Measurement and analysis of intracellular ATP levels in metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures

Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.     

Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK₁₇

The ethanol production capacity from sugars and lignocellulosic biomass hydrolysates (HL) by Thermoanaerobacterium strain AK(17) was studied in batch cultures. The strain converts various carbohydrates to, acetate, ethanol, hydrogen, and carbon dioxide. Ethanol yields on glucose and xylose were 1.5 and 1.1 mol/mol sugars, respectively. Increased initial glucose concentration inhibited glucose degradation and end product formation leveled off at 30 mM concentrations. Ethanol production from 5 g L(-1) of complex biomass HL (grass, hemp, wheat straw, newspaper, and cellulose) (Whatman paper) pretreated with acid (0.50% H(2) SO(4)), base (0.50% NaOH), and without acid/base (control) and the enzymes Celluclast and Novozyme 188 (0.1 mL g(-1) dw; 70 and 25 U g(-1) of Celluclast and Novozyme 188, respectively) was investigated. Highest ethanol yields (43.0 mM) were obtained on cellulose but lowest on hemp leafs (3.6 mM). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The influence of various factors (HL, enzyme, and acid/alkaline concentrations) on end-product formation from 5 g L(-1) of grass and cellulose was further studied to optimize ethanol production. Highest ethanol yields (5.5 and 8.6 mM ethanol g(-1) grass and cellulose, respectively) were obtained at very low HL concentrations (2.5 g L(-1)); with 0.25% acid/alkali (v/v) and 0.1 mL g(-1) enzyme concentrations. Inhibitory effects of furfural and hydroxymethylfurfural during glucose fermentation, revealed a total inhibition in end product formation from glucose at 4 and 6 g L(-1), respectively.     

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.     

Citric acid production by Candida lipolytica Y 1095 in cell recycle and fed-batch fermentors

The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed-batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h(-1), a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L . h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell . h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L . h) were obtained in the fed-batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed-batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed-batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed-batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed-batch fermentor were dependent on the cycle time and dissolved oxygen concentration. (c) 1995 John Wiley & Sons, Inc.     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus

A 5 l packed bed bioreactor was used to study the effect of initial lactose concentration and hydraulic retention time (HRT) on cell growth, lactose utilization and lactic acid production. Up to 95% of the initial lactose concentration was utilized at longer HRTs (30-36 h). The study showed that lactic acid production increased with increases in HRT (12-36 h) and initial lactose concentrations. The highest lactic acid production rate (3.90 g l(-1) h(-1)) was obtained with an initial lactose concentration of 100 g/l and an HRT of 18 h, whereas the lowest lactic acid production rate (1.35 g l(-1) h(-1)) was obtained with an initial lactose concentration of 50 g/l and an HRT of 36 h. This suggested that optimal lactic acid production can be achieved at an HRT of 18 h and initial lactose concentration of 100 g/l.     

Lipid production and fatty acid composition of selected strains belonging to Mucorales

The lipid accumulation, fatty acid composition and γ-linolenic acid (GLA) production by 28 strains belonging to Mucorales were investigated. The lipid content varied from 5 to 30% on dry biomass and the percentage of γ-linolenic acid in total intracellular lipid was in a range from 2.5 to 15.4% (w/w). The best yield of γ-linolenic acid (expressed as mg GLA per 1 g biomass) was found for Mucor mucedo CCF – 1384 (28.4) and Cunninghamella echinulata CCF – 103 (25.1).     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.     

Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.     

Lactic acid production from agricultural resources as cheap raw materials

Agricultural resources such as barley, wheat, and corn were hydrolyzed by commercial amylolytic enzymes and fermented into lactic acid by Enterococcus faecalis RKY1. Although no additional nutrients were supplemented to those resources, lactic acid productivities were obtained at >0.8 g/l h from barley and wheat. When 200 g/l of whole wheat flour was hydrolyzed by amylolytic enzymes after the pre-treatment with 0.3% (v/v) sulfuric acid and sterilized by filtration, E. faecalis RKY1 efficiently produced lactic acid with 2.6 g/l h of lactic acid productivity and 5.90 g/l of maximal dry cell weight without additional nutrients. Lactic acid productivity and cell growth could be enhanced to 31% and 12% higher values than those of non-adapted RKY1, by adaptation of E. faecalis RKY1 to CSL-based medium. When the medium contained 200 g/l of whole wheat flour hydrolyzate, 15 g/l of corn steep liquor, and 1.5 g/l of yeast extract, lactic acid productivity and maximal dry cell weight were obtained at 5.36 g/l h and 14.08 g/l, respectively. This result represented an improvement of up to 106% of lactic acid productivity and 138% of maximal dry cell weight in comparison to the fermentation from whole wheat flour hydrolyzate only.     

Continuous itaconic acid production by immobilized biocatalysts.

The continuous itaconic acid production from sucrose with Aspergillus terreus TKK 200-5-3 mycelium immobilized on polyurethane foam cubes was optimized in column bioreactors using statistical experimental design and empirical modelling. The highest itaconic acid product concentration calculated on the basis of the obtained model was 15.8 g l-1 in the investigated experimental area, when sucrose concentration was 13.5%, aeration rate 150 ml min-1 and residence time 178 h. From sucrose with immobilized A. terreus TKK 200-5-3 mycelium itaconic acid production was stable for at least 4.5 months in continuous column bioreactors. In comparison, using glucose as substrate and immobilized A. terreus TKK 200-5-1 mycelium as biocatalyst similar stability was obtained with higher product concentration. The omission of copper sulphate from the production medium gave the highest itaconic acid product concentration (26 g l-1) from 9% glucose with 0.25% ammonium nitrate and 0.095% magnesium sulphate.     

Levels of sulfhydryls and disulfides in proteins from Neurospora crassa conidia and mycelia

Proteins extracted with 6 M guanidine at 90 degrees C from conidia (asexual spores) of Neurospora crassa contained ca. 25% more total protein thiol and a fivefold-higher content of disulfide bonds than proteins extracted from mycelia, as determined by labeling with iodo[14C]acetic acid. The total thiol content was 88 mumol/g of protein in conidia and 70 mumol/g of protein in mycelia. The level of protein disulfide was 18.5 mumol/g of protein in conidia and 3.5 mumol/g of protein in mycelia, by the iodo[14C]acetic acid labeling method. Confirmatory results were obtained with 5'5-dithio-bis-2-nitrobenzoic acid titration of protein thiol groups in 1% sodium dodecyl sulfate as well as by amino acid analysis of cysteic acid derivatives. Buffer-extracted proteins from conidia, but not mycelia, were found to contain enriched levels of protein thiols and disulfides per gram of protein as compared with guanidine hydrochloride extracts. It was demonstrated that the high disulfide content of crude conidial extracts was not due to measurable levels of mixed disulfides formed between protein sulfhydryl groups and cysteine. During germination of the conidia, the high disulfide levels of the conidial proteins remained constant. These data suggest that, unlike the disulfides of glutathione, the bulk of conidial protein disulfides were not reduced, excreted, or extensively degraded during germination.     

Effects of medium glucose concentration and pH on docosahexaenoic acid content of heterotrophic Crypthecodinium cohnii

The effects of medium glucose concentration and pH on growth and docosahexaenoic acid (DHA, C22:6 ω-3) content of Crypthecodinium cohnii were investigated. Over a range of glucose concentrations (5–40 g l⁻¹) investigated, the highest specific growth rate (0.12 h⁻¹), highest cell dry weight concentration (3.13 g l⁻¹) and highest growth yield on glucose (0.6 g g⁻¹) were obtained at 20 g l⁻¹ glucose. However, the highest degree of fatty acid unsaturation (3.2) and highest DHA proportion (53.4% of total fatty acids) were achieved at 5 g l⁻¹ glucose. Low glucose concentrations enhanced the degree of fatty acid unsaturation and DHA formation. Medium pH also affected cell growth, fatty acid unsaturation and DHA proportion. When medium pH was 7.2, the highest specific growth rate (0.089 h⁻¹), highest cell dry weight concentration (2.73 g l⁻¹), highest growth yield on glucose (0.564 g g⁻¹), highest degree of fatty acid unsaturation (3.4) and highest DHA proportion (56.8% of total fatty acids) were obtained. Results suggest that glucose concentration and pH value could be effectively manipulated to achieve maximum DHA production by C. cohnii.     

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO₂. The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05–2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.     

A rotational drum fermentation system with water flushing for enhancing hydrolysis and acidification of solid organic wastes

In this work, fresh soybean meal was used as the substrate for both batch and continuous experiments in a rotational drum fermentation (RDF) system to characterize the acidogenic process of solid organic waste degradation at high unionized volatile acid (U-VA) level and evaluate the effect of water flushing on the acidogenic performance. The experiments were conducted under mesophilic condition with a reaction time of 20 days. The results of the batch experiment showed that U-VA had a growing adverse effect on the volatile acid (VA) production and hydrolysis of the substrate as the initially added U-VA concentration increased (0, 5, 15, 25 g/L). VA formation deteriorated drastically when the initial U-VA concentration exceeded 5 g/L. VS degradation ratios decreased from 43.8% to 7.3%, and the hydrolysis rate constants varied between 28.8 and 3.8 x 10(-3)/d in response to the initial U-VA concentration. In the continuous experiment, two cascade process configurations (CP1 and CP2) without and with VA removal by water flushing, respectively, were developed. The results showed that the hydrolysis rate constants and VS degradation ratios were 13.1 x 10(-3)/d and 23%, respectively, in CP2, while only 9.1 x 10(-3)/d and 16.7% in CP2. Compared to CP1, the VA spectrum varied little in CP2 with water flushing. It suggested that the higher U-VA level had a significant inhibition on the acidogenic process of solid organic waste degradation, and the VA removal by water flushing improved the acidogenic performance.     

The trout (Salmo trutta) population of the Afon Cwm, a small tributary of the Afon Dyfi, mid-Wales

Data are presented from a 10-year (1984 to 1993) study of a Salmo trutta population in the Afon Cwm, a small tributary of the Afon Dyfi, mid-Wales. The stream is a spawning and nursery area for sea trout. Growth of trout within the stream can be summarized by a von Bertalanffy growth coefficient ( K ) of 0·310, with asymptotic length (1∞) 21·6 cm and with length at age 1 of 7·6 cm. Mean population density in the whole stream varied from year to year between 0·05 and 0·60 0-group trout m⁻² and between 0·05 and 0·70 older trout m⁻². Mean biomass varied, between years, from 0·1 to 3·5 g m⁻² for 0-group and from 1·3 to 10·4g m⁻² for older trout. Loss between 3 and 5 months of age appeared to be proportionate at about 50 to 60% and instantaneous loss rate from 5 to 53 months of age varied from 0·04 to 0·10 month⁻¹ and was positively correlated with cohort number at 3 months of age. Production between 3 and 53 months of age varied between cohorts from 3 to 8 g m ⁻² live weight.     

酶法合成头孢环己二烯

以环己二烯甘氨酸甲酯盐酸盐为酰基供体,7-氨基脱乙酰氧基头孢烷酸为酰基受体,γ－氧化铝为载体的固定化巨大芽孢杆菌胞外青霉素G酰化酶为酰化剂,合成了头孢环己二烯。5％酰基供体,2％酰基受体,每毫升反应物加44IU固定化酶,pH7．5,25℃振荡反应5h,头孢环己二烯产率为81％。苯乙酸、苯氧乙酸和头孢霉素G对酶法合成有不同程度的抑制作用。     

Occurrence and Distribution of 5-Hydroxytryptophol in the Rat

Abstract: The distribution of the serotonin metabolites 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was determined in the rat by a sensitive and specific gas chromatography-mass spectrometric assay. 5-HTOL occurred in all tissues assayed, with highest concentrations in small intestine (mean ± S.E.M. = 193 ± 13 mg/g), lung (78.8 ± 13.2 mg/g), and liver (64.1 ± 4.9 mg/g). Brain 5-HTOL concentrations (9.80 ± 0.36 mg/g) were only 1% of brain 5-HIAA levels. Conjugated 5-HTOL accounted for a significant fraction of the total 5-HTOL concentrations in all tissues and varied from 20% in heart to 70% in kidney. In plasma and urine, 5-HTOL occurred almost completely in conjugated form. Except for liver, 5-HIAA concentrations were substantially greater than 5-HTOL in all tissues, plasma, and urine. Highest 5-HIAA concentrations occurred in brain (787 ± 28 mg/g), lung (744 ± 52 mg/g), and small intestine (424 ± 35 mg/g). 5-HTOL concentrations in plasma and urine were about 25% of the respective 5-HIAA levels. It is concluded that significant biotransformation of serotonin to 5-HTOL in the rat occurs in the intestine, liver, and lung while in brain formation of 5-HTOL represents a minor pathway of serotonin metabolism.     

Interconversions and production of volatile fatty acids in the sheep rumen

1. Sheep fed at a constant rate were infused intraruminally with [1-(14)C]-acetate, -propionate or -butyrate during 5hr. periods. 2. Volatile fatty acids were estimated in the rumen contents and steady-state conditions were obtained. 3. Of the butyric acid carbon 60% was in equilibrium with 20% of the acetic acid carbon, and 2-3g.atoms of carbon were interconverted/day. 4. Little interconversion took place between propionic acid, acetic acid or butyric acid. 5. The net production rates for acetic acid, propionic acid and butyric acid were 3.7, 1.0 and 0.7moles/day respectively. 6. The production of volatile fatty acids accounted for 80% of the animal's energy expenditure.