Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Metabolism of a phenylcoumaran substructure lignin model compound in ligninolytic cultures of Phanerochaete chrysosporium

The degradation of the phenylcoumaran substructure model compound methyl dehydrodiconiferyl alcohol by the white-rot wood decay fungus Phanerochaete chrysosporium was investigated using culture conditions optimized for lignin oxidation. Initial attack was in the cinnamyl alcohol side chain, which was oxidized to a glycerol structure. This was subsequently converted by loss of the two terminal carbon atoms, C and C, to yield a C-aldehyde structure, which was further oxidized to the C-acid compound. The next detected intermediate, a phenylcoumarone, was produced by double bond formation between C and C, and oxidation of the C-alcohol to an aldehyde group. Further oxidation of C to an acid yielded the next intermediate. The final identified degradation product was veratric acid. No products from the 5-substituted aromatic ring, and no phenolic products, were found. The initial glycerol-containing intermediate was a mixture of the threo and erythro forms, and no optical activity could be found, suggesting that its formation might have involved nonstereospecific C-C epoxidation followed by non-enzymatic hydrolysis of the epoxide.Abbreviations TLC thin layer chromatography - LDA lithium diisopropyl amide - DDQ 2,3-dichloro-5,6-dicyanobenzoquinone - MS mass spectrometry - UV ultraviolet spectroscopy     

Simultaneous recognition of two chiral centers in α,β-diastereomeric amino acids by an N-carbamoyl-L-α-amino acid amidohydrolase from Alcaligenes xylosoxidans

Cells of Alcaligenes xylosoxidans containing N-carbamoyl-L--amino acid amidohydrolase strictly distinguished the configuration of not only the -carbon but also the -carbon of N-carbamoyl--methylphenylalanine, and produced threo-l--methylphenylalanine specifically from a mixture of the four stereoisomers.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Physiology of α-amylase production by immobilized Bacillus amyloliquefaciens

Summary Bacillus amyloliquefaciens 321S cells were immobilized with 3.4% -carrageenan gel in bead form, and -amylase production by the immobilized cells was studied. Cells in the gel, after the population reached maximum were restricted to a layer of 50 m thickness, from the surface of the gel, suggesting that oxygen diffusion is the growth limiting factor. The specific respiratory activity and the growth rate of the entrapped cells under such conditions were 1/2 and 1/5 1/10, respectively, that of free cells. In spite of the repressed respiration and growth, the specific rate of -amylase production of the entrapped cells reached the maximum value of free cells or higher.In continuous culture, in an aerated vessel with a volume ratio of gel beads to medium of 1:2, the maximum production rate of -amylase was obtained at a dilution rate of 1.0 h^–1, which was double the maximum specific growth rate of the strain.These results showed that bacterial -amylase production, which is a nongrowth-associated type of synthesis was achieved with the use of immobilized cells.     

Isothermic variation of the specific growth rate of Saccharomyces cerevisiae in batch culture

The specific growth rate () of a respiration-deficient mutant of Saccharomyces cerevisiae growing under defined experimental conditions in batch culture (mineral medium plus glucose and vitamins at 25°C) varied from experiment to experiment over a wide range (0.10–0.24 h^-1) and showed a normal distribution. Neither the age of the culture, the history of the inoculum, nor experimental error accounted wholy for the variability of . The variation was positively correlated with the specific rate of glucose transfer and negatively with the specific rate of production of non-fermentative CO₂. The yield decreased with implying higher maintenance requirements in batch culture (4.7 mmoles g^-1 h^-1) than in continuous culture (0.8 mmoles g^-1 h^-1). It was concluded that the strain is capable of establishing any one of several steady states of growth under the same experimental conditions, each steady state displaying some buildin inertia with respect to change. The variations of the specific rates of glucose transfer and non-fermentative CO₂ production, and of the yield appeared to be consequences rather than causes of the variation of . The ultimate causes of the variation of remained unidentified.Part of a doctoral thesis submitted by J. Martinez-Peinado to the University of Navarra Spain     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Movements and diurnal behaviour patterns in a Baltic grey seal (Halichoerus grypus)

A sub-adult grey seal was caught and tagged with a satellite relay data logger at Tihällan (61°2800N, 17°2540E), Sweden, on 21 August 1992. We tracked it for 115 days during which time we recorded 841 locations, data on 3556 dives and 80 haulout periods. It travelled 730 km in the first 12 days to the southern Archipelago Sea. It stayed most of the remaining time within 25 km of a haulout site, with a major exception of an 8-day trip of 520 km. Dive shape and swimming activity were used to distinguish dive types. Square-shaped foraging dives were more common over the midday period. Haulout periods were more common during the night.     

A method for the estimation of gene flow parameters from a population structure caused by restricted gene flow and genetic drift

Summary A method has been developed which enables the estimation of the plant gene flow parameters _p (pollen dispersal), _s (seed dispersal) and t (outcrossing rate) from a selection-free continuously structured population in equilibrium. The method uses Wright's F-coefficients and introduces a new F-function which describes the genetic similarity as a function of the spatial distance. The method has been elaborated for wind pollinated plant species but can be modified for insect pollination and for animal species. In practice allozymes will provide for the necessary neutral genetic variation. The more loci used and the more intermediate the gene frequencies, the more reliable the results. For the estimation of _p and t together (when the outcrossing rate is not known) at least two chromosomally unlinked loci are required. The method for estimating _s depends on whether the plant species is annual or perennial. The mechanism of selfing has been analysed by the explanation of the value of t by three components: population density (d), pollen flow (_p) and relative fertilization potential of own pollen (Z). The concepts of neighbourhood size and isolation by distance, developed by Wright, who used a single gene flow parameter , have been extended to the situation which is realistic for seed plants, using all three parameters _p, _s and t. When _p is large with respect to _s, _s largely determines the value of the neighbourhood size, whereas _p is the most dominating factor in isolation by distance. The use of local effective population size and mean gene transport per generation instead of neighbourhood size and neighbourhood area, respectively, is proposed to avoid confusion. Computer simulations have been carried out to check the validity and the reliability of the method. Populations of 200 plants, using two or three loci with intermediate allele frequencies, gave good results in the calculation of _p with known value of t and of _s and N_e. With unknown t, especially with lower values of t, larger populations of at least 1,000 plants are necessary to obtain reasonably accurate results for _p and mean gene transport per generation M.Grassland Species Research Group Publication No. 81     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

A novel technique for the study of bacterial cell mechanical properties

A micromanipulation method is described for measuring the bursting forces of bacteria and relating them to cell size. At a compression speed of 6.2 m s^–1, bursting forces of three samples of rapidly growing Staphylococcus epidermis from a batch culture varied from 3 to 34 N with an average value of 13.8 N (standard error 0.8 N). Escherichia coli grown in continuous culture at a specific growth rate of 0.5 h^–1 had bursting forces varying from 1 to 9 N with an average value of 3.6 N (standard error 0.4 N). In squeeze-hold experiments, force relaxation was observed, which was attributed to water loss from the cells, or viscoelasticity, or both. At high compression speed, such as 6.2 m s^–1, this relaxation could be neglected. Micromanipulation strength measurements might be used in studies of cell mechanical disruption and of the dependence of cell strength on cell physiology.     

Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

Oligomerization reactions of deoxyribonucleotides on montmorillonite clay: The effect of mononucleotide structure,phosphate activation and montmorillonite composition on phosphodiester bond formation

2-d-5-GMP and 2-d-5-AMP bind 2 times more strongly to montmorillonite 22A than do 2-d-5-CMP and 5-TMP. The dinucleotide d(pG)₂ forms in 9.2% yield and the cyclic dinucleotide c(dpG)₂ in 5.4% yield in the reaction of 2-d-5-GMP with EDAC in the presence of montmorillonite 22A. The yield of d(pC)₂ (2.0%) is significantly lower but comparable to that obtained from 5-TMP. The yield of dimers which contain the phosphodiester bond decreases as the reaction medium is changed from 0.2 M NaCl to a mixture of 0.2 M NaCl and 0.075 M MgCl₂. A low yield of d(pA)₂ was observed in the condensation reaction of 5-ImdpA on montmorillonite 22A. The cyclic nucleotide (3,5-cdAMP) was obtained in 14% yield from 3-ImdpA. The yield of d(pA)₂ obtained when EDAC is used as the condensing agent increases with increasing iron content of the Na⁺-montmorillonite used as catalyst. Evidence is presented which shows that the acidity of the Na⁺-montmorillonite is a necessary but not sufficient factor for the montmorillonite catalysis of phosphodiester bond formation.     

Pullulan production from brewery wastes by Aureobasidium pullulans

The production of pullulan from brewery wastes by Aureobasidium pullulans in shake flask culture was investigated. The maximum pullulan concentration (6.0g/l) was obtained after 72h of fermentation. The external addition of nutrients into the spent grain liquor improved significantly the production of pullulan. In this case, the highest values of pullulan concentration (11.0±0.5g/l), pullulan yield (48.2±1.5%), and sugar utilization (99.0±0.5%) were obtained in the medium (pH 6.5–7.5) supplemented with K₂HPO₄ 0.5%, l-glutamic acid 1%, olive oil 2.5%, and Tween 800.5%.     

Some observations on cyclodextrin-mediated boosting of secreted amylolytic enzymes byLactobacillus amylovorus

The amounts of a 160-kDa amylase and a 140-kDa -amylase (A. Burgess-Cassler and S. H. Imam, Curr. Microbiol. 23:207–213, 1991) secreted into culture medium by the starchutilizingLactobacillus amylovorus were enhanced by the use of cyclodextrin (CD) as the carbon source. The levels of total extracellular -amylase obtained with glucose as the carbon source could be boosted severalfold by use of CD. The best enhancer was -CD, and the rank order of best to least effective was -CD>-CD=-CD>glucose.Another amylase, a 65-kDa -amylase, which degraded para-nitrophenyl-(1,4)-d-glucopyranoside, was also detected in this study. The most effective enhancer in this case was -CD, and the rank order was -CD>-CD>-CD glucose. Despite its ability to degradep-nitrophenylated glucose, this enzyme did not convert maltose to glucose. It showed a cleared zone on starch zymograms and did degrade short maltodextrins to maltose. Neither this new -amylase nor the 140-kDa -amylase exhibited any detectable ring-decyclizing (cyclodextrinase) activity against -or -CD.Other extracellular amylases (not characterized here) appeared to be similarly enhanced by CDs. Although the precise mechanism by which this effect is accomplished remains undefined, CDs can be useful inducing agents, boosting the expression and/or secretion of otherwise low-level enzymes, either as additives to growth media or as sole carbon source.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of bakers yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol. For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, , is fixed at a level below the point of ethanol production, i.e., _crit. Optimally, growth should be maintained at _crit, but in practice, this is difficult because _crit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just above _crit by regulating ethanol concentration in the bioreactor. The models used for control design are shown, as are the experimental results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an overflow type metabolism.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo