The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.
The composition of the medium used for cultivation of micro-organisms is directly reflected in their physiological phenotype and their fermentation performance, which in turn affects the results of strain analyses and strain performance in industrial applications. For this reason, the successful development of strains for large scale industrial production of heterologous proteins [1, 2] and low-value fuels, chemicals and materials [3, 4] merits the composition of cultivation media in various steps of strain development to be reconsidered.
Introducing novel recombinant strains into industrially relevant cultivation media may reveal that the strain has not been properly designed for this environment. For example, it was found that a strain of the lactic acid bacterium Streptococcus thermophilus engineered for enhanced exopolysaccharide production – a trait highly desirable in yoghurt production – failed to express the phenotype in milk without the addition of an extra nitrogen source . Similarly, a genetically modified strain of the yeast Saccharomyces cerevisiae, which had been communicated as the ultimate solution to the fermentation of lignocellulose derived xylose [6, 7], was found to require yeast extract, additional hexose sugar and oxygenation to efficiently ferment the xylose fraction in spent sulphite liquor . Furthermore, heterologous protein production in yeast is strongly influenced by the nitrogen-composition of the production medium [9, 10]. Thus the final industrial environment must be considered throughout the strain development process to avoid unfounded expectation and – more importantly – to prevent costly investment into premature production facilities.
A cultivation medium is designed to reflect the elemental composition and the biosynthetic capacity of a given microbial cell (see e.g. ). While the elemental composition of microbial cells is relatively similar, their biosynthetic capacity varies widely. The yeast S. cerevisiae and the bacterium Escherichia coli have extensive biosynthetic capacity and grow in defined mineral media . In contrast, the biosynthetic capacity of many lactic acid bacteria is limited and they require rich or extensively supplemented medium for efficient growth . Furthermore, economic constraints make the large-scale production of low-cost products reliant on cheap sources of carbon and nitrogen, such as molasses from the sugar industry, corn steep liquor from the starch industry, spent sulphite liquor from the forest products industry and cheese whey from the dairy industry [14, 15]. In addition to providing carbon, nitrogen, vitamins and trace elements necessary for cell growth and metabolite production, such industrial media may also contain substances which inhibit growth and metabolite production.
This paper reviews the influence of the composition of cultivation media on the development of novel industrial production strains with the view that it is necessary to consider the final cultivation conditions in every stage of strain development. Primarily two types of recombinant strains of S. cerevisiae are used as examples: strains which produce heterologous proteins and strains with an expanded substrate range to include pentose sugars. Experience from other organisms is included to complement the discussion. Finally, genetic engineering approaches to overcome industrial media constraints are also exemplified.
Traditionally, novel production strains have been developed by mutagenesis , breeding , and the lately revived concept of evolutionary engineering (Figure 1; [18–20]). Strains with novel traits are now also developed by life science technologies including genetic and metabolic engineering (Figure 2; [21–23]). In recent years these engineering concepts have been expanded in the context of systems biology to also include information and system science technologies [24–26]. In metabolic engineering, cells are iteratively analyzed, designed and synthesized (Figure 2) using molecular tools such as recombinant DNA technology and genomic information, [27, 28]. Evolutionary engineering (Figure 1) relies on carefully designed selection protocols, i. e. media and cultivation conditions [18–20] for the development of strains with industrially interesting characteristics. Metabolic and evolutionary engineering technologies may also be combined to generate novel traits [29–33]. The multitude of data generated in the analysis of the genome, transcriptome, proteome and metabolome  requires the use of information and system science technologies to translate these data into design strategies for next rounds of metabolic and evolutionary engineering . Several studies have pointed out that the cultivation conditions and media composition used for the analysis of novel engineered strains strongly influence the data generated [34–39]. Since such data form the basis for the design strategy for the following rounds of strain development, it is evident that choice of cultivation media is a fundamental and integral part of strain development.
Principle of Evolutionary Engineering [18-20].
Principe of Metabolic Engineering (adapted from ).
Whereas strain development by recombinant techniques is usually performed in genetically defined laboratory strains harboring markers suitable for selection of transformed cells in chemically defined cultivation media, the typical industrial production strain is genetically undefined and adapted to perform in rather poor, toxic, viscous, and nutrient-limited media. Once desired novel traits have been established in recombinant laboratory strains, the novel strains are either directly transferred to the industrial production environment or – as occurs much more frequently – a potential production strain has to undergo a new round of metabolic engineering procedures. In both cases, the medium in which the novel pathways are developed differs substantially from the medium in which the final production strain is expected to perform.
The genetic stability of strains is an absolute requirement for utilization in industrial processes. Due to the adaptive nature of microorganisms, attention should also be directed towards the stability of any novel traits in recombinant or mutant strains. In industry, rich or undefined media are often used, which may result in unexpected loss of plasmids and even chromosomal modifications. Prolonged cultivation, for example in continuous fermentation set-ups, increases the probability of detrimental genetic instability. Even in mineral medium, loss of plasmids with auxotrophic marker has been reported in prolonged continuous cultures . This was caused by released amino acids from the dying cells, and probably also by spontaneous chromosomal insertion of the marker gene .
In S. cerevisiae, both episomal plasmids (YEp; ) and integrative plasmids (YIp; ) are used as expression vectors for heterologous protein expression and metabolic engineering. The advantage of YIp vectors, despite their low copy numbers, is their robust genetic stability even in unselective medium due to the integration of the vector into the yeast genome [43–51]. The benefit of using YEp plasmids is the high gene copy number of up to 70 copies per cell  resulting in high expression levels of the desired proteins, although their high segregational instability often results in plasmid loss especially in rich medium [53, 54]. However, the stability of YEp-type vectors can be improved by autoselection systems, such as the fur1 ura3 system , where the deletion of FUR1 together with the use of a plasmid containing the URA3 marker results in stable plasmid expression even in continuous culture . Without such autoselection systems it is necessary to use a selective medium to overcome the instability of YEp plasmids, which may pose a limitation to the industrial use of such strains especially with low-cost products.
Generally, microorganisms grow more vigorously in rich media than in mineral media, because rich media contain biosynthetic precursors that can be channeled directly into anabolic pathways, reducing the need to produce biosynthetic precursors and saving metabolic energy. This has a significant effect on growth and production characteristics.
For example, a three-fold increase in production levels of heterologous laccase by recombinant Yarrowia lipolytica was reported when switching from yeast nitrogen base (YNB) to complex medium . When autoselective strains of S. cerevisiae expressing heterologous xylanase or α-L-arabinofuranosidase genes were cultivated in complex YPD medium, 24-fold higher xylanase and up to 70-fold higher levels of α-L-arabinofuranosidase were produced [57, 58]. Similarly, production levels of the potent thrombin-specific inhibitor, hirudin, by recombinant S. cerevisiae was improved 20 fold in complex medium , demonstrating the substantial impact of medium composition on heterologous protein production.
Aoki et al.  elegantly demonstrated single-step purification of recombinant cysteine proteinase (NsCys) from Pichia pastoris by switching medium composition during cultivation. The recombinant P. pastoris was first cultivated in glycerol complex medium to generate biomass in a short time. The cells were harvested and resuspended in minimal medium for induction of NsCys production. The minimal medium faciliated protein secretion and subsequent purification.
Strains of E. coli with altered levels of pyruvate decarboxylase and alcohol dehydrogenase displayed a reduced flux of pyruvate into the native fermentation pathways when cultivated in defined medium . In addition, the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway and biosynthesis was restricted, which dramatically reduced growth yields in defined medium compared with complex medium. The observations demonstrated that inherent limitations in the metabolism of engineered strains can be masked by the presence of complex nutrients in the medium and are often not observed without cultivation in defined medium.
To illustrate the influence of media composition on strain performance, we compared the growth of baker's yeast and two recombinant strains: a laboratory strain of S. cerevisiae, TMB3001  and an industrial strain of S. cerevisiae, TMB3400 . Both recombinant strains have been engineered for xylose utilization with the introduction of the XYL1  and XYL2  genes encoding xylose reductase and xylitol dehydrogenase, respectively, from the yeast Pichia stipitis. In addition the endogenous gene XKS1  encoding xylulokinase has been overexpressed.
We evaluated the influence of four commonly used media on growth and product formation under aerobic and oxygen limited conditions: yeast extract-peptone (YP [66, 67]); a defined mineral medium (DM; ); yeast nitrogen base (YNB; ); and synthetic complete (SC) medium equivalent to supplemented YNB  (Table 1). YP is an undefined rich complex medium composed of yeast extract (YE) and peptone. YE is prepared by autolysis of whole yeast cells at around 50°C [66, 69–71] and peptone is an acid- or enzymatic hydrolysate of a protein-rich by-product from the food and feed industry . YP contains all components necessary for propagation of yeast cells, including biosynthetic building blocks, and it is frequently used in the initial stages of fermentation when a large inoculum is required. YNB is a chemically defined medium that can be supplemented to satisfy auxotrophic requirements of yeast mutants used in metabolic engineering, then referred to as SC medium. DM medium contains almost all components of YNB medium (Table 1), however, some components are present in higher and even an order of magnitude higher concentration than in YNB medium. The DM medium and variants thereof are commonly used to obtain quantitative physiological data for yeast strains. It has been designed to assure that concentrations of vitamins and trace elements do not exercise growth limitation . Sodium chloride, riboflavin and folic acid were not found to be necessary for growth of S. cerevisiae, whereas cobalt apparently supported growth (Table 1). EDTA seems to be required to dissolve elevated concentrations of trace elements.
YP supported growth of a commercial baker's yeast strain even in the absence of additional carbon source (Figure 3). The maximum specific growth rate under these conditions was 0.29 h-1 with a final OD620 of 3–4 after 24 hours. With additional 20 g/l glucose an OD620 of 22 was reached at a maximum specific growth rate of 0.45 h-1. Sugars present in YE may explain this phenomenon. Yeast accumulates storage carbohydrates such as glycogen and trehalose, the amount of which is strongly dependent on cultivation conditions . During the preparation of YE these compounds are fully or partially hydrolyzed to monomer glucose. YE also contains lactate, which can serve as carbon source in yeast cultivation . Lactate is a consequence of non-sterile cultivation conditions in baker's and brewer's yeast production . In addition to these auxiliary carbon sources, YE also contains a number of other compounds, which strongly influence fermentation performance .
The maximum specific growth rate of TMB3001 and TMB3400 in the four different media (Table 2) under aerobic and oxygen limited conditions varied considerably. Highest growth rates were obtained in aerobic YP medium and significantly lower growth rates were observed in the three defined media both for glucose and xylose as carbon source (Table 2). The results support previous observations that complex media components can mask inherent limitations in the metabolism of recombinant strains as demonstrated for E. coli  and S. cerevisiae .
Growth on glucose and xylose, respectively, and xylose uptake rate for recombinant xylose-utilizing S. cerevisiae strains, TMB 3001  and TMB 3400 . Cells were pre-grown (16 h) in DM medium with 20 g l-1 glucose, harvested by centrifugation and washed three times before inoculation at 0.01 g l-1. Aerobic growth: 100 ml medium in 1000 ml baffled E-flask at 180 rpm. Oxygen-limited growth: 78 ml medium including 1.25 ml l-1 Ergosterol/Tween® 80 in 80 ml flask with rubber septum and cotton-filled needle for CO2 outlet, stirring speed 150 rpm by a 2 cm magnetic stirrer bar. Media containing 20 g/l glucose and 20 g/l xylose, respectively, were buffered to pH 5.5 with 50 mM phthalate and the temperature was set at 30°C.
In all media TMB3400 displayed significantly higher specific growth rate on xylose than TMB3001 confirming previous results [29, 32]. Reducing the oxygen supply emphasized this difference. Both strains displayed significantly lower specific growth rate on xylose than on glucose (Table 2) confirming previous observations . Also in this comparison, oxygen limitation increased the difference. DM and YNB medium resulted in almost identical specific growth rates indicating that the level of trace elements and vitamins in YNB were not limiting growth under the presently chosen cultivation conditions. Amino acid supplementation in YNB slightly increased the specific growth rates during xylose utilization but not under glucose utilization (Table 2).
The volumetric xylose uptake mirrored the growth rate in all four media with both strains and under the two levels of oxygenation (Table 2). Figure 4 shows the time course for xylose consumption and product formation for TMB3400 under oxygen limited conditions using YP and YNB medium. Results from only one of the mineral media are displayed since growth and product formation was identical in all three mineral media. Whereas the results emphasize the strong growth promoting influence of the YP medium they also show that the media composition did not influence the distribution of products under the chosen conditions (Figure 4).
Xylose utilisation and product formation during oxygen-limited cultivation of TMB 3400  in YP and YNB media. Xylose YP-●-, Xylose YNB -○-, Ethanol YP -▴-, Ethanol YNB -△-, OD620 YP -■-, OD620 YNB -□- Sugars and products were analyzed with HPLC (Bio-Rad, Aminex 87-H column).
Strain development may require large numbers of strains to be evaluated for their performance in simple screening set-ups [74, 75], where neither oxygen availability nor pH is controlled. When microorganisms grow in defined mineral medium with ammonium as the sole nitrogen source, the medium quickly acidifies due to proton excretion during active transport of nutrients into the cell . Acidification quickly inhibits cell growth and metabolism . Therefore, the media must be buffered around the optimal pH for the microorganism to be investigated. For example in industrial yeast fermentations, it is relevant to maintain pH around 5.5. We compared the influence of 50 mM buffering salts on growth of TMB3001 in YP and in DM media (Figure 5). The suitability of citrate, citrate/phosphate, phosphate and phthalate to buffer the growth medium at pH 5.5 were compared.
YP had an inherent buffering capacity, while pH in non-buffered DM decreased to 2.5 when maximum OD620 was reached. The presence of citrate and citrate/phosphate severely inhibited growth in YP, whereas the inhibition was somewhat less severe in DM. With three carboxyl groups, citrate is a chelating compound and complexes with trace elements in YP. In DM, where the concentration of trace elements has been enhanced (Table 1), the inhibition of citrate was less severe. Phthalate showed the best buffering capacity, however, the price of this buffering compound may limit its use in large amounts. With phosphate buffer, pH of DM medium dropped to around 3 in the late stationary phase, but no growth inhibition was observed. Thus depending on the scale of strain screening either phthalate or phosphate buffer should be used for yeast development work.
The construction of recombinant strains requires selectable marker genes for efficient detection and selection of transformed cells. For S. cereviciae, mutant and deletion strains having one or several auxotrophic requirements are the most commonly used tools in the development of recombinant strains . The use of auxotrophic mutants relies on the assumption that complementing auxotrophy by plasmid expression makes the strain equivalent to its prototrophic counterpart. However, this is not always the case, as was shown for strains carrying the LEU2 gene on a multicopy plasmid .
In addition to the auxotrophic markers used for plasmid retention, uncomplemented auxotrophic mutations often remain present in the transformed yeast strains, requiring the addition of the necessary amino or nucleic acids to the cultivation medium. The use of such auxotrophic strains has recently been critically reviewed . Based on the complications involved in translating experimental data obtained with auxotrophic strains into quantitative physiological data, the author concluded that auxotrophic strains should be avoided unless auxotrophy itself was under investigation. A solution to this problem is genetic complementation of the remaining auxotrophic markers, which is quite simple (see e.g. [32, 37]) and recovers the prototrophic genotype.
Uncomplemented auxotrophic mutations can also affect production levels of recombinant proteins . This was recently confirmed when growth and extracellular protein production were compared for an auxotrophic and a prototrophic S. cerevisiae strain expressing the Trichoderma reesei β-1, 4-xylanase XYN2 gene . Only excessive amino acid supplementation allowed the auxotrophic strain to produce the heterologous protein at levels comparable to the prototrophic strain. Other studies have confirmed that excessive auxotrophic markers in transformed S. cerevisiae strains often result in overconsumption of the required metabolite and decreased growth, protein production and genetic stability [9, 34, 45, 79–84]. These studies clearly demonstrated that physiological data obtained with auxotrophic strains have to be evaluated with great caution and should not form the basis for future strain design strategies.
Expression of proteins is an inherent strategy of metabolic engineering, whether it is performed for the production of the protein itself or for redirecting a metabolic pathway. It was early observed that high-level protein expression influenced cell physiology of both prokaryotic and eukaryotic microorganisms (lit. reviewed in [37, 38, 85]). The most prominent effect was reduced cell growth. The phenomenon was named "metabolic" [53, 86] and later "protein" [40, 87] burden since it was discriminated from the influence of the catalytic activity of the protein . The magnitude and the cause of the protein burden were estimated by comparing recombinant strains of S. cerevisiae expressing T. reesei β-1, 4-xylanase encoded by the XYN2 gene . Both the introduction of the glycolytic promoter without the structural gene, and the structural gene itself, exercised a metabolic burden on the host. The reduction of the maximum specific growth rates, the biomass yields and the specific glucose consumption rates were much larger than expected from the amount of heterologous protein produced . When the cultivation medium was supplemented with a balanced mixture of preferred amino acids (Ala, Arg, Asn, Glu, Gln and Gly) or succinate, the detrimental metabolic effect could partially be relieved . Amino acids enhanced cell growth and heterologous protein production, which supported the observation that recombinant yeast expressing heterologous proteins experience depletion of amino acids and biosynthetic precursors .
The latter observation was the basis for genome-wide transcription analysis of two isogenic strains of S. cerevisiae harboring either a multicopy plasmid with the T. reesei XYN2 gene under control of the S. cerevisiae PGK1 promoter  or the plasmid with neither the structural gene nor the promoter. Transcription data (available at [89, 90]) are summarized in Table 3. Transcriptional profiles during the expression of the heterologous xylanase strongly resembled severe amino acid limitation resulting in up-regulation of amino acid transport and synthesis, complemented by the induction of the general stress response and respiration, and the repression of ribosomal and glycolytic gene expression. The transcriptional response to heterologous xylanase expression thus closely resembled the stringent stress response, apparently due to amino acid limitation [38, 91, 92]. Similar stringent stress response has been reported for strains of the bacterium E. coli overproducing heterologous proteins. The E. coli stringent stress response normally involves the repression of ribosome synthesis and the derepression of respiration, amino acid uptake and amino acid biosynthesis due to nutrient limitation . The stringent stress response in S. cerevisiae has previously been associated with nitrogen limitation and a nutritional downshift [94–97]. The fact that amino acid supplementation of the cultivation medium also improved heterologous protein production under control of an oxygen regulated promoter in the yeast Pichia stipitis  seems to further support that the induction of a nitrogen starvation response due to heterologous protein expression is general.
The choice of nitrogen source in cultivation media for the production of heterologous proteins is crucial as has been amply illustrated by observations with various complex nitrogen sources for industrial protein production. Inconsistency in complex components such as yeast extract can limit the reproducibility of industrial fermentation performance, resulting in 2–3 fold differences in heterologous protein production levels . For industrial production, the proteins of complex cheese whey can be hydrolysed by proteases to allow for utilization by micro-organisms , which has been shown to improve the heterologous protein production compared with mineral medium [99, 100]. However, other reports have indicated slower growth and lower production of heterologous proteins in cheese whey compared to mineral medium containing lactose .
The presence of nitrogen components in cultivation media may also be important to protect heterologous proteins in the extracellular medium from proteolysis. Extracellular proteolysis of heterologous proteins is affected by nutritional conditions, and may increase due to glucose exhaustion or carbon starvation [45, 102, 103]. The addition of complex nitrogen sources, such as casamino acids, peptides, amino acids, skim milk or bovine serum albumin has been shown to decrease the degradation of the heterologous proteins by S. cerevisiae and P. pastoris, probably by providing large amounts of protein substrate or reducing the production of extracellular proteases [81, 104–110]. Addition of the amino acids arginine and lysine to cultures of S. cerevisiae in defined medium has decreased proteolysis of extracellular recombinant proteins, most likely due to the inhibition of proteolytic enzymes specific for peptide bonds including basic amino acid [111, 112]. Buffering the cultivation medium to a pH where protein degradation is minimized can also reduce the breakdown of heterologous proteins [104, 107, 113–116].
Particular carbon sources may also be required to support heterologous protein production during specific growth phases. The production of recombinant antigens during gluconeogenesis in S. cerevisiae required additional medium components such as lactate and trehalose to ensure sufficient availability of metabolic energy . In addition, too high concentrations of salts may reduce heterologous protein production .
For industrial production of high-value heterologous proteins such as bio-pharmaceuticals, the higher costs associated with the use of a defined mineral medium may be justified on the basis of increased reproducibility, productivity, and requirements for regulatory approval . For both defined and complex media, the negative effect of nutrient limitations can be minimized by optimizing the concentrations of the medium components. This should preferably be done by the response surface methodology (see e.g. [118, 119]). Such an empirical procedure is required separately for each heterologous protein, for each of which the optimal medium compositions may differ substantially.
Media components have a very strong impact on economics of industrial fermentation processes and can account for up to 30% of the total production cost [120, 121]. Large scale production of cheap commodities such as fuels, chemicals and materials requires very cheap raw material [14, 15]. Such processes use by-products from the agricultural, forestry and chemical industry as carbon and nitrogen sources. Carbon sources include sugar beet and sugar cane molasses, residues from sugar production, spent sulphite liquor (SSL) from the paper pulping industry, and cheese whey from the dairy industry. Spent yeast biomass can be processed to obtain valuable medium supplements (see e.g. [120, 121]) and may serve as replacement for the more expensive yeast extract. A frequently used nitrogen source in industrial fermentation processes is corn steep liquor formed during starch production from corn [14, 15].
Economic constraints in large-scale industrial processes rigorously limit the utilization of additives and pretreatments prior to cultivation. For the production of cell mass and ethanol using molasses and SSL, salts of ammonia are often the only additive providing the desired pH, a nitrogen source and possibly phosphate. Production of cell mass and ethanol are usually performed under non-sterile conditions  at a low pH, which allows yeast to grow while the growth of contaminating microorganisms is inhibited. It is obvious that production strains working efficiently in such media are widely different from laboratory-strains initially used to develop novel metabolic traits. The environmental constraints of industrial fermentation media will be summarized under the following headings: (i) multiple sugar substrates to be converted into the final product; (ii) by-product formation; (iii) nutrient limitation; and (iv) inhibitors.
In addition to easily metabolized sugars, industrial substrates may also contain a mixture of more unusual sugars. For example, beet and cane molasses contain galactose, raffinose, and melibiose; starch derived substrates contain maltose; and hemicellulose-derived substrates contain galactose, mannose, xylose and arabinose. For maximum process economy all sugars should be converted to the desired product. The simultaneous presence of multiple sugars in the industrial media may pose limitations such as incomplete substrate utilization and inhibition of sugar utilization pathways. Some sugars such as galactose  and mannose are metabolized by S. cerevisiae, whereas the utilization of other sugars such as raffinose [123–125], melibiose , xylose  and arabinose [30, 32] requires that a new metabolic pathway is genetically introduced. In addition, "natural" sugar utilization by S. cerevisiae is governed by carbon catabolite repression  and pathway induction , such that glucose and mannose are utilized first and other sugars are consumed only when these carbon sources are depleted. To circumvent this phenomenon, recombinant yeasts engineered in key signaling elements of the carbon catabolite repression cascade have been developed [130, 131], which resulted in enhanced total sugar consumption rate. However, the feasibility of such engineered strains in industrial environments remains to be demonstrated. Carbon catabolite repression can also be overcome by using fed-batch fermentation regimes , which are easily applicable in industrial processes.
Glycerol is formed in relatively small amounts during anaerobic ethanolic fermentation . However, considering the scale of ethanol production this unwanted by-product represents product losses in the million € range. Glycerol production during ethanolic fermentation is a consequence of surplus NADH formation in biosynthetic reactions [134–136]. During anaerobic growth of S. cerevisiae in the absence of an active respiratory pathway, biosynthetic NADH can only be oxidized through the reduction of dihydroxyacetone-phosphate to glycerol 3-phosphate, which ultimately leads to glycerol secretion. It was experimentally demonstrated that glycerol secretion is directly linked to amino acid synthesis in S. cerevisiae , Glycerol production was reduced when ammonium in the cultivation medium was substituted with amino acids. However, amino acid supplementation of industrial substrates for large-scale ethanol production is presently not considered economically viable even with relatively cheap protein hydrolyzates such as yeast extract and peptone.
Reducing glycerol formation during ethanolic fermentation has also been approached with metabolic engineering strategies. Bacteria harbor transhydrogenase enzymes, which convert NADH into NADPH in response to cellular requirements. Attempts to express these enzymes in S. cerevisiae have met with limited success [137, 138]. Instead, endogenous redox reactions of the ammonia and amino acid metabolism in S. cerevisiae have been engineered to create artificial transhydrogenase functions [139, 140]. In anaerobic cultivation, ethanol formation increased at the expense of glycerol formation in the engineered strains . The use of such engineered strains in industrial applications remains to be demonstrated.
Anaerobic fermentation of xylose results in xylitol formation as a consequence of the difference in co-factor preference in the xylose reductase and xylitol dehydrogenase reactions, respectively (reviewed in ). Xylose reductase can use both NADPH and NADH as cofactor, whereas xylitol dehydrogenase exclusively uses NAD+. Xylitol is secreted and lost for ethanol production as a consequence of intracellular NAD+ depletion. Several strain design strategies have been explored to increase ethanol formation during xylose fermentation including modulations of intracellular co-factor availability [141–143] and expression of a mutated xylose reductase with reduced affinity for NADPH . None of the engineered strains have so far been reported to be exposed to an industrial substrate.
An unexpected, yet fully explainable observation is that industrial cultivation media sometimes decrease unwanted by-product formation. For natural xylose fermenting yeast it was recognized that the reduction of an external electron acceptor such as acetoin provided NAD+ for the xylitol dehydrogenase reaction, which prevented xylitol formation [145–147]. For recombinant S. cerevisiae the same phenomenon was quantified with metabolic flux analysis . The fact that recombinant laboratory strains of S. cerevisiae produced more ethanol in a lignocellose hydrolysate was interpreted in terms of lignocellulose derived components acting as external electron acceptors (; see further discussion below).
Ethanol is an unwanted by-product in baker's yeast production [150, 151]. Baker's yeast is industrially produced using a fed-batch regime, where the carbon substrate is fed into the production vessel at a rate which prevents "overflow" metabolism at the level of pyruvate and thus limits ethanol formation . Since S. cerevisiae is also used for large-scale heterologous protein production , the unwanted ethanol formation during cell mass production has been approached by genetic engineering. The affinity of a S. cerevisiae hexose transporter has been reduced by gene shuffling  as well as by chemostat selection . For both yeast strains, reduced ethanol formation during batch growth at high glucose concentration could be demonstrated as a consequence of the reduced glucose uptake rate. Such engineered strains are advantageous in the field of heterologous protein production, but it is more doubtful whether such strains ever can replace the simple fed-batch fermentation regime in baker's yeast production. For baker's yeast the characteristics of the product inherently include efficient carbon dioxide formation under non-growing oxygen limited conditions and it has not yet been demonstrated that this feature remains un-impaired in strains with reduced glycolytic rate.
Nutrient limitation and starvation with respect to industrial yeast fermentation has mainly been discussed in relation to the classical processes of beer, wine and baker's yeast production. It may lead to "stuck" fermentation, which translates into large economic losses to the industry. New insight into the molecular mechanisms of nutrient limitation and starvation  makes this field of research develop rapidly [156–158]. Whereas media and strain modification in the production of beer, wine and baker's yeast may be limited by legislation and the final organoleptic quality of the product, the large scale fuel, materials and chemical industry is limited by economic constraints. Therefore, it remains to be demonstrated that recent research on nutrient starvation in yeast can be translated into novel fermentation strategies and novel industrial fermentation substrates. In ethanol production for the fuel and chemical markets, one rather relies on natural strain isolates, which have fully adapted to nutritional variation.
Fermentation substrates for the production of fuels, materials and chemicals will be produced from lignocellulosic raw materials rather than from starch and sugar. The liquefaction of lignocellulose inherently leads to the formation of weak acids, furan derivatives and phenolic derivatives [74, 159]. It is well known that weak acids can act as uncouplers and stimulate ethanol production [68, 160]. Similarly, furan and phenolic compounds often appear carbonylated and as such function as external electron acceptors, which in the case of xylose fermentation is beneficial for ethanol formation (see (ii) By-products; [148, 149]). However, the beneficial effect of these compounds is strongly concentration dependent and they more often act synergistically to inhibit yeast fermentation . Therefore, the majority of reports on the fermentation of hydrolysates derived from lignocellulosics deal with the inhibitory characteristics of such fermentation substrates.
Lignocellulose hydrolysates have to be detoxified prior to fermentation [161, 162]. However, the detoxification adds cost to the process  and should therefore be avoided. An elegant solution was demonstrated by applying a fed-batch regime to the fermentation of lignocellulosic hydrolysate . Numerous yeast strains have been evaluated for their ability to ferment non-detoxified lignocellulose hydrolysate [165–169] The results of these studies are not always coherent, which reflects the profound influence of fermentation conditions such as media composition, oxygenation and fermentor set-up. However, it emerges that most laboratory strains used in the early stages of strain development cannot be used for an industrial raw material such as hydrolyzed lignocellulosics, whereas strains isolated from industrial environments generally perform much better. This was illustrated by comparing three different recombinant Saccharomyces strains expressing the XYL1, XYL2 and XKS1 genes for their ability to grow and ferment sugars in non-detoxified northern spruce hydrolysates (NSH). The laboratory strain TMB3001  could not even tolerate 20% NSH, whereas the industrial strain TMB3400  could grow in 33% NSH after supplementation with yeast extract. TMB3006  derived from an acetic acid-tolerant Saccharomyces industrial strain isolated from a continuous spent sulfite liquor fermentation plant , could sustain growth in 40% NSH.
Strains tolerant to industrial media can be further improved by evolutionary engineering [18–20]. After exposing TMB3006 to continual selection to a NSH gradient of 40 – 70%, a NSH-adapted strain was obtained that could sustain growth in 70% NSH. This strain could be maintained in steady state at a dilution rate of D = 0.1 h-1 with an ethanol yield of 0.41 g/g on consumed glucose, which illustrated the importance of strain background to achieve the necessary robustness to ferment harsh sugar syrups, such as NSH.
The current literature on media composition in different stages of strain development for large scale industrial yeast fermentation has been summarized with a view that media composition is an integral part of strain development. In particular the final industrial environment must be carefully considered throughout the strain development process in order to assure the successful introduction of novel engineered strains into large-scale industrial processes.
The Swedish foundation for International Cooperation in Research and Higher Education (STINT), the Swedish Energy Agency (STEM), the Swedish Institute (SI), the International Foundation for Science (IFS) and the National Research Foundation (NRF; South Africa) are gratefully acknowledged for financial support.


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Year 2021 Ushers in a Ray of Hope!
by Gollamudi Radha Krishna Murty
As the year 2020 has come to an end, everyone heaved a sigh of relief as though the worst — the challenges of the Covid-19 pandemic and the resultant woes of the economy, the pain, incivility and grief of communal isolation, the economic deprivation of the marginalized wrought by the lockdowns/slowdown of economy, a kind of helplessness that ran unchecked amidst the death of over 1.8 million people around the world that shook the last 12 months — is over. And the glimmer of hope is the arrival of Covid-19 vaccines with around 95% effectiveness. The unflappable optimists are therefore hoping for a return to normality soon.
But the reality appears to be otherwise, for there is a clear uncertainty encasing this hope. Health experts say that all that we know about vaccines is that "rather than preventing a person from getting infected", they "appear to keep people from getting dangerously sick". It is still not known if the vaccinated people who do not exhibit symptoms can still spread the virus to others. There is also the threat of mutants to the vaccines. Then comes the challenge of manufacturing of vaccines meeting the demand. Even the distribution of two-dose vaccine across the geography of India is in itself a big challenge. So, in all likelihood, vaccinating the whole population and achieving 'herd-immunity' may as well extend into 2022. It means, we still cannot abandon wearing masks, social distancing and avoiding indoor gathering in the immediate future.
Now, turning to the economic crisis inflicted by the pandemic, we witness an upward trajectory: Indian economy that declined by 23.9% in the first quarter of 2020-21 could limit its contraction to 7.5% in Q2. It is the 3.4% growth in agriculture that buoyed the economy as a whole. Similar improvement is seen in manufacturing sector that entered the positive territory in Q2 in tune with a simultaneous improvement in core sectors such as electricity consumption growing at 0.6% as against –39.3% in Q1. There is an appreciable narrowing of investment slump from –98% in Q1 to –7.2% in Q2. But the continuing slump in consumption expenditure (–11.3% vis-à-vis –25% in Q1) reveals paucity of incomes, uncertainty-sentiment and none-too-happy job scenario. Amidst these green shoots what is disturbing is the squeeze in government spending by 22% y-o-y as against a rise of 16% in Q1 and yet the rising fiscal deficit owing to revenue contraction.
No doubt, there is a fall in growth in almost all the countries that mattered, but what is more disturbing with our economy is that the fall in our growth rate did not start with the pandemic: right from 2017-18 there is a continuous fall in our growth rate — each year's growth rate being lower than the previous year right up to 2020-21. Therefore, a section of economists strongly advocate that a fiscal deficit policy with a focus on investment spending rather than putting money in the hands of people be adopted to not only lift the growth rate to 8-9% in the new year but also to sustain it on a long-term basis. And, there is also a concern about the already widened fiscal deficit, which is estimated to be around 7% GDP. So, reconciling the demand for stimulus and containing fiscal deficit is going to be a big challenge. Again, all this depends on how we manage the Covid-19 pandemic.
Interestingly, the pandemic has hastened the usage of digital tools significantly: online shopping, video-conferencing, online-teaching and remote working have become the new-normal. Remote working for knowledge workers bestowed many benefits for companies: it reduces real estate costs, enables them to hire globally without the hassles of immigration issues and ensures enhanced productivity besides offering employees geographic-flexibility. There are however risks embedded in it such as communicating with workers in different time-zones, security risk of customer data, etc. Pandemic has also given a boost to the use of robots not only in the industry but also for carrying out house-hold chores. Indeed, with the advances made in AI and machine learning technologies, it is becoming increasingly evident that usage of digital tools is going to be the in-thing in the near future and it will be up to companies how they gear up to benefit under this emerging new norm. No wonder if e-learning and e-health emerge as new drivers of growth. And the real challenge would be: it creates disruption in employment, while making fresh demand for reskilling the existing employees.
Turning to geo-political scenario of the globe, it is yet not clear how much the new incumbent in the White House will be able to restore the crumbling rule-based international order. So is the case of its trade war with China: Mr. Biden may, by fostering healthy relationships with the allies, continue it rather more effectively. Amidst these global tensions, we have our own border tension with China: as the reports indicate, China may not restore the status quo ante of April 2020 in Ladakh soon, calling for dexterous handling of it by India, for it will have an economic-fallout too.
So, given the yet to be controlled pandemic, unknown prospects of economic recovery in the wake of widening fiscal deficit, and the border tensions, the year 2021 appears to be unpredictable. But optimism is the greatest trait of mankind and science and scientists are always there to kindle hope and steer us out of uncertainty.
More by : Gollamudi Radha Krishna Murty
Top | Analysis

redpajama_set_name: RedPajamaCommonCrawl


With exciting new features and performance enhancements, you'll learn how to accelerate from Go Karts to Grand Prix cars and Midgets to NASCAR. Race faster than ever in our improved racing game, played by 5 million people.
Enter the new Advanced Academy to fast-track to Grand Prix and NASCAR with tips from Jenson Button and Tony Stewart on how to make championship-winning maneuvers and understand from the drivers how the technology of Mobil 1 motor oil helps.
Test your skills in all-new mini games and earn points to upgrade your engine and customize your cars. Learn how to make the perfect Grid Start, take the perfect racing line and overtake your opponents.
To experience the Mobil 1 Racing Academy, which features 3D graphics, multiple car racing, and an amazing full-screen mode, you must have the Unity software plug-in on your computer.


I probably don't need to remind you the clocks go back tonight............an extra hour in bed woohooo! Unless of course you have a little person in your house who doesn't get this at all. Or a cat tapping your face because it's breakfast time and you are 2 minutes late! Why stay in bed when you can wake mummy and daddy up and get them to play with you?!!
Many of us underestimate the importance of sleep when it comes to our health. We look forward to the weekends as an opportunity to catch up (if you are one of the lucky ones) but if we were able to sleep more consistently through the entire week, we would be so much better off.
We are sleeping less than ever yet we ignore the fact that mental and physical health problems become more pronounced when we have less than 6 hours sleep a night. Around 1/3 of the UK population are sleep deprived, but how much sleep do we really need? 8hrs a night is really a myth. Experts believe most adults need ~6-9 hours a night in order to feel refreshed and be able to function the following day; it is a very personal thing.
I bet most of us know from personal experience that just one bad night's sleep affects our mood and productivity. Catching up on that bad night however is easier said than done! Our to do lists are longer than ever and of course when we are tired, we achieve less. We procrastinate more, find it harder to focus and so it becomes less likely we will get everything done we wanted to............so we work for longer, stay up later, get more tired, the cycle goes on.
Long term sleep deprivation is well known to be associated with several serious health concerns - heart disease, diabetes, stroke and depression but more recently there's been much more focus on the link between sleep, eating and body weight. Disrupted sleep is linked to changes in appetite and metabolism; the less we sleep, the more likely we are to gain weight. Just one night of poor sleep slows our metabolism the following day with many other negative repercussions.
Sleep-deprived people tend to seek more calories, are more impulsive with their food choices and also eat more (portion sizes tend to be larger). One of the reasons for this is that sleep loss causes hormonal shifts that cause us to feel more hungry. We release a hormone called grehlin that stimulates appetite. This isn't a true hunger but a response our body often uses when we are tired; we go for more Calorie rich foods (particularly fatty foods) in an effort to boost energy. When we were running about in loin cloths it was a mechanism to increase survival. Today however, we live in a society where food is plentiful, accessible and cheap for most so it's easy feed those hunger signals.
And when we feel tired, we are also less likely to want to exercise. Energy expenditure falls which can also have a negative impact on our motivation, 'get up and go' and waist line.
Our bed times will also have a significant effect on our food intake too. Nearly half of us go to bed after 11pm and that time may well be spent watching TV, playing on mobile phones and tablets, checking emails; being busy doing nothing really!
The later we stay up, the more likely it is we will snack and of course, eating late at night is not very conducive to good sleep. We may snack out of habit whilst watching a film. It could be out of boredom or due to stress / emotion. I think the evening time is one of the most challenging times for eating foods we might have resisted all day.
Get active! Inactivity is linked to poor quality sleep and fewer hours too.
Avoid caffeine after 3pm, particularly if you find it difficult to drop off at bed time.
Find a routine that works for you - try to stick to a consistent bed time and wake up time - our body clock loves routine!
Have a 30 minute wind down period before you go to bed to help your body prepare for sleep -turn down the lights and switch off your electronic devices. Reading or listening to music are ideal.
Ensure your room is a sleep haven; dark, quiet and free from distractions - leave your phone charging downstairs. Get rid of the TV and any other unnecessary electronics.
Next time I'll be sharing some tips on which foods can be good for sleep and help you calm down before bed.


As part of the Incredible Adventures of Cinnamon Girl blog tour, I am pleased to welcome author, Melissa Keil, onto the blog, to tell us seven secrets about herself and her writing.
My favourite book of all time, since I was a kid, is The Hitchhikers Guide to the Galaxy. The last line of the 'Guide's prologue is, 'It begins with a house'. The almost-last line in The Incredible Adventure's of Cinnamon Girl's prologue is, 'It begins with a house'. This may not have been purely coincidental.
I love travelling, but have a pretty severe car phobia and have never been able to drive a car. I adore long train trips though.
I am a hopeless fangirl, and have a total melt-down whenever I have to meet anyone remotely famous who I admire. I was a stammering idiot the time I (briefly) met Neil Gaiman. Oh, and I once followed Elton John around a department store in London, literally stalking him whilst hiding between display racks of clothes, but was too terrified to say hello.
My favourite movie when I was about five was The Sound of Music. I used to watch it on repeat, every single day, until one of my family members got so irritated with it that she recorded over the VHS tape (this was in the days before everything was downloadable, so, for five-year-old me, was a HUGE calamity). Now, whenever the movie is on TV, I make sure to call this particular family member, with the volume up loud while singing really loudly down the phone. I don't think it's made the movie any more appealing for her. (Alba and her mum in Cinnamon Girl also happen to be big musical fans).
I used to collect pet mice when I was young, but never managed to figure out how to secure them in a proper hutch. Consequently, the Great Escaped Mice Hunt was a frequent feature of my childhood.
My favourite food in the world is cheeseburgers. If not for the threat of scurvy (and heart disease), I could easily eat cheeseburgers for every meal, every day.
The Incredible Adventures of Cinnamon Girl is published by Stripes Books on the 11th of February 2016.
If you enjoyed this post, make sure you check out all the other blog posts below which are in the tour.


You have the most innovative software ideas in your particular market, but you're not entirely sure how to get from the product development to getting it on the shelf — online. Product roadmapping is a very beneficial process that "maps" out how to go from A to Z. While you might not think that creating a roadmap is a very important use of your time, your investors and shareholders will ask for this information to determine whether or not the project would be feasible to begin (and often overlooked…maintained). The more professional, thorough and focused your product roadmap is, the more likely you'll receive the proper permissions, funding, and resources to proceed. Beyond distinct buy-in, there's also a lot less risk when you align your product to an end goal or path.
When looking for the right company to build your product roadmap, it's best to find a team that can distill all of your ideas into a single document. One of the services that we provide is an onsite intensive workshop or shall we say, brainstorm session? This will effectively get all of the ideas and requirements out on the table and facilitate essential out-of-the-box thinking. Once the product is defined, every detail is clearly presented in a unified document.
The best part about leveraging the CURTIS Digital team for this process is that we don't have to stop at assisting you with just the ideation. We can build your ideas that we helped you define. The last thing you need is a consulting firm with ideas that are unrealistic or add to your operating constraints.


همونطور که میدونید خوراک خرچنگ و لابستر یا صدف بیشتر درسفره های فرنگی باب هست و در ایران کمتر به این غذاهای دریایی توجه میشه.
There are many ways to enjoy lobster. We have compiled a small collection of recipes for you to try and enjoy.
Over medium heat in a medium sized saucepan, heat butter and add garlic, onions, and mushrooms. Saute until soft. Add walnuts, black pepper, lemon, spinach and white sauce. Blend thoroughly and remove from heat. In individual shallow baking dishes, assemble crepes. Place 2 oz. of lobster in each crepe, top with spinach mixture, and wrap each crepe. Place in baking dish, approximately 2 to a dish, with folded side down. Bake in preheated oven for 10 minutes at 350 degrees. While crepes are baking, make Hollandaise sauce. Remove crepes from oven, top with Hollandaise, garnish with chopped parsley and paprika and serve.
Lobster Recipes: Lobster Bisque, Newburg and More.
Lobsterclambake.com takes pride in our fresh, succulent lobster. We also pride ourselves in delicious lobster recipes . Below are some of our favorite lobster recipes.
We hope you enjoy these lobster recipes as much as we do. We are always in search of new lobster recipes. If you have a favorite lobster recipe, please email email your lobster recipes to us. If you like our lobster recipes, please share them with a friend.
Click on these links to visit our Live Lobsters Live Lobsters and New England Clambakes New England Clambakes pages for selections and prices.
Remove all meat and tamalley from lobster shell. Place in bowl and chill. Chop lobster shells as fine as possible. Heat a medium size saute pan until hot and add butter. As butter starts to smoke, add shells, reduce heat and cook shells for 1-2 minutes tossing the shell pie fragments frequently. Add all the minced vegetables and garlic and cook for 5 more minutes. Slowly sprinkle in the flour and cook for another 2-3 minutes. Transfer everything into a 2 quart saucepan and deglaze the saute pan with the sherry adding everything to the saucepan. Heat the mixture until hot then slowly add the lobster stock until the soup thickens. Add the tomato paste, thyme, bay leaf, and paprika.
In a 2 qt. double boiler, bring 1 qt. of milk to 180 degrees. While the milk is heating, melt the margarine in a small saucepan. Add the flour and mix thoroughly to the melted margarine with a wire whip . Slowly add this roux to the scalded milk, stirring constantly with the whip. Keep whipping to avoid lumping as the sauce thickens. Add the Dijon mustard mix and then the lobster fumet, slowly whipping until smooth. Set aside. In a large sauté pan, put the clarified butter and the minced shallot and cook on low heat until the shallot is slightly browned. Cut the lobster meat into bite sized pieces and add butter and shallots. Cook on heat for about 1 minute then add Worcestershire and black pepper. While stirring constantly with a wooden spoon, add the sherry and the lobster tomalley and cook until the sherry starts to boil approximately 2 min. Sprinkle in the paprika and add the white sauce. Reduce heat and simmer for 2 minutes. Remove from heat and serve on a golden brown patty shell.
Follow the cooking method for sautéed lobster. Keep the shell intact and carefully remove the meat from the body. Reserve the tamalley in a bowl and place in oven. Cook for approximately 45 minutes or until top of quiche is golden brown. Remove from heat and let stand for 15 minutes. and cut the meat into small bite sized pieces. In a medium sized sauté pan, heat olive oil over medium-high heat. Add scallions, bell peppers, chopped mushrooms, tarragon and black pepper. Cook 2-3 minutes until vegetables just start to soften, then add brandy and wine and turn heat to high. When liquid is boiling, add lobster stock and reduce to almost a glaze by lowing heat to medium. Add the white sauce and Dijon mustard and blend thoroughly. Add the lobster pieces and tomalley and mix together. When mixture starts to bubble, remove from heat. Place mixture in shells, top with parmesan cheese and sprinkle with paprika. Set under a preheated broiler and brown. Serve immediately.
In large bowl mix melted butter into cracker crumbs and garlic powder. Split the lobsters down the center and remove the stomach and vein. Save the tamale (liver) and add to cracker crumb mixture. Stuff lobsters with cracker mixture. Bake for 20 minutes at 450 degrees.
Using a medium sized wooden salad bowl, mash and spread the anchovies and the garlic with a fork and rub the inside of the bowl with it. Adding the vinegar and mustard, blend thoroughly. Using the fork, add the olive oil and the coddled (meaning to cook in simmering water for 1 minute) egg yolk, mix together and add the Worcestershire, lemon, and parmesan cheese. Add the romaine lettuce and croutons and toss the salad until all the lettuce is coated with the dressing. Serve in individual bowls adding a lobster claw to each. Serve immediately.
4 cups peeled & cubed potatoes, 1/2" cubes 2 tsp.
Bring chicken broth to a boil in a small stock pot. Add leeks, potatoes, thyme, pepper and Worcestershire sauce. Bring to a boil again, cover and let simmer for 1 hour. Remove pot from heat and drain off one cup of the liquid. Puree rest of mixture (with blender, food processor, etc.). Add cream to each batch that you puree. On the last batch you puree add mustard. Place pureed soup in container and add lobster meat mixing thoroughly. Chill for four or more hours. Serve in chilled bowls and top with parsley and scallions. Great with your favorite wine!
In a food processor using the knife attachment, place the cooked meat of one quarter lobster. Start motor and run until meat is finely chopped. Remove lobster and repeat process with the crackers. In a medium sized bowl, combine all ingredients and mix thoroughly.
Preheat oven to 375 degrees. In a medium sized mixing bowl, blend eggs, sour cream, Worcestershire, pepper and Dijon mustard together. (This is the quiche batter.) Arrange pie dough in a pie shell. In a separate bowl, toss the two cheeses with the scallions and lobster. Place the lobster mixture into the pie shell. Add the quiche batter, sprinkle with paprika.
Boil water in large pan and immerse lobsters to kill them. Remove from water, lay them on their backs and cut them in half lengthwise with a chef knife. Remove the intestinal tract that runs down the back of the tail section and the stomach. Save the tamalley and the roe in a separate bowl to add later on. In a large skillet, place olive oil and garlic. Cook on high heat until garlic starts to brown. Add the onions, celery, peppers, parsley, pepper, Tabasco, Worcestershire, oregano and basil. Cook over medium heat for 4-5 minutes then add lobsters split side down. Pour the tomatoes, marsala wine, tomalley and roe over lobsters, cover and cook on medium heat 8-10 minutes. In a large shallow bowl, place 8 oz. of the pasta. Remove lobsters from pan and place split side up on pasta. Cover with sauce, sprinkle with parmesan cheese and serve.
Over medium heat, in a medium size sauce pan, melt the whole butter and add the lobster fumet and the heavy cream and turn heat up to medium high. When cream is hot, just before boiling, add the parmesan cheese and whisk briskly until all the cheese is melted and dissolved into the cream. Add the Worcestershire, Tabasco, black pepper and Dijon and whisk thoroughly again. Reduce heat again to a fast simmer and allow mixture to simmer 20 minutes. While the sauce is simmering, cook pasta to your preference, drain and set onto plates. Cut the lobster meat into small pieces and add to sauce, add the egg yolks and turn heat to medium high. Sauce should be of medium thickness. Ladle sauce over pasta, sprinkle with fresh chopped parsley and serve.
Over medium heat in a medium sized saucepan, heat butter and add garlic, onions, and mushrooms. Sauté until soft. Add walnuts, black pepper, lemon, spinach and white sauce. Blend thoroughly and remove from heat. In individual shallow baking dishes, assemble crepes. Place 2 oz. of lobster in each crepe, top with spinach mixture, and wrap each crepe. Place in baking dish, approximately 2 to a dish, with folded side down. Bake in preheated oven for 10 minutes at 350 degrees. While crepes are baking, make Hollandaise sauce. Remove crepes from oven, top with Hollandaise, garnish with chopped parsley and paprika and serve.
Bring a large pot of salted water to a boil. Cook lobsters for 10 minutes. Turn the cooked lobsters onto their backs. With a sharp knife, split down the center. Remove the stomach and discard. Crack claws with flat side of knife. In a large bowl, mix crab meat, onion, garlic, egg and herbs. Season with salt and pepper. Steam rice over medium heat, then fluff with fork.
Salsa: In a bowl, mix red onion, red pepper, cilantro, oil, balsamic vinegar and mango. Add rice. Reserve until ready to serve.
Stuff the cavity of the lobster with the crab mixture. Pour butter over the stuffing. Place under the broiler for 10 minutes or until lobster is cooked. Serve with the rice mixture.
In a large pot of boiling salted water, cook lobsters for 10 minutes. Cool. Split the lobsters in half with a sharp knife. Remove tail meat and save. Break the arms free of the body. With a sharp knife, cut into the claw and twist the knife to break the shell. Twist the lower claw to remove cartilage, snap and pull. Remove the claw meat and chop. Leave the tail meat whole.
Assemble the purses: In a heavy pan over medium heat, melt the butter and sautT the leeks for 10 minutes or until soft. Add the garlic and cook for another three minutes. Add the wine, increase the heat to high and reduce until almost no liquid remains. Add salt and pepper to taste. Stir in basil and set aside to cool.
Bring a pan of water to a simmer. Add spinach leaves and blanch for 10 seconds. Remove with slotted spoon; immediately plunge in ice water. Squeeze out excess water. Place a circle of the rice paper on the lower half of a towel and fold the top half over the rice paper, enclosing it. Press down gently and let rest for two minutes. Unfold the towel and transfer the paper to a work surface. Place three spinach leaves in the center of each circle, overlapping slightly. Place 2 tbsp of the leek mixture into the center. Place 1 lobster tail and one claw on top of the mixture. Lift the sides of the rice paper and gather together at the top. Tie tops with the chive strips. Preheat oven to 400o F. Brush a pan with butter. Place the lobster purses in the pan. Bake the purses for 8 to 10 minutes or until golden brown. Place 4 slices each of avocado and mango on individual plates and place the purse on top. Serve hot.
Combine white wine, wine vinegar, salt, pepper and shallots in a saucepan. Reduce liquid over medium heat until only 3 tablespoons remain. Add the cream and peppercorns and reduce again by one half.
Steam Clearwater Lobster in water and a little white wine. Remove the tail meat from the shell and cut in half lengthwise.
Stand head up, arrange body meat on plate with tail meat on top. Place claws on either side. Top meat with Sauce Beurre Rouge. Garnish with whole pink peppercorns.
The eggs often found in female lobsters are called lobster roe. They are bright red and firm when cooked and are considered a gourmet delicacy. Here's a quick and easy recipe designed to make almost any meal more Roe-mantic!
The smooth green substance found in a cooked lobster's body cavity is called tomalley, and is also considered a gourmet delight. Makes a great spread!
Mix all ingredients together and chill. Serve with crackers or fresh vegetables.
Shells are to lobsters what bones are to other animals. Like soup bones, they can serve as the basis of delicious broths and bases. Use them in stocks to add color and flavor to sauces, stews and bisques.
Simply simmer with onions, carrots, celery and seasonings in water, for about 30 minutes. Strain. Reserve liquid.
Planning lobster meals is easy because you can purchase your fresh Clearwater lobster in every size and quantity to suit all your cooking and menu needs, from romantic dinners to family style get-togethers.
A 1-2 lb (454 g – 908 g) whole lobster serves one person. But you probably won't be able to stop at just one!
For maximum fresh-flavor, cook and serve lobsters as soon as possible after you receive your order.
Store refrigerated, covered with a layer of wet newspaper, for no more than 12 hours.
Do not store live lobsters on ice, in fresh water or in the bathtub.
Store cooked lobsters on their backs to keep in the juices.
Everyone knows how delicious lobster is . But were you aware of how good lobster is for you? Nutrition-wise, lobster is a valuable low-fat source of protein, calcium, zinc, phosphorus and a good source of vitamin B 12. Lobster is lower in cholesterol than you think. In fact, lobsters contain Omega 3 Fatty Acids, substances that researchers believe may actually fight cholesterol build up, reducing the risk of heart disease.


text:
Picard: "Is There Anyone On Romulus He Might Know, Or Choose To Contact?
Picard: "Is there anyone on Romulus he might know, or choose to contact?"
Sarek: "Pardek?" Picard: "Who is Pardek?" Sarek: "It could be...Pardek!"
Picard: "Who is Pardek?" Sarek: "He is a Romulan senator. Spock has
maintained a relationship with him over the years. I don't know
how they met. At the Khitomer Conference, I'd imagine." Picard:
"Pardek represented Romulus." Sarek: "Yes! I'm sure he did. In fact,
I recall Spock coming to me with optimism about maintaining a continual
*dialogue* with the Romulans. I told him that it was illogical to
maintain such an expectation. Spock was always so impressionable.
This Romulan Pardek had no support at home! Of course, in the end,
I was later proven correct."
-- "Unification I", Stardate 45235.4
Picard: "But you believe that he might have gone there to see Pardek.
Sarek: "The Romulan senator? How do you know Pardek?...
Sarek: "You're here, on Vulcan." Picard: "I need your help.
I must find Spock." Sarek: "He's not here, I know....
Picard: "Sarek! I've come a long way to see you." Sarek
I will not answer!" Picard: "I must talk to you about your son....
Pardek: "How could they know of this location? Someone has betrayed us!
Spock: "Yes, you did." Pardek: "Spock, we've been friends for eighty years....
Picard: "Sarek, we are a part of each other. I know that he has caused you pain.
But I also know...that you love him." Sarek: "Tell him, Picard....
Neral: "What do you know of this human? Jean-Luc Picard.
A Starfleet captain?" Pardek: "I know nothing of him....
Spock: "For some time now, I've been aware of a growing movement here of people who seek to learn the ideals of the Vulcan philosophy.
They've been declared enemies of the state, but there are a few in the Romulan hierarchy like Pardek, who are sympathetic....
Riker: "How well did you know Spock?" Picard: "I only met him once.
What I know of him comes from history books. And, of course, a mind meld with his father....
Spock: "I know of your mind meld with my father, which enabled him to complete his last mission.
Picard: "It was an honor. He's a great man." Spock...


Canadians report a surge in national pride in the afterglow of the Olympics.
Kevin Roberts on the enduring power of English football's Play-Offs...the stuff 'sporting dreams are made of'.
Kevin Roberts on great ideas and creativity in sport... so long as we remember to keep sport at the heart of any new project.


Welcome to ByJeannie Jewelry's official blog! It's about time!
I wanted to personally share with you my inspirations, happenings, and favorite fashion trends. Also, stay tuned for new jewelry collections, sneak peeks, and press features!


"Where's Inkspot?" The readers grow restive.
"Been long since his verse was tempestive."
Can't blame him - the season's too festive!
Newsmericks wishes everyone Shubho Bijoya and Happy Diwali (in advance). Blogging will resume after November 9th.
i have posted 50 blogs all are related to KERALA .It includes culture, festivals, temples, tourist places, trekking, hiking etc. Promote the blog and the kerala cultre.
Then open my PROFILE page, here i have posted all 50 blogs.
but return on november nine!
ps: so lame but what to do? mind's already gone for the holidays!
Creative blog!!! I enjoyed it!!


Note: I've made the dots much bigger than they need to be, just so they show up easily in the photo. By the way, there should be a dot on the center-line, right near the bottom - but the photo cuts it off.
Firstly, mark the the top dot on the center line, then lay the vertical spar down beside the plastic and mark the bottom dot.
Measure 1.25SL (36.3cm, 14 3/8") down from the top dot and mark the middle dot.
Next, mark each end of the fold-line plus the mark which is part-way along the trailing edge, as in the photo - judging the 90 degree angle by eye is quite OK since any small error will be exactly the same on the other side.
Lay a leading-edge spar over the fold-line, and shift the lower dot a little if necessary, so the fold line is exactly the same length as the bamboo spar.
Next, mark the 2 corners of the long narrow flap along the leading edge - after seeing what 0.05SL (1.5cm, 5/8") looks like, just put these dots in by eye.
Finally, rule lines between the marks, as in the photo.
Cut out a roughly rectangular section of the bag containing the kite sail, open it out and lay it flat on the table - you can now see the complete sail outline, as in the photo.
Lay a piece of clear sticking tape along the short nose section with the line showing through the center of the tape - but avoid getting any tape on the fold-line.
Lay 4 pieces of clear sticking tape along the trailing edge, with each line showing through the center of the tape - again, avoiding the fold-line.
With scissors, cut all around the sail outline.
Place the leading edge spars, the ones with just one short reinforcer, over the fold lines - with the reinforcers facing the center-line.
Lay one of the remaining spars over the 2 leading edge spars, so it crosses them at the top end of the 2 reinforcers - this will be the spreader.
Making sure the spreader is centered over the sail center-line, make marks where it crosses the leading edge spars.
Re-check the fit, since you will probably have to snip a few more times to get it just right - the tips tuck neatly in, with the spreader lying flat against the table.
When it all looks good, drip on some wood glue, as in the photo.
Now wait for this to dry!
The black pieces of electrical tape mentioned below should all be roughly 3 times longer than they are wide. You can't see the full length in the photo since they are folded under.
Place the remaining spar over the center line of the sail, and underneath the spreader - this is the vertical spar.
Secure the upper end of the vertical spar to the sail with a piece of electrical tape, wrapping it under to the other side of the sail.
Secure the lower end of the vertical spar to the sail with a similar piece of electrical tape, wrapping it under to the other side of the sail as before - gently pull any slackness out of the sail as you do this.
Next, fold the sail over the leading edge spars and cap the tips with black tape, as in the photo - the tape should be folded around the tip of the spar so it can't slip through the plastic.
Add more tape like the yellow pieces in the photo - right over top of the short reinforcers.
Add 4 long pieces of clear sticky tape to complete the taping down of the sail near the leading edges of the kite.
Secure the spreader to the vertical spar, using drops of glue where they cross.
If you want to, trim any bits of tape that stick out, with scissors.


On a stretch of road outside Palm Springs lies a small white building, filled with dates prepared in every imaginable way. Covered in chocolate. Spun into milkshakes. Refined into sugar. It's because of this place that when I think of Palm Springs, I imagine wrinkled brown dates nearly as much as I do palm trees, pools, and boozy Rat Pack era stars.
Among my last haul of goods from that Palm Springs date shop was a bottle of date syrup that I'd intended to use to flavor some oatmeal, but have since realized I'd rather use to sweeten up drinks. Today it's providing that touch of sweetness to a variation on the Martinez cocktail, a darker and sweeter alternative to the martini that may even pre-date its drier, tuxedo-wearing cousin.
When it comes to sweetening a cocktail, sugar is just the starting point. Honey is one alternative; dates are another. When rendered into a thick, sticky syrup sometimes called date molasses, dates offer a warmer and more complex flavor than your regular granulated stand-by, with less sweetness. A few years ago, date syrup was limited to the aisles of Middle Eastern specialty stores and health food groceries, but these days it's more available in supermarkets and, of course, online.
The syrup's rich, deep date flavor pairs wonderfully with a nutty but dry amontillado sherry. Here, my fall-inspired riff on the Martinez mixes the sherry with equal parts gin to dry out the drink and lend some contrasting juniper and pine flavors. The cocktail gets a sweet and spicy kick from a few dashes of Peychaud's bitter, and is brightened up with a twisted orange peel that doubles as a garnish. If you're knee-deep in dates, they can make a nice garnish, too, but I'm saving mine for snacking.


Philippines 2019 Informational meeting on Sunday, April 22. We will meet immediately after the 11am service at the 46 Campus. Interested? No need to register, just come to the meeting for more details. This potential mission trip will be open to medical and non-medical team members.


text:
UNSETTLING / DOK.aroundtheclock am 26. Januar
Overview calls 2020
Regulations DOK.education
DOK.network Africa
Competitions and sections
VR Pop Up Cinema
Awards and Juries
Focus Music
DOK.forum Industry
DOK.forum Podcast
DOK.education
DOK.art/fair
Virtual Reality Pop Up Cinema
Virtual Reality creates new visual experiences and opens up new ways of storytelling.
On the occasion of the 30th anniversary of the cooperation between Québec and Bavaria, the VR POP UP KINO presents seven current documentary VR/360° experiences from Bavaria and Québec in the Space-Age Kunststoffhaus Futuro at the Pinakothek der Moderne.
We go on a space mission with NASA astronauts, ride our motorcycles from Ho Chi Minh City to Bangkok, float on a red velvet chair through the Munich National Theater, immerse ourselves in the world of a bipolar young woman and much more - in an outstanding selection of forms of representation and narrative.
MANIC VR
Canada 2018, directed by Kalina Bertin, in collaboration with Sandra Rodriguez, Nicolas S. Roy and Fred Casia, Produced by Sandra Rodriguez EyeSteelFilm_Creative Reality and Dpt.co, 12 min.
Explore the exuberant and chaotic bipolar world of Felicia and François. The user will embark on a journey to decipher the whirlwind of mania, psychosis and depression.
ROXHAM
Canada 2018, created by Michel Huneault, in collaboration with Maude Thibodeau and Chantal Dumas, produced by NFB, in collaboration with Le Devoir, Phi and Dpt., 15 min.
Photographer Michel Huneault takes you to Roxham Road, the location with the largest number of irregular border crossings by asylum seekers moving from the United States to Canada. Based on 32 true stories.
SPACE EXPLORERS: A NEW DAWN
Canada 2018, written and directed by Félix Lajeunesse und Paul Raphaël, produced by Felix & Paul StudiosFelix & Paul Studios, 20 min.
Witness the lives of NASA astronauts as they navigate the trials and sacrifices of their training and missions. Explore the camaraderie of space programs around the world, and discover the latest advancements and plans for deep space exploration.
TRAVELING WHILE BLACK
USA, Kanada 2018, directed by Roger Ross Williams, in collaboration with Félix Lajeunesse and Paul Raphaël, co-directed by Ayesha Naradajah, produced by Ayesha Naradajah, Johan Robinson and Felix & Paul Studios, 21 min.
TRAVELING WHILE BLACK is a cinematic VR experience that immerses the viewer in the long history of restriction of movement for black Americans and the creation of safe spaces in our communities. Visit historic Ben's Chili Bowl and join patrons as they share and reflect on their experiences. Confronting the way Americans understand and talk about race, TRAVELING WHILE BLACK highlights the urgent need to facilitate a dialogue about the challenges minority travelers still face today.
Germany 2019, directed by Emil Spiewok, produced by Caroline Muhl, Fimcrew Media GmbH, Vtopia360, 20-40 min.
A travel documentary set on one of the most beautiful motorcycle routes in South East Asia. Meandering from Ho Chi Minh City to Bangkok we cross countries shaken by wars and political turmoil – Local perspectives, problems and dreams are center stage in this VR experience.
FAT CAP
Germany 2018, directed and produced by Christian Felder, 7 min.
The mini documentary "Fat Cap" tells the story of the artists of the "Scale" Festival. Graffiti artist Daniel Man brought the international stars of the Mural scene to Munich for this project.
V-ARIA
Germany 2018, directed by Daniel Moshel, produced by Bavarian State Opera, 5 min.
With V-ARIA everyone can dive into the dazzling and overwhelming world of opera. On a red velvet chair from the Munich National Theatre you travel through the opera house, meet musicians, dancers and great singers.
Space-Age Kunststoffhaus Futuro,
Die Neue Sammlung, Pinakothek der Moderne,
Barer Str. 40, 80333 München
Monday to Sunday, 10:00 am to 6 pm
Thursday until 8 pm
Thursday, 9th May from 3 pm
Myriam Achard, Centre Phi Montréal
A driving force in communications, Myriam Achard has more than 20 years of experience in public relations and press relations. For the past 10 years, she has worked with Phoebe Greenberg to develop and promote the Canadian and international avant-garde arts scene. She has been Director of Public Relations and Communications at the Phi Centre since it opened in 2012, and is Director of Communications for the DHC/ART Foundation for Contemporary Art.
Astrid Kahmke, Bayerisches Filmzentrum, Creative Director
As Creative Director of the Bavarian Film Centre, Astrid has also been Head of the "European Creators' Lab" for artistic VR developers since 2017. For several years she has been curating the "i4c-Events", which grows into the international competition for narrative VR - "Virtual Worlds" in July under her direction. Astrid also works as a VR producer for INVR.Space.
Oliver Seibert, Bayerisches Filmzentrum, Deputy of the Management Board
Studied media informatics at the Brandenburg University of Applied Sciences, developed numerous learning apps on topics such as negotiation and team management. At the Film Center he is responsible for project planning, cost controlling, IT management, press and public relations.
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The City of Vinton will celebrate it's Annual Heritage Festival on the second Saturday of October. So, come out to the Vinton City Park on the corners of Center and Horridge Streets. There will be lots of good food, arts and crafts, a petting zoo, free games and jumps for all the kids and live Music. Free Admission and parking, no ice chests, alcoholic beverages or glass containers please. Bring the whole family out for the Vinton Heritage Fest on the second Saturday in October. From I-10 take Vinton exit 8, head north to the Vinton City park. Sponsored by the Town of Vinton and the Louisiana Convention and tourism bureau.


As a great resource for all of our classes' discussions on AI I want to point out first the AI Now annual report. AI Now Institute is a research institute (within NYU! we can go talk to them) focused on interdisciplinary research on the social impacts of AI.
A list of books fleshing out a variety of important background information as well as providing analysis. I have most of these if anyone wants to borrow.


There are many ways to create income from mutual funds. But there are only a few that are really safe over the long-run (which we'll get to). Many people think that they can buy mutual funds that buy bonds or preferred stocks and that by so doing they'll safely get the income they want.
It's true that such funds generate income but they expose investors to risks they may not be aware of. I say this because people who pile into bonds give up the potential to grow their money. They also make the mistake of locking in to today's ridiculously low rates. That means that when interest rates rise, the value of that bond mutual fund is going to sink faster than the Titanic. That's because as interest rates rise bond values drop. That's just what bonds do.
So if you are interested in creating safe income from mutual funds – especially growth mutual funds – there is a far better way. Let's look at such an alternative known as systematic withdrawals.
Let's assume you have $100,000 and want to take an income check every year from your account. You've decided that bonds aren't for you. You like the idea of investing in the S&P 500 because you like the long-term potential for growth.
Neal's Notes: By the way, you can also use similar techniques to get income from stocks even if they don't pay a dividend.
Can you see that the balance of your initial $100,000 is worth almost $170,000 (hypothetically) by 1991? Feels good…right? Now, if you set up your account to withdraw 4% of the balance, your income increased from $4000 a year in 1988 to more than $6700 in 1991. Sweet. That's an increase of over 50%.
And by 1999 the value of your original investment grew to more than $540,000. That means your income grew from $4000 to over $21,000. If that's not fantastic, I don't know what is.
Sadly, the direction isn't only up. By 2002, your account value dropped to $292,000 – enough to ruin anyone's barbeque. And to make things worse, your income dropped from $21,000 in 1999 to $11,703 in 2002. That's more than a 50% drop. Terrible. Of course, it's still almost 3 times greater than the $4000 income you started getting 11 years ago but most people forget about that. They focus on the "loss" rather than the relative gain.
Which would you prefer? A fixed income of $4,000 a year or one that went from $4000 to as high as $21,000 and then back "down" to $14,000? While the emotional roller coaster was greater with option 2, so was the income.
While it's nice to see your principal rise every single year – it's not important. You aren't spending your capital (hopefully). The income is what's important. Am I right?
It's important to grow your capital if you want to grow your income. There is no other way to do that. And if you want to grow your capital you must accept short-term risks.
If you want to create "safe" income from mutual funds you must first define "safe". Is it more important that your income is fixed? If that's how you define "safe" you will struggle with inflation down the line and ultimately this decision may jeopardize your financial future. I can make the case that this route is anything but "safe" over the long run.
If you define "safe" as having sufficient income over the long-run you'll turn to equity growth. The downside will be short-term fluctuation of your capital and your income.
How do you define "safe" income?
You said, "Look at column "C" and see what theoretically happens to the value of your $100,000 investment – after the market does it's thing and after you withdraw 4% of the value of the account value at year end." Actually, it looks like you calculated 4% of the value of the account at the beginning of the year. Example: At the beginning of the year, you calculated 4% of $112,610.00 which is $4504, let the $112,610.00 increase by 31.69% and then withdrew the $4504 leaving the account with $143,791.71. Even though you seem to have misstated how you arrived at the numbers, I like calculating the 4% at the beginning of the year so you know for a fact how much you will be withdrawing at year's end.
Nicely explained. Truly, the benefits outweigh the risks and losses.
This analysis is very misleading. How about starting from 1999 with the first year a 9.1% decline and take it from there? The next 13 years leaves someone a much smaller balance for the years going forward to appreciate from at this current point and that's if there's much appreciation from the currently richly priced market (average p-e but with historically high profit margins). Bottom line; the results are enormously different than your rosy scenario.
Steve, you make a good point but I also take issue with one statement. First, bring light to the fact that there can be extended periods of poor performance and that is important. A 10-year period isn't a long-enough to make this work always.
Sadly, I do not understand finances very well. My (new and first) broker quickly invested $100,000 I inherited from my Mom in a variable annuity last year. I just went along not knowing anything. Brokers get paid well for selling annuities. This money is as important to me as millions are to others!! I am trying to read and learn. The more I read the less I understand. But, wouldn't it be wiser to invest this money in a mutual fund (of some sort) than an annuity? I understand annuities are expense but I get a "guaranteed" stream of income? I am currently 60 yrs old and I will have to retire in a couple of years due to poor health.
The variable annuity may or may not be appropriate for you. Generally speaking, I'm not a fan. Trying reading this on variable annuities and this on a possible law suit against your agent.
Just curious, what if you took the 4% from a three year moving average rather than the value of just one year. Would that even out the income somewhat?
Trina, this is amazing question. I have never seen this analysis. It certainly would even things out. I don't know about the "survivability" because it would slow the reductions down when those adjustments need to be made. Also, to be fair, it would slow the increases. I really love this idea. Great thinking! I would have to look into this further though….
Great article. Many investors don't get the concept of income. All they understand is what they see as the balance on their statements every month. There is entirely too much focus on performance, and not enough emphasis on income. Articles like this bring that back into perspective.


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	<title>Interface danperron\danmvc\Core\Config\IConfiguration | danmvc</title>

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		Overview

Namespaces

danperron

danmvc

Core

Config

Log

Http

PHP

Classes

ArrayConfiguration

JSONConfiguration

XMLConfiguration

Interfaces

IConfiguration

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				Overview

				Namespace

Class

				Tree

Interface IConfiguration

Direct known implementers

			danperron\danmvc\Core\Config\ArrayConfiguration,
			danperron\danmvc\Core\Config\JSONConfiguration,
			danperron\danmvc\Core\Config\XMLConfiguration

		Namespace: danperron\danmvc\Core\Config

				Author:
				dan

		Located at danperron/danmvc/Core/Config/IConfiguration.php

Methods summary

			 public
			stdClass

		#
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Returns

					stdClass
attributes

		danmvc API documentation generated by ApiGen 2.8.0

redpajama_set_name: RedPajamaGithub