Comments on the “Maintenance coefficient” changes during alcohol fermentation

Summary The concept of maintenance is discussed in terms of the biological meaning and the applicability of the maintenance coefficient, m, in bioengineering for optimization of yields in fermentation. A method of calculation is proposed for the evaluation of m in the course of fermentation in the case of a metabolite (e.g, ethanol). During alcoholic fermentation m is not constant and decreases with the growth rate.The phenomena involved in maintenance are numerous and complex and there is a semantic problem in its definition which can be generalized by the apparently non-finalized substrate consumption.Nomenclature a specific maintenance rate (defined by eq. (9)) - m maintenance coefficient - X cell mass concentration (as a dry weight) - S substrate concentration - P product concentration - r rate of reaction - r_x,r_s,r_p rate of reaction related to biomass, substrate and product - r_sm,r_sg,r_spi rate of reaction related only to the consumption by maintenance, growth and the synthesis of the ith product - r_xe maintenance rate defined by eq. (10) - q_s,q_Pi specific rate of substrate consumption and i^th product production - Y yield coefficient - Y^o, Y^o apparent yield coefficient related to the cell and i^th product - Y _xs ^xs , Y _Pis ^Pis maximum theoretical yield coefficient related to the cell and i^th - specific growth rate produce 420     

Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Dα in soybean seed

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild‐type (WT) seeds stored for 33 months were non‐viable, 30%–50% of PLDα‐knockdown (PLD‐KD) soybean seeds stored for 33 months germinated. WT and PLD‐KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD‐KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long‐term storage.     

Stability during fermentation of a recombinant α-amylase plasmid in Bacillus subtilis

Summary We have studied the stability during fermentation of a hybrid plasmid carrying a Bacillus -amylase gene in Bacillus subtilis. In the absence of antibiotic selection plasmid loss was associated largely with the post-exponential phases of growth and decline. In fermentations containing selective antibiotics, various deleted plasmids were recovered during late stationary phase, regardless of whether the host was rec ⁺ or recE. We therefore propose that the plasmid loss observed during late growth in antibiotic-free fermentations is due to deletion events which include the origin of plasmid replication. The structure of the deleted plasmids was determined and the sequences in the vicinity of the end-points analysed. When the deleted plasmids were subjected to further fermentations in the absence of selective antibiotics, they were completely stable.     

Are changes in maximal squat strength during preseason training reflected in changes in sprint performance in rugby league players?

Because previous research has shown a relationship between maximal squat strength and sprint performance, this study aimed to determine if changes in maximal squat strength were reflected in sprint performance. Nineteen professional rugby league players (height = 1.84 ± 0.06 m, body mass [BM] = 96.2 ± 11.11 kg, 1 repetition maximum [1RM] = 170.6 ± 21.4 kg, 1RM/BM = 1.78 ± 0.27) conducted 1RM squat and sprint tests (5, 10, and 20 m) before and immediately after 8 weeks of preseason strength (4-week Mesocycle) and power (4-week Mesocycle) training. Both absolute and relative squat strength values showed significant increases after the training period (pre: 170.6 ± 21.4 kg, post: 200.8 ± 19.0 kg, p < 0.001; 1RM/BM pre: 1.78 ± 0.27 kg·kg(-1), post: 2.05 ± 0.21 kg·kg(-1), p < 0.001; respectively), which was reflected in the significantly faster sprint performances over 5 m (pre: 1.05 ± 0.06 seconds, post: 0.97 ± 0.05 seconds, p < 0.001), 10 m (pre: 1.78 ± 0.07 seconds, post: 1.65 ± 0.08 seconds, p < 0.001), and 20 m (pre: 3.03 ± 0.09 seconds, post: 2.85 ± 0.11 seconds, p < 0.001) posttraining. Whether the improvements in sprint performance came as a direct consequence of increased strength or whether both are a function of the strength and power mesocycles incorporated into the players' preseason training is unclear. It is likely that the increased force production, noted via the increased squat performance, contributed to the improved sprint performances. To increase short sprint performance, athletes should, therefore, consider increasing maximal strength via the back squat.     

Influence of specific fermentation conditions on natural microflora of pomace in “Grappa” production

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective.     

Time-dependent changes in membrane excitability during glucose-induced bursting activity in pancreatic β cells

In our companion paper, the physiological functions of pancreatic β cells were analyzed with a new β-cell model by time-based integration of a set of differential equations that describe individual reaction steps or functional components based on experimental studies. In this study, we calculate steady-state solutions of these differential equations to obtain the limit cycles (LCs) as well as the equilibrium points (EPs) to make all of the time derivatives equal to zero. The sequential transitions from quiescence to burst-interburst oscillations and then to continuous firing with an increasing glucose concentration were defined objectively by the EPs or LCs for the whole set of equations. We also demonstrated that membrane excitability changed between the extremes of a single action potential mode and a stable firing mode during one cycle of bursting rhythm. Membrane excitability was determined by the EPs or LCs of the membrane subsystem, with the slow variables fixed at each time point. Details of the mode changes were expressed as functions of slowly changing variables, such as intracellular [ATP], [Ca(2+)], and [Na(+)]. In conclusion, using our model, we could suggest quantitatively the mutual interactions among multiple membrane and cytosolic factors occurring in pancreatic β cells.     

Seasonal changes in zooplankton abundance in the lower Rhine during 1987–1991

Zooplankton in the River Rhine was surveyed for five years at the Dutch sampling stations, Lobith (German/Dutch border) and Maassluis (at the point of discharge of the river into the North Sea). The zooplankton abundance showed an apparent seasonal pattern at both stations, characterized by low densities during the winter period, and higher densities during the summer period, with a spring peak. Zooplankton was dominated by rotifers at both stations, although during the winter periods the contribution of copepods was considerable. The rotifers were dominated byBrachionus angularis, B. calyciflorus, Keratella cochlearis andK. quadrata; the copepods by cyclopoid nauplii; the cladocerans by small-sized species mainly belonging toBosmina. At Maassluis the relative contribution of copepods was higher than at Lobith. Furthermore, the zooplankton at Maassluis included the speciesEurytemora affinis, characteristic for estuarine conditions. In spring, the rotifer density and water temperature and rotifer density and chlorophylla concentration were positively correlated. Furthermore, both rotifer density and chlorophylla were inversely correlated with discharge. The possible role of environmental factors (water temperature, chlorophyll content, discharge and biotic factors) controlling the river zooplankton dynamics is indicated.     

Yeast assessment during alcoholic fermentation inoculated with a natural “pied de cuve” or a commercial yeast strain

The present study has been carried out in an organic winery established in 2003 in the Denomination of Origin “Sierras de Málaga” (Southern Spain) region during the 2007 vintage. The aim of this work was to ascertain the yeast microflora present in the winery and during the vinifications and to obtain a collection of autochthonous S. cerevisiae strains from this area. Yeast populations from three vats containing fermenting musts from different grape varieties were analysed. Two of them were inoculated with a natural “pied de cuve” while the third one was sown with a rehydrated commercial yeast strain. A total of 382 yeasts were isolated and identified, initially by restriction analysis of ribosomal DNA and further by sequencing of this region. Non-Saccharomyces yeasts were found in all three musts but they practically disappeared as the fermentations progressed. Analysis of mitochondrial DNA RFLP revealed 13 different restriction patterns of Saccharomyces cerevisiae strains, five of them similar to those of commercial strains used in the winery. Commercial strains were found even in vats inoculated with a “pied de cuve” generated by spontaneous fermentation of a must sample. The analysis of samples recovered from different winery surfaces and equipments demonstrated that non-Saccharomyces and both commercial and autochthonous Saccharomyces strains were part of the resident microflora in the winery. Biodiversity of autochthonous S. cerevisiae in fermentation vats was low but two of them were able to compete with the commercial ones and they were isolated even at the end of the fermentation.     

Physical changes of β-sitosterol crystals in oily suspensions during heating

The aim of this research was to describe the thermal behavior of β-sitosterol crystals in oil-suspensions with a focus on the role of water during heating. The suspensions were prepared by recrystallization in order to achieve a microcrystalline particle size. The structural changes together with the mechanical properties of the suspensions during heating were studied by using variable temperature X-ray powder diffractometry (VT-XRPD), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). Hydrated β-sitosterol crystals in an oil-suspension, dehydrated, despite the composition of the suspensions, at low temperatures. At high β-sitosterol concentration, the monohydrate crystal form changed partially to a hemihydrated form, and when only a small amount of water was initially incorporated, the hemihydrate crystal form dehydrated to a mostly anhydrate crystal form. The released water, which was immiscible in the surrounding oil, caused the recrystallization of hydrated β-sitosterol during cooling. This procedure indicated a reversible dehydration process. Structural and thermal analysis of β-sitosterol crystals in suspensions, together with mechanical analysis made it possible to understand various physical changes during heating. Published: October 19, 2005     

Microbiological characterization of yam fermentation for ‘Elubo’ (yam flour) production

Yams (Dioscorea rotundata) were processed and fermented to the traditional West African dried yam flour Elubo. Yam slices were blanched at 60°C for 10min and then fermented at 30°C for 24h. The microbial population increased with fermentation time during the first 24h of natural fermentation with a fall in the pH from 6.2 to 5.4. The dominant aerobic mesophilic bacteria consisted of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii and Bacillus subtilis. Significant contributions were also made by Lactococcus lactis, Klebsiella pneumoniae, Citrobacter freundii and the yeasts Pichia burtonii and Candida krusei. Accelerated natural lactic fermentation was achieved by repetitive use of the previous fermentation batch as an inoculum through back-slopping at a rate of 10% (w/v). After three consecutive fermentation cycles, the microflora was dominated by L. plantarum and L. brevis. Fermentation of blanched samples with pure cultures of the isolates showed L. plantarum L. brevis and B. subtilis to be the main species responsible for pH reduction and in changes in the browning levels of the reconstituted flour paste (amala). The typical colour of amala is disliked by many individuals and it seems possible that amala with a lighter colour which will be more acceptable can be produced by fermentation.     

αMSH-like peptides in rat brain: Identification and changes in level during development

MSH-like peptides were extracted from rat brains and separated by high pressure liquid chromatography. Using C and N terminally directed antibodies, and a bioassay for melanotropic activity, two major melanotropic peptides were detected. One peptide was identified as αMSH and the other as des-acetyl αMSH, a form which has not been previously reported in brain. Analysis of the level of αMSH-like peptides in brain and pituitary gland during development, showed steady increases of pituitary αMSH from day 18 fetuses to adults, whereas in brain, significant increases were not observed until one day post partum. This difference in the time of onset of αMSH production in the two tissues suggests the presence of biosynthetically independant pools of αMSH-like peptides in pituitary and brain.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Production of higher alcohols during indonesian tapé ketan fermentation

A study was made of the higher alcohols (fusel oils) produced during the Indonesian tapé ketan fermentation using Amylomyces rouxii as the principal mold, alone or in combination with yeasts belonging to genera commonly found in the tapé ketan fermentation (Endomycopsis, Candida, and Hansenula). Total fusel oils increased with length of fermentation. Fusel oils detected in the product distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts. A. rouxii alone produced nearly the same quantity of fusel oils (total production, 275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total production, 292 mg/liter at 192 h).A. rouxii and Endomycopsis fibuliger produced fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in combination with Candida yeasts produced somewhat more fusel oils, ranging from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During the first 36 h, production of fusel oils was higher at 30 and 35 degrees C than at 25 degrees C. At 48 h fusel oil production was slightly higher at 30 degrees C than at 35 degrees C. Beyond 48 h, production of fusel oils was higher at 25 degrees C. A. rouxii in combination with Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192 h.     

Zooplankton community changes in Lake Kinneret (Israel) during 1969–1985

Long-term records (1969–1985) of zooplankton density in Lake Kinneret indicated significant reduction of biomass (Copepods, Cladocera, Rotifera) and production (Copepods, Cladocera). Nauplius and adult copepod densities decreased but those of copepodites did not change. Mesocyclops was suppressed more than the smaller Thermocyclops and males of both genera became more abundant relative to the larger females. Ratios of small/large Cladocera densities became higher. Numbers of total cladocerans were stable, and therefore reduction of Cladocera grazing capacity is assumed. The abundant Keratella spp. were reduced. It is likely that intensification of fish visual-attack-predation pressure shifted the size-class structure towards smaller adult copepods and cladocerans. Reduction of Keratella spp. and copepod nauplii was probably affected by increasing pressure of fish filter-feeders. Data on fish food consumption, feeding behaviour and fisheries management suggested their direct impact on long-term changes of zooplankton in Lake Kinneret.     

Ultrastructural changes in wheat embryos during a “presowing drought hardening” treatment

Summary Changes in the ultrastructure of the shoot apex of wheat embryos were studied during a reputed drought hardening treatment consisting of 48 hours of hydration followed by dehydration to air dry weight and during a subsequent second hydration for 48 hours. In the initial dry state lipid droplets are closely packed against the cell walls and the mitochondria and plastids have little internal structure. During the first hydration the lipid droplets are reduced in number and disperse while the mitochondria and plastids assume a normal appearance. The nucleus does not change appreciably. Under the conditions used in this work dehydration to air dry weight is not lethal to the embryos although notable changes in cellular structure occur: 1. Massive condensation of the normally loosely organized chromatin, 2. some loss of internal mitochondrial structure, 3. specific association of short elements of rough ER with vacuoles and lipid droplets, and 4. formation of fibrillar elements in the cytoplasm. In some respects then, embryos dehydrated after a 48 hour soaking have a very different structure to that of embryos that have dried during maturation of the seed. Since these changes are, in the case of viable embryos, reversible upon rehydration they cannot be linked, as such, to any long term drought hardening effect of the treatment. It is suggested that some changes, particularly the condensation of chromatin, may indicate a protective response to water stress. The kind of information needed to sustain this suggestion is discussed.This project is supported by grants from the Rural Credits Development Fund and the Australian Research Grants Committee.     

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.     

Extracellular enzyme activities during cassava fermentation for ‘fufu’ production

Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for fufu production, attaining their peak activities after 12 and 24h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period.     

Post-translational processing of β-d-xylanases and changes in extractability of arabinoxylans during wheat germination

Endo-1,4-β-d-xylanase (EC 3.2.1.8, β-d-xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3–4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I β-d-xylanase as established by immunodetection. This suggests that other, yet unidentified β-d-xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like β-d-xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher β-d-xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa β-d-xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa β-d-xylanase occurred and was associated with a drastic increase in β-d-xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the β-d-xylanase is processed into more active forms to achieve extensive AX breakdown.     

Circadian rhythmicity in a fermentation process?

Summary An idea is proposed for the role of the circadian rhythmicity in the control of the oscillatory behavior observed in the growth and product formation during the cell-retention continuous culture of Clostridium acetobutylicum. C. acetobutylicum is highly sensitive to the permeability of the cell membrane. A physical mechanism for the variability of the cytoplasmic membrane has been proposed suggesting that the performance of the cell membrane, due to its liquid crystalline structure, is influenced by the external forces (e.g. earth's magnetic field). A previously developed Physiological State Model was extended by incorporating the effect of external forces on the cell membrane permeability. The new mathematical model could simulate the observed oscillatory behavior of the microbial culture. Some experimental results in support of the theoretical predictions have been presented.Nomenclature a Anisotropy - B Butanol concentration in the fermentation broth (g/l) - B _i Intracellular butanol concentration (g/l) - B _ex Extracellular butanol concentration (g/l) - Mean value of the butyric acid solution concentration (g/l) - BA _i Intracellular butyric acid concentration (g/l) - BA _ex Extracellular butyric acid concentration (g/l) - D Dilution rate (l/h) - H Magnetizing force (oersted) - K Constant in Equation (1) - k B Constant in Equation (15) - K _BA Saturation constant - k _{BA 1} Constant in Equation (13) - k _{BA 2} Constant in Equation (13) - K _D Constant in Equation (13) - k _{G 1} Constant in Equation (8) - k _{G 2} Constant in Equation (8) - k _{G 3} Constant in Equation (9) - K _I Inhibition Constant - k _p Constant in Eq. (11) - K _S Monod constant - n Number of the active sugar transport sites - P Cellular membrane permeability (l/g wet cell·h) - q _S Specific rate of substrate utilization (g substrate/g biomass·h) - S Substrate concentration in the fermentation broth (g/l) - S _O Substrate concentration in the feed solution (g/l) - t Time (h) - X Total biomass concentration (g/l) - X ₁ Active biomass concentration (g/l) - X ₂ Non-active biomass concentration (g/l) Greek Letters Ratio of the dry to wet cell weight (g dry cell/g wet cell) - ₁ Constant in Equation (6) - ₂ Constant in Equation (6) - ₃ Constant in Equation (6) - Specific culture growth rate (1/h)