and 70% of total P from effluent water samples containing approximately 400 μg l⁻¹ combined N and 60 μg l⁻¹ P. Nutrient removal was not severely impaired by an altered temperature gradient, doubled light intensity, or DIC limitation. Recycling 75% of the biomass at the end of each light period resulted in unimpaired NO₃⁻ + NO₂⁻ removal, 38–45% P removal and no net NH₃ removal. Diurnally varying P removal, averaging 50–60%, and nearly constant > 80% N removal, are therefore projected for a full-scale process with continuous biomass recycling.     

A numerical and experimental study of compliance and collapsibility of preterm lamb tracheae

Knowledge of the mechanical behaviour of immature tracheae is crucial in order to understand the effects exerted on central airways by ventilatory treatments, particularly of Total Liquid Ventilation. In this study, a combined experimental and computational approach was adopted to investigate the compliance and particularly collapsibility of preterm lamb tracheae in the range of pressure likely applied during Total Liquid Ventilation (−30 to 30 cmH₂O). Tracheal samples of preterm lambs (n=5; gestational age 120–130 days) were tested by altering transmural pressure from −30 to 30 cmH₂O. Inflation (S_i) and collapsing (S_c) compliance values were calculated in the ranges 0 to 10 cmH₂O and –10 to 0 cmH₂O, respectively. During the tests, an asymmetric behaviour of the ΔV/V₀ vs. P curves at positive and negative pressure was observed, with mean S_i=0.013 cmH₂O⁻¹ and S_c=0.053 cmH₂O⁻¹. A different deformed configuration of the sample regions was observed, depending on the posterior shape of cartilaginous ring. A three-dimensional finite-element structural model of a single tracheal ring, based on histology measurements of the tested samples was developed. The model was parameterised in order to represent rings belonging to three different tracheal regions (craniad, median, caudal) and numerical analyses replicating the collapse test conditions were performed to evaluate the ring collapsibility at pressures between 0 and −30 cmH₂O. Simulation results were compared to experimental data to verify the model's reliability. The best model predictions occurred at pressures −30 to −10 cmH₂O. In this range, a model composed of median rings best interpreted the experimental data, with a maximum error of 2.7%; a model composed of an equal combination of all rings yielded an error of 12.6%.     

Matching unrelated stimuli with same discriminative functions: training order effects

Previous research has shown that after training simple discriminations (A1+/A2−, B1+/B2−), bringing these tasks under conditional control (J1–A1, J2–A2) leads to transfer of discriminative control (J1+/J2−) and to generalized matching on the basis of same discriminative functions (e.g. J1–B1, J2–B2). The same occurs when conditional discriminations are trained (D1–E1, D2–E2; F1–G1, F2–G2). When the subjects are then trained to demonstrate correct relations (D1–E1, D2–E2) when given X1 and to demonstrate incorrect relations when given X2 (XD–E), transfer of discriminative control (X1+/X2−) and generalized matching on the basis of same discriminative functions emerges (e.g. X1F1–G1, X2F1–G2). The present study investigated if these performances are dependent on the training and/or testing order. In Experiment 1, the lower-order contingency tasks were trained before the higher-order contingency tasks (A1+/A2−, B1+/B2− before J–A, and D–E, F–G before XD–E). Half the subjects received the J–B test before the more complex XF–G test (Condition A), while for the other subjects, this testing order was reversed (Condition B). Finally, all subjects received additional tests in which they were given the opportunity to demonstrate the discriminative properties of the J and X stimuli (J1+/J2−, X1+/X2−), and to match the A, J, and X stimuli with newly introduced stimuli of same discriminative properties (e.g. J1-POLITE, J2-RUDE). Experiment 2 was the same except that the training order was reversed (J–A before A1+/A2−, B1+/B2−, and XD–E before D–E, F–G). The results were affected by the training order but not by the testing order. Transfer of discriminative functions and generalized matching on the basis of same functions only occurred reliably when the lower-order contingency tasks were trained first. A stimulus-control account of the data is offered.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.     

Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor

A membrane bioreactor was developed to perform an extractive bioconversion aimed at the production of isovaleraldehyde by isoamyl alcohol oxidation with whole cells of Gluconobacter oxydans. A liquid/liquid extractive system using isooctane as extractant and assisted by a hollow-fiber hydrophobic membrane was chosen to recover the product. The aqueous bioconversion phase and the organic phase were maintained apart with the aid of the membrane. The extraction of alcohol and aldehyde was evaluated by performing equilibrium and mass transfer kinetic studies. The bioprocess was then performed in a continuous mode with addition of the substrate to the aqueous phase. Fresh solvent was added to the organic phase and exhausted solvent was removed at the same flow rate. The extractive system enabled a fast and selective in situ removal of the aldehyde from the water to the organic phase. High conversions (72–90%) and overall productivity (2.0–3.0 g l⁻¹ h⁻¹) were obtained in continuous experiments performed with different rates of alcohol addition (1.5–3.5 g l⁻¹ h⁻¹). Cell deactivation was observed after 10–12 h of operation.     

Growth, nodulation and nitrate reductase activity in the aquatic legume Neptunia plena (L.) benth. At different external nitrate concentrations

The effects of different external nitrate concentrations (0 (control), 1, 50, 100, 500, 1000 and 20 000 mmol m⁻³) on growth, nodulation and nitrate-reductase activity (NRA) of inoculated Neptunia plena (L.) Benth. were examined.

Plants given 500 and 1000 mmol m⁻³ nitrate had greater (P < 0.05) shoot length, leaf, stem and root dry mass, and carbon and nitrogen contents than the controls and plants given 20 000 mmol m⁻³ nitrate. Nodule number was not significantly affected by nitrate concentration up to 50 mmol m⁻³, but 100 mmol m⁻³ nitrate reduced nodulation by 68% and concentrations above 100 mmol m⁻³ completely inhibited nodule development. Plants given 100–20000 mmol m⁻³ nitrate had a greater nitrate content per g leaf, stem and root dry mass (DM) than controls. Nitrate per g root DM did not increase with external nitrate concentration above 500 mmol m⁻³, but levels in leaf and stem were greater at 20 000 mmol m⁻³ nitrate than at all other concentrations. NRA per g leaf, stem and root fresh mass (FM) was greater for plants given 500–20000 mmol m⁻³ than for controls, but there was no significant increase with nitrate concentration above 500 mmol m⁻³. Substantial proportions of total plant nitrate and NRA were found in both root and shoot over the entire range of external nitrate concentrations given.

Findings for N. plena are compared with data obtained previously for terrestrial legumes.     

High moisture twin-screw extrusion of sago starch: 1. Influence on granule morphology and structure

Effects of barrel temperature (81–149°C) and screw speed (315–486rpm) on extrusion processing of sago starch in a co-rotating twin-screw extruder under a high moisture system (34–47%) were investigated using response surface methodology. Structural changes were characterised by measuring water solubility index (WSI), water absorption index (WAI), degree of gelatinisation (DG), dextrose equivalent (DE) and high performance size-exclusion chromatography (HPSEC) profiles of the extradates. Thermomechanical processing of sago starch in the twin-screw extruder at the high moisture (34–47%) system led to shearinduced limited degradation and starch phase transitions (a composite melting gelatinisation process). Strong positive correlations between WAI, WSI and DG showed that gelatinisation was the fundamental mechanism in this high moisture system rather than dextrinisation. Processing-induced solubility increased at the expense of water absorption. Low WSI (4.5–18.1%) is ascribed to the presence of structures of either granular crystallite remnants or rearrangement of bonds during extrusion.     

Elements in autopsy liver tissue samples from Greenlandic inuit and Danes. V. Selenium measured by X-ray fluorescence spectrometry

The content of selenium in normal liver tissue samples from Greenlandic Inuit was measured and the results compared with those obtained in normal liver tissue samples from Danes. Normal liver tissue samples were obtained at autopsy from 50 Greenlandic Inuit (27 men, 23 women) with a median age of 61 years (range 23–83) and from 74 Danes (44 men, 30 women) with a median age of 60 years (range 15–87). Total liver selenium content was measured by X-ray fluorescence spectrometry. The content of selenium (median) was in Inuit 26.6 mol/kg dry liver (5–95 percentile: 15.2–49.4) and in Danes 17.7 mol/kg dry liver (5–95 percentile: < 3.8–36.5) (p < 0.0001). Liver selenium content displayed no significant gender difference, either in Inuit or Danes. In Inuit men, there was a negative correlation between liver selenium content and age (r_s = −0.39, p < 0.05), whereas Danish men displayed a positive correlation between liver selenium content and age (r_s = 0.37, p = 0.02). There was no correlation in Inuit or Danish women. In Inuit, the median hepatic selenium index (liver selenium content divided by age) was 0.48 and in Danes 0.33 (p = 0.001). There was an inverse correlation between hepatic selenium index and age both in Inuit (r_s = −0.77, p < 0.0001) and in Danes (r_s = −0.47, p < 0.0001). In conclusion, Inuit had a higher liver content of selenium and a higher hepatic selenium index compared with Danes. The more favourable selenium status is due to a higher nutritional selenium intake with fish and meat from sea mammals.     

Effect of vibration magnitude, vibration spectrum and muscle tension on apparent mass and cross axis transfer functions during whole-body vibration exposure

Twelve seated male subjects were exposed to 15 vibration conditions to investigate the nature and mechanisms of the non-linearity in biomechanical response. Subjects were exposed to three groups of stimuli: Group A comprised three repeats of random vertical vibration at 0.5, 1.0 and 1.5 m s⁻² r.m.s. with subjects sitting in a relaxed upright posture. Group B used the same vibration stimuli as Group A, but with subjects sitting in a ‘tense’ posture. Group C used vibration where the vibration spectrum was dominated by either low-frequency motion (2–7 Hz), high-frequency motion (7–20 Hz) or a 1.0 m s⁻² r.m.s. sinusoid at the frequency of the second peak in apparent mass (about 10–14 Hz) added to 0.5 m s⁻² r.m.s. random vibration. In the relaxed posture, frequencies of the primary peak in apparent mass decreased with increased vibration magnitude. In the tense posture, the extent of the non-linearity was reduced. For the low-frequency dominated stimulus, the primary peak frequency was lower than that for the high-frequency dominated stimulus indicating that the frequency of the primary peak in the apparent mass is dominated by the magnitude of the vibration encompassing the peak. Cross-axis transfer functions showed peaks of about 15–20% and 5% of the magnitudes of the peaks in the apparent mass for x- and y-direction transfer functions, respectively, in the relaxed posture. In the tense posture, cross-axis transfer functions reduced in magnitude with increased vibration, likely indicating a reduced fore-aft pitching of the body with increased tension, supporting the hypothesis that pitching contributes to the non-linearity in apparent mass.     

Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Bioethanol production from barley hull using SAA (soaking in aqueous ammonia) pretreatment

Barley hull, a lignocellulosic biomass, was pretreated using aqueous ammonia, to be converted into ethanol. Barley hull was soaked in 15 and 30 wt.% aqueous ammonia at 30, 60, and 75 °C for between 12 h and 11 weeks. This pretreatment method has been known as “soaking in aqueous ammonia” (SAA). Among the tested conditions, the best pretreatment conditions observed were 75 °C, 48 h, 15 wt.% aqueous ammonia and 1:12 of solid:liquid ratio resulting in saccharification yields of 83% for glucan and 63% for xylan with 15 FPU/g-glucan enzyme loading. Pretreatment using 15 wt.% ammonia for 24–72 h at 75 °C removed 50–66% of the original lignin from the solids while it retained 65–76% of the xylan without any glucan loss.

Addition of xylanase along with cellulase resulted in synergetic effect on ethanol production in SSCF (simultaneous saccharification and co-fermentation) using SAA-treated barley hull and recombinant E. coli (KO11). With 3% w/v glucan loading and 4 mL of xylanase enzyme loadings, the SSCF of the SAA treated barley hull resulted 24.1 g/L ethanol concentration at 15 FPU cellulase/g-glucan loading, which corresponds to 89.4% of the maximum theoretical yield based on glucan and xylan.

SEM results indicated that SAA treatment increased surface area and the pore size. It is postulated that these physical changes enhance the enzymatic digestibility in the SAA treated barley hull.