Ethanol production in multistage continuous, single stage continuous, Lactobacillus-contaminated continuous, and batch fermentations

Saccharomyces cerevisiae-based ethanol fermentations were conducted in batch culture, in a single stage continuous stirred tank reactor (CSTR), a multistage CSTR, and in a fermentor contaminated with Lactobacillus that corresponded to the first fermentor of the multistage CSTR system. Using a glucose concentration of 260 g l^–1 in the medium, the highest ethanol concentration reached was in batch (116gl^–1), followed by the multistage CSTR (106gl^–1), and the single stage CSTR continuous production system (60gl^–1). The highest ethanol productivity at this sugar concentration was achieved in the multistage CSTR system where a productivity of 12.7gl^–1h^–1 was seen. The other fermentation systems in comparison did not exceed an ethanol productivity of 3gl^–1h^–1. By performing a continuous ethanol fermentation in multiple stages (having a total equivalent working volume of the tested single stage), a 4-fold higher ethanol productivity was achieved as compared to either the single stage CSTR, or the batch fermentation.     

Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove-fringed estuary,Khung Krabaen Bay (Thailand) using dual stable isotope techniques

Stable carbon (¹³C) and nitrogen (¹⁵N) isotopes were used to elucidate primary food sources and trophic relationships of organisms in Khung Krabaen Bay and adjacent offshore waters. The three separate sampling sites were mangroves, inner bay and offshore. The ¹³C values of mangrove leaves were –28.2 to –29.4, seagrass –10.5, macroalgae –14.9 to –18.2, plankton –20.0 to –21.8, benthic detritus –15.1 to –26.3, invertebrates –16.5 to –26.0, and fishes –13.4 to –26.3. The ¹⁵N values of mangrove leaves were 4.3 to 5.7, seagrass 4.3, macroalgae 2.2 to 4.4, plankton 5.7 to 6.4 , benthic detritus 5.1 to 5.3, invertebrates 7.2 to 12.2 , and fishes 6.3 to 15.9. The primary producers had distinct ¹³C values. The ¹³C values of animals collected from mangroves were more negative than those of animals collected far from shore. The primary carbon sources that support food webs clearly depended on location. The contribution of mangroves to food webs was confined only to mangroves, but a mixture of macroalgae and plankton was a major carbon source for organisms in the inner bay area. Offshore organisms clearly derived their carbon through the planktonic food web. The ¹⁵N values of consumers were enriched by 3–4 relative to their diets. The ¹⁵N data suggests that some of aquatic animals had capacity to change their feeding habits according to places and availability of foods and as a result, individuals of the same species could be assigned to different trophic levels at different places.     

Polar bears make little use of terrestrial food webs: evidence from stable-carbon isotope analysis

Summary The mean stable-carbon isotope ratios (¹³C) for polar bear (Ursus maritimus) tissues (bone collagen –15.7, muscle –17.7, fat –24.7) were close to those of the same tissues from ringed seals (Phoca hispida) (–16.2, –18.1, and –26.1, respectively), which feed exclusively from the marine food chain. The ¹³C values for 4 species of fruits to which polar bears have access when on land in summer ranged from –27.8 to –26.2, typical of terrestrial plants in the Arctic. An animal's ¹³C signature reflects closely the ¹³C signature of it's food. Accordingly, the amount of food that polar bears consume from terrestrial food webs appears negligible, even though some bears spend 1/3 or more of each year on land during the seasons of greatest primary productivity.     

Production of high molecular weight pullulan by Aureobasidium pullulans using glucosamine

Pullulan productivity was optimized in Aureobasidium pullulans ATCC 42023 with 54 g glucose l^–1. Pullulan with its higher molecular weight (>1000000) was produced using 2% (w/v) glucose and 3% (w/v) glucosamine together. The maximum concentration of pullulan was 8 g l^–1 at 140 h with shake-flask culture.     

Thermal stability and thermodynamic analysis of native and methoxypolyethylene glycol modified trypsin

Four methoxypolyethylene glycols (MPEG, molecular masses 350, 750, 2000 and 5000 Da), each activated by nitrophenyl chloroformate, were used to modify trypsin. Compared with the native trypsin, the MPEG-modified trypsin was more stable against temperature between 30°C and 70°C, longer chain of MPEG moiety corresponding to higher thermal stability. The T for the native and the modified trypsin (0.4 mg ml^–1) was increased from 47°C to 66°C. The stabilization effect caused by MPEG modification was the result of decreasing in both the autolysis rate and the thermal denaturation rate. The thermodynamic analysis of the thermal denaturation process showed that the activation free energy (G^*) of the native and the modified trypsin at 60°C was increased from 102.9 to 109.3 kJ mol^–1; the activation enthalpy (H^*) was increased from 57.4 to 86.9 kJ mol^–1; the activation entropy (S^*) was increased from –136 to –67 J molK^–1. A possible explanation for the decreased thermal denaturation rate caused by MPEG modification was also discussed.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Mixed-acid fermentation and polysaccharide production by Lactobacillus helveticus in milk cultures

Lactobacillus helveticus grown in milk with pH control at 6.2 had a slower growth rate (=0.27 h^–1) and produced less exopolysaccharide (49 mg l^–1) but increased lactic acid production (425 mM) compared to cultures without pH control (=0.5 h^–1, 380 mg exopolysaccharide l^–1, and 210 mM lactate), respectively. Both cultures displayed a mixed-acid fermentation with formation of acetate, which is linked not only to citrate metabolism, but also to alternative pathways from pyruvate.     

Comparison of laboratory- and field-derived soil water retention curves for a fine sand soil using tensiometric,resistance and capacitance methods

The approximate range from 100 to 50% of plant-available water in Apopka fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult) is 0.08–0.04 cm³ cm^–3 soil water content () or –5 to –15 kPa of soil water matric potential (). This narrow range of plant-available soil water is extremely dry for most soil water sensors. Knowledge of the soil water retention curves for these soils is important for effective irrigation of crops in fine sand soils of subtropical and tropical regions of the world. The primary objective of this study was to compare sandy soil water retention curves in the field as measured by tensiometer and resistance block values and capacitance sensor . The second objective was to compare these curves to one developed on a Florida fine sand soil using a pressure plate apparatus. Tensiometer and resistance block values were compared to values from capacitance sensors calibrated gravimetrically. The effective range of both tensiometers and resistance sensors in fine sand soils is between –5 and –20 kPa . Soil water potential values for both sensors were within 2 kPa of the mean for each sensor. Change in was similar over the range of 0.04–0.08 cm³ cm^–3 . Curves for the two sensors were different by 4 kPa at 0.04 cm³ cm^–3. The relationship between and were similar at 10–20, 20–30 and 40–50 cm depths. This was not true for a laboratory determined soil water retention curve for the same soil type. These differences are significant in soils with very low water holding capacities. Differences between laboratory- and field-determined retention curves could be due to a combination of entrapped air in the field soil and/or alteration in bulk density in the laboratory samples.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

Net N input and riverine N export from Illinois agricultural watersheds with and without extensive tile drainage

Some of the largest riverine N fluxes in the continental USA have been observed in agricultural regions with extensive artificial subsurface drainage, commonly called tile drainage. The degree to which high riverine N fluxes in these settings are due to high net N inputs (NNI), greater transport efficiency caused by the drainage systems, or other factors is not known. The objective of this study was to evaluate the role of tile drainage by comparing NNI and riverine N fluxes in regions of Illinois with similar climate and crop production practices but with different intensities of tile drainage. Annual values of NNI between 1940 and 1999 were estimated from county level agricultural production statistics and census estimates of human population. During 1945–1961, riverine nitrate flux in the extensively tile drained region averaged 6.6kgNha^–1year^–1 compared to 1.3 to 3.1kgNha^–1 for the non-tile drained region, even though NNI was greater in the non-tile drained region. During 1977–1997, NNI to the tile-drained region had increased to 27kgNha^–1year^–1 and riverine N flux was approximately 100% of this value. In the non-tile-drained region, NNI was approximately 23kgNha^–1year^–1 and riverine N flux was between 25% and 37% of this value (5 to 9kgNha^–1year^–1). Denitrification is not included in NNI and, therefore, any denitrification losses from tile-drained watersheds must be balanced by other N sources, such as depletion of soil organic N or underestimation of biological N fixation. If denitrification and depletion of soil organic N are significant in these basins, marginal reductions in NNI may have little influence on riverine N flux. If tile drained cropland in Illinois is representative of the estimated 11 million ha of tile drained cropland throughout the Mississippi River Basin, this 16% of the drainage area contributed approximately 30% of the increased nitrate N flux in the Lower Mississippi River that occurred between 1955 and the 1990s.     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Batch cultivation and astaxanthin production by a mutant of the red yeast,Phaffia rhodozyma NCHU-FS501

Phaffia rhodozyma cells were treated with the mutagenic agent NTG several times and plated on yeast-malt agar containing -ionone as a selective medium. This mutagenesis of the yeast yielded a mutant (NCHU-FS501) with a total carotenoid content of 1454 g g^–1 dry biomass. Temperature and pH had only a slight effect on the volumetric pigment production by the red yeast, however astaxanthin yield and specific growth rate were influenced more significantly by temperature and pH. The optimum inoculum size, temperature and air flow rate for astaxanthin formation by the mutant in a bench-top fermentor were 7.5% (v/v), 22.5°C and 3.6 vvm, respectively. Glucose (1%, w/v) as carbon source yielded the highest volumetric astaxanthin production (6.72 g ml^–1). Peptone (15.8% total nitrogen) was the best nitrogen source for astaxanthin production (6.72 g ml^–1). Pigment formation by the mutant was further improved by increasing the glucose concentration to 3.5%, where the astaxanthin concentration was 16.33 m ml^–1. At 4.5% glucose or above astaxanthin formation was inhibited. Control of the pH of the fermentation broth did not improved pigment production.     

Exopolysaccharide biosynthesis and related enzyme activities of the medicinal fungus, Ganoderma lucidum, grown on lactose in a bioreactor

Exopolysaccharide (EPS) production and biosynthesis were studied in Ganoderma lucidum, a fungus used in traditional Chinese medicine, grown with lactose in a bioreactor. -Galactosidase activity, which implies the existence of a lactose permease system, was induced by lactose. Lactose feeding also increased -phosphoglucomutase activity and EPS accumulation but decreased phosphoglucose isomerase activity and lactate concentration in the culture broth. A maximum cell density of 22 g l^–1 and EPS at 1.25 g l^–1 were obtained in fed-batch bioreactor culture.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Radial transport of water across cortical sleeves of excised roots ofZea mays L.

The stationary radial volume flows across maize (Zea mays L.) root segments without steles (sleeves) were measured under isobaric conditions. The driving force of the volume flow is an osmotic difference between the internal and external compartment of the root preparations. It is generated by differences in the concentrations of sucrose, raffinose or polyethylene glycol. The flows are linear functions of the corresponding osmotic differences ( ) up to osmotic values which cause plasmolysis. The straight lines obtained pass through the origin. No asymmetry of the osmotic barrier could be detected within the range of driving forces applied ( =±0.5 MPa), corresponding to volume-flow densities of j_{v, s}=±7·10^–8 m·s^–1. Using the literature values for the reflection coefficients of sucrose and polyethylene glycol in intact roots (E. Steudle et al. (1987) Plant Physiol.84, 1220–1234), values for the sleeve hydraulic conductivity of about 1·10^–7 m·s^–1 MPa^–1 were calculated. They are of the same order of magnitude as those reported in the literature for the hydraulic conductivity of intact root segments when hydrostatic pressure is applied.Abbreviations and symbols a _s outer surface of sleeve segment - c concentration of osmotically active solute - j _{v, s} radial volume flow density across sleeve segment - Lp_s hydraulic conductivity of sleeves - Lp_r hydraulic conductivity of intact roots - _N thickness of Nernst diffusion layer - reflection coefficient of root for solute - osmotic value of bulk phase - osmotic coefficient     

Composition and mean residence time of molecular weight fractions of organic matter extracted from two soils under different forest species

The organic matter extracted from various mineral horizons of two forest soils, one under silver fir (Abies alba Mill.), the other under European beech (Fagus sylvatica L.), was fractionated by dialysis into three fractions, 100–1000, 1000–8000, and >8000Da. On a C basis, in all horizons the recovered organic matter amounted to less than a half of the total and was mainly composed of molecules >8000Da. The 100–1000Da fraction had a principal elemental composition profoundly different from the other two fractions, which, instead differed from each other significantly only for the S content and the molar ratio of C with N. No significant difference in this regard was found between soils. The richness in O and some typical absorption bands in the FT-IR spectra indicated that the 100–1000Da fraction had a lot of carboxyl moieties. The spectroscopic (¹³C NMR) investigation showed that the 1000–8000 and >8000Da fractions had a prevalently aliphatic nature and signals attributable to polysaccharides (O-alkyl C) revealed overall a high presence of non-humic biopolymers. These latter were significantly more abundant, suggesting a lower degree of humification, in the >8000Da fraction than in the 1000–8000Da fraction. Comparing soils, that under beech appeared significantly richer in O-alkyl C than that under fir. The organics extracted from the A horizon of both soils had positive ¹⁴C values, indicating recent synthesis mainly due to the present forest cover. The mean residence time (MRT) of the combined 100–1000Da and 1000–8000Da fractions and the >8000Da fraction increased with depth, even to about 5000 years in the more than 1-m deep BC horizons under beech. In some cases, and especially in the soil under fir, despite higher values of ¹³C denoting stronger microbial decomposition, the 100–8000Da fraction showed a higher MRT than that of the >8000Da fraction, perhaps due to its ascertained lower content of non-humic biopolymers.     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.     

Qualitative Analysis of the Carbohydrate Composition of Apolipoprotein H

The specific binding of digoxigenin-labeled lectins to carbohydrate moieties is used to characterize the carbohydrate chains bound to apolipoprotein H. Our results show that apolipoprotein H is rich in sialic acid linked (2–6) to galactose or N-acetylgalactosamine. Sialic acid is not (2–3)-linked to galactose. Galactose is (1–4)-linked to N-acetylglucosamine and (1–3)-linked to N-acetylgalactosamine. High-mannose N-glycan chains are barely detectable. After N-glycosidase F treatment the molecular weight is substantially reduced. The main band is 32,500 daltons. Carbohydrate O-linked chains, which are mainly represented by sialic acid, are (2–6)-linked to galactose or N-acetylgalactosamine. Galactose is also organized in O-linked chains and it is (1–4)-linked to N-acetylglucosamine and (1–3)-linked to acetylgalactosamine. Biochemical analysis of carbohydrate structures reveals that no specific carbohydrate complex is bound to a single isoform.