The citric acid which is used in the manufacture of lemonade and presently it is produced by a Aspergillus niger and it is done in an effort to offer a sensible foundation for the optimization of citric acid production by Aspergillus niger, and developed a mathematical replica of the metabolism of this filamentous fungus when in situation of citric acid accretion. The widespread appraisal of the dependability and value of the model put us in a situation to address questions of optimization of the scheme with reverence to improved citrate production. The controlled optimization of A. Niger metabolism with the objective of predicting an enzyme action outline yielding the utmost rate of citrate production, whilst at the same time, keeping all enzyme performance within predestined, physiologically tolerable ranges. When the total enzyme concentration is endorsed to twofold its basal value, the citric acid production rate can be amplified. Citric acid is a product chemical produced and inspired throughout the world. It is used largely in the food and beverage industry, principally as acidulate. While it is one of the oldest industrial fermentations, its world production is tranquil in fast growing. Worldwide production of citric acid in 2007 was above 1.6 million tones. Biochemistry of citric acid fermentation, different microbial strains, as well as different substrates, technological processes and product revival are accessible. Global production and economics aspects of this advantageously product of bulk biotechnology are discussed. Citric acid is produced by inundated fermentation of starch or sucrose-based medium, using the filamentous fungus Aspergillus niger. Citric acid (a tricarboxylic acid) is of industrial significance because; it is broadly used in dairy, medicine and biochemical industries. Significant interest has been revealed in using agricultural wastes for citric acid production. The major manufacturing technique to citric acid used nowadays is cultures of Aspergillus niger which are fed on sucrose to produce citric acid. Once the mold is filtered out of the foremost solution, citric acid is isolated by precipitating it with lime (calcium hydroxide) to capitulate calcium citrate salt, opening where citric acid is regenerated by accomplishment with sulphuric acid.
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1.1 Current Production: The oldest microbial process for production of a high volume, low cost organic acid is the production of citric acid by the filamentous fungus Aspergillus niger. Currently the yearly production of citric acid is approximately 1.6 million tons (t). Unlike most of the other bio-derived acids that are considered industrial products, citric acid was produced industrially before the development of a microbial process. The industrial production relied on extraction from Italian lemons until it was discovered that Aspergilli accumulate this acid in high amounts under certain conditions. The crucial parameters resulting in efficient production of citric acid by A. niger have been determined empirically and include high substrate concentration, low and finite content of nitrogen and certain trace metals, thorough maintenance of high dissolved oxygen, and low pH. The correct definition of these parameters enabled the development of highly efficient biotechnological processes. However, many of the biochemical and physiological mechanisms underlying the process remain unknown. These processes are currently undergoing investigation to enable improvement of the citric acid production process, for which vital improvement is no longer possible through traditional means, such as mutagenesis or cultivation optimization. Citric acid was originally extracted from lemons and limes, but it is now produced commercially by a fermentation process. The mould Aspergillus niger is used to ferment a carbohydrate source such as Molasses.(Lakshya Unatwal, 2008)
Citric acid fermentation is only of the incomparable examples of industrial fermentation technology. The active world market estimates recommend that aloft of 4.0 x 105 tonnes citric acid per year may be produced (Kristiansen et al. 1999). Citric acid is a key product but the increasing trend in its use seen over numerous years is a yearly 2-3% raised. The requirement for this particular metabolite is mounting day by day which requires a much more proficient fermentation method for superior yield product (Moreira et al. 1996). The vital substrates for citric acid fermentation using covered technique of fermentation are beet or cane-molasses (Pazouki et al. 2000). Cane-molasses was engaged as the basal fermentation medium in the stirred fermentor under the sunken fermentation circumstances. Citric acid is also obtained by fermentation of glucose with the facilitate of Aspergillus Niger. The citric acid cycle too is renowned as the Krebs cycle. Part of the citric acid produced currently is used by the soft drink industry. Citric acid is used as a flavouring means and as an additive. In the pharmaceutical field, citric acid is used with bicarbonates to manufacture carbon dioxide in sparkling medicines and cosmetics. The cleaning properties of citric acid make it ideal for numerous of the cleaning products found in our homes and in industrial cleaners. It is used for biological, cleaning, cosmetics, chemical, dyeing, industrial and construction foods, beverages and personal care and in photography.
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Strategy to maximise production:
Citric acid is formed by Krebs cycle. As Pyruvic acid enters the mitochondrion, carbon is separated, forming CO2, and electrons are isolated, changing NAD+ to NADH. Co-enzyme A adds to 2-carbon molecule, forming acetyl-CoA. Acetyl-CoA then joins 2 carbon acetyl groups to a 4-carbon compound, forming citric acid. Prior to Krebs cycle there is the glycolysis cycle. In this cycle the product is pyruvic acid. So following that the Krebs cycle (Citric acid cycle starts), which starts off with pyruvic acid. Then the election transport cycle follows.
2.1Knock-down Genes: The genes which are knocked down are listed as Fructose Bisphosphatase (FBP1), Phosphoenol Pyruvate Carboxykinase (PCK1) which follows Gluconeogenesis pathway in the metabolic network pathway, while Aconitase (ACO1) follows TCA cycle in the network and hence gets knocked down. By genetic modification of use of SiRNA and converting it into degradation of mRNA.
2.2 Up-regulated Genes: The genes which are needed to be up regulated are listed as Hexokinase (HXK2), Phospho-Gluco-Isomerase (PGI1), Phospho-Fructo-Kinase (PFK1), Pyruvate Kinase (PYK1) follows Glycolysis pathway in the metabolic network pathway, and Pyruvate Dehydrogenase (PDA1/PDB1) follows Pyruvate Dehydrogenase Complex pathway, Citrate Synthase (CIT1) follows TCA cycle in the network, while Malate Dehydrogenase(MDH2) follows Gluconeogenesis pathway in the network. By genetic modification of insertion of extra copies of genes we have up regulated genes.
3.0 Modelling of metabolic networks:
This is the central carbon metabolism pathway for yeast (Saccharomyces cerevisiase). Glucose enters the cytoplasm goes through the process of glycolysis. At the end of glycolysis pathway pyruvate is produced. Pyruvate now in the mitochondria where acetyl-coenzyme A is produced and reacts with oxaloacetate to initiate the citric acid cycle. Once the citric acid in the cell is high enough it is secreted out of the cell.
3.1 Gene Deletion: The genes which could be deleted are
From the above diagram, based on our own considerate on what each gene does and research from papers, we are proposing these introductory set of genetic modifications. We recommend deleting three out of the eight genes as indicated by the model. Deleting the other five genes compromises cell stability or their expression is not important considering the situation we are operating under. The three genes we propose to delete are glycerol-3-Phosphate Dehydrogenase (GPD1), Aldehyde Dehydrogenase(ALD6), and Ribose-5-phosphate Ketol-Isomerase(RKI1) and significance of these deletions are that we want to split apart glycerol biosynthesis, glucose fermentation and PPP and express the flux of carbon towards the product.
The best optimization solutions are experimentally observed by over expressing genes that are on the glycolytic pathway, some key ones are Phospho-Fructo-Kinase and Pyruvate Kinase. In the mitochondria we want to enhance enzyme activities of pyruvate dehydrogenase and citrate synthase. The most significant of this is the Pyruvate Dehydrogenase complex. This is because of the anaplerotic reaction where CO2 formed by the pyruvate dehydrogenase reaction is used by Pyruvuate carboxylase to produce oxalacetate, which results in soaring citric acid yield.
We want to down regulate the genes that have a negative effect on citric acid manufacture and involved in recycle effect. The most vital of this is anonitase which is concerned in isocitrate production, which is an unwanted product. The genetic modification used is Homologous recombination.
Throughout metabolic engineering concerning main metabolism (e.g. glycolysis, TCA cycle and pentose phosphate pathway, etc.), the carbon flux distributions at input branch points (called 'nodes') is frequently completely redirected from the flux distributions that are usually linked with balanced growth. Such flux alterations are repeatedly openly conflicting by mechanisms that have evolved to retain original flux distributions for best growth.
The metabolic flux distributions (using glucose as a substrate) for balanced growth, for 30-40% molar yield and for maximum 'yield (75% molar yield), where glucose uptake was used as a standard indicated by 100 units.It will be seen that flux distribution for maximum overproduction represents a significant deviation from that for balanced growth or from that for 30-40% overproduction. If pathways leading to production of byproducts (e.g. pyruvate dehydrogenase complex or PDC leading to TCA cycle) are blocked, one would expect a shift in metabolic pathway leading to maximum over production of lysine. But the metabolic network does not respond to such alterations in metabolic network due to metabolic rigidity at key branch points or nodes. Such metabolic rigidity must be identified and overcome before results of metabolic engineering can be exploited.Input metabolites of a metabolic pathway are called substrates and output metabolites are called products.
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4.0 Monitoring Techniques: The methods used are follows as under
4.1 Levels of Gene Expression:
We have developed a polymerase chain reaction (PCR)-based method to measure glutathione peroxidase (GSH-Px) mRNA levels. Expression was measured by multiplex competitive PCR amplification of (a) cDNA from GSH-Px and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (b) two internal standards consisting of single-base mutants of GSH-Px and GAPDH cDNA that cause either a loss (GSH-Px) or a gain (GAPDH) of an EcoRI restriction endonuclease recognition site. RNA extracted from a human papillomavirus-immortalized human bronchial epithelial cell line (BEP2D) was reverse transcribed. Serial dilutions of cDNA were PCR amplified in the presence of GSH-Px and GAPDH primers and quantified amounts of mutated internal standards. The amplified DNA was restriction digested with EcoRI and electrophoresed on an agarose gel stained with ethidium bromide, separating native from mutated products. Densitometry was performed to quantitate the bands. Our studies demonstrate that this technique measures the relative expression of GSH-Px to GAPDH precisely and reproducibly for studies done with the same master mixture and dilution of internal standards. Ratios of relative gene expression varied less than 25% from the mean. This technique will be useful to measure changes in gene expression, particularly when the amount of study sample is limited or the level of gene expression is low.
4.2 Monitoring the transcription of relevant genes:
A method of cluster analysis for genome-wide expression data from DNA microarray hybridization is shown that uses standard statistical algorithms to organize genes according to resemblance in outline of gene expression. The DNA microarrays are used to learn the transcriptional responses of an organism to genetic and ecological changes. The promising yeast Saccharomyces cerevisiae that clustering gene expression data groups collectively of economically genes of known alike function, and we find a similar affinity in human data .A usual basis for organizing gene expression data is to group jointly genes with similar patterns of appearance. The initial step to this end is to assume a mathematical explanation of resemblance. For any succession of magnitude, a number of rational method of similarity in the performance of two genes can used. As we have modest, a previous knowledge of the entire repertoire of expected gene expression patterns for any circumstance, we have favoured unsupervised methods or hybrid (unsupervised followed by supervised) approaches. Always merge clustering methods with a graphical illustration of the primary data by demonstrating each data point with a colour that quantitatively and qualitatively reflects the unique experimental explanation. The end product is a depiction of complex gene expression information that, during statistical association and graphical demonstrate, allows biologists to understand and investigate the data in an expected intuitive manner.
Materials and methods:
Sources of Experimental Data:
Data analyzed here were collected on spotted DNA microarrays (6, 7). Gene expression in the budding yeast Saccharomyces cerevisiae was studied during the diauxic shift (8), the mitotic cell division cycle (9), sporulation (10), and temperature and reducing shocks (P.T.S., P.O.B., and D.B., unpublished results) by using microarrays containing essentially every ORF from this fully sequenced organism (8). After hybridization and appropriate washing steps, separate images were acquired for each flu or, and fluorescence intensity ratios were obtained for all target elements.
Ref: Genetics: Eisen et al., 1998
Metrics: The gene resemblance metric we use is a type of association coefficient.
Hierarchical Clustering: The hierarchical clustering algorithm used is based intimately on the average-linkage method of Sokal and Michener (5), which was developed for clustering correspondence matrixes such as those used here.
Ordering of Data Tables: For whichever dendrogram of n fundamentals, there are 2n-1 linear orderings dependable with the arrangement of the tree.
Display: With any ordering, the chief data table is represented graphically by colouring each cell on the origin of the calculated fluorescence proportion.
Result: A prominent property of the clustered images in outline is the existence of large contiguous patches of colour demonstrating groups of genes that share like expression patterns over numerous conditions.
Redundant Representations of Genes Cluster Together: At the optimum level, we have establish frequently that genes represented by more than single array element or genes with elevated degrees of sequence individuality are clustered next to, or in the instant vicinity of, each other. Thus the exact depiction of a gene on the array (vary cDNA clones of inconsistent length in the case of human arrays or extremely homologous genes in S. cerevisiae) makes slight variation in the observed pattern of gene expression.
Ref: Schena et al., 1995
A soaring capacity scheme was developed to supervise the expression of many genes in parallel .Microarrays equipped by rapid robotic printing of corresponding DNAs on glass were used for quantitative expression dimensions of the equivalent genes. Because of miniature format and high concentration of the arrays, hybridisation volumes of 2 micro-litres might be used that enabled recognition of rare transcripts in probe mixtures imitative from 2 micrograms of total cellular messenger RNA .Discrepancy appearance magnitude of 45 Arabidopsis genes were prepared by means of concurrent, two-colour fluorescence hybridization.
4.3 Quantifying the production of relevant genes:
A proceed of chief importance to the advance of 2-D technology occurred by the use of isoelectric focusing (IEF) techniques. The initial aspect parting could now be based on the charge properties of the proteins and might be used in combination with particular concentration or gradient polyacryl amide gels. When IEF was joined with a next dimension parting in polyacrylamide gels containing the anionic detergent sodium dodecyl sulphate (SDS), a method proficient of separating proteins on the basis of molecular weight, it formed a 2-D means which determined proteins according to two truly autonomous parameters, i.e. charge and size . For this 2-D method to be appropriate to a broad choice of samples with incompatible solubility properties, customized IEF measures had to be developed. The most significant of these modifications to the IEF scheme was the addition of urea in the gels and the use of a mixture of non-ionic detergent (e.g. Triton X-100).
Two-dimensional gel electrophoresis (2DE), the topmost resolution of the protein gel electrophoresis methods, has made exceptional development in modern duration. Two-dimensional gel electrophoresis, shortened as 2-DE or 2-D electrophoresis, is a type of gel electrophoresis usually used to analyze proteins. Mixtures of proteins are removed by two properties in two dimensions on 2D gels. To split the proteins by iso-electric point is called iso-electric focusing (IEF).Thus, an incline of pH is functional to a gel and an electric potential is useful athwart the gel, building one end more positive than the former. At all pHs former than their iso-electric point, proteins will be stimulating. If they are positively stimulating, they will be pulled near the more negative end of the gel and if they are negatively stimulating they will be pulled to the more positive conclusion of the gel. The proteins applied in the primary aspect will shift along the gel and will amass at their iso-electric point; i.e., the tip at which the largely charge on the protein is 0 charge (neutral). This is finest evidenced by the decree of numerous thousand spots in human myocardium, the quantification of their discoloration intensities, recognition of numerous of the protein spots, and purpose of their main series. State-of-the-art 2DE technology is based on the use of immobilized pH gradients (IPGs), instead of transporter ampholytes, to let exact couture of gradients in thin or broad pH choice and parting of mg quantities of proteins lacking loss of decree. With IPG gels in the primary dimension and SDS-PAGE in the next dimension, the consequential parting is enormously reproducible, and the patterns can be enthusiastically analyzed using computer algorithms. Reproducibility was also enhanced by using pre-mixed polyacrylamide solutions or pre-cast gels. An intra- and inter laboratory contrast of the positional reproducibility of protein spots was ready in two diverse laboratories using 2 dissimilar protein samples, human cardiac and barley leaf proteins. Inter laboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient 2DE was also reported. The elevated decree of 2DE using IPG is generating 2DE maps of proteins from numerous tissues which are being compiled into 2DE databases. Among its many applications, 2DE has been used to analyze cellular protein synthesis, and its contrasting response, protein degradation, by showing for protease substrates in protein mixtures use diagonal 2DE.
2D Gel Examination Software:
The automatic based software analysis includes:
partly detached (overlapping) spots (less-defined and/or split)
pathetic spots / sound (e.g., "ghost spots")
successively differences amid gels (e.g., protein migrates to dissimilar positions on unlike gels)
matchless/unnoticed spots, foremost to missing values
errors in quantification (numerous different spots may be incorrectly detected as a solo spot by the software and/or parts of a spot may be expelled from quantification)
differences in software algorithms and consequently investigation tendencies
4.4 Determining the levels of metabolites and flux through the central carbon metabolism:
It describes the division of the carbon-13 isotope (13C) at the unlike carbon positions of metabolites in cells fed with 13C-enriched substrates. The representation allows the purpose of fluxes during diverse metabolic pathways from and 1H-NMR spectroscopy and mass spectrometry information. The use of substrates enriched with carbon-13 (13C) in combination with 13C-NMR spectroscopy has proved to be a expensive technique for investigating the metabolism of existing systems by analyzing the tag division in their metabolites. 13C-NMR spectroscopy is therefore an effective technique for monitoring the parameter of the intermediary metabolism. The calculated metabolic network includes glycolysis, gluconeogenesis, the citric acid cycle and amount of reactions equivalent to protein or fatty acid metabolism. The definite enrichments of glutamate, aspartate and alanine carbons were resolute from 1H-NMR spectroscopy, or mass spectrometry data.
1T-NMR spectra were used at 100.6 MHz with a Bruker AM 400 spectrometer in a 10-mm broad-band probe head, using a 55" flip angle and a 1.65s interpulse delay (0.65-s acquisition time). A bi-level proton decoupling was useful during acquisition (6 W) and recreation delay (1 W). Spectra were accumulated overnight. Chemical shifts were articulated relative to the resonance at 63.7 ppm of 20 pmol ethylene glycol used as a domestic reference. 1H-NMR spectra were used at 400MHz using the similar probe head, with a 30" flip angle and a 6-s interpulse delay.
The principle of the present work was to verify whether it is probable to explain the allocation of 13C within the metabolites typically detected by 13C NMR spectroscopy and, from that division, to estimate metabolic fluxes during the intermediary metabolism (with glycolysis, gluconeogenesis and citric acid cycle and associated pathways). The uniqueness of the model used in this study relies on the thought of mainly utilizable resonances in 13C-NMR spectra.
4.5 Determination of the Final Product Concentration:
A flow injection analysis (FIA) system with potentiometric detection has been developed for the determination of citric acid in commercial fruit juices using a copper-selective tubular electrode.
Automated systems based on flow injection analysis (FIA) have been used for citrate determination. The results generated by the computerized system were compared with those of an enzymatic convectional method used for the investigation of citric acid in food and a relative deviation of less than 4 % was establish. Citric acid is of primary significance to living animals and plants. It is directly accountable for the production of energy by way of the Krebs cycle.
Diagram: Flow injection manifold for the determination of citric acid in soft drinks
Ref: Lima et.al, 1999
The samples injected into the FIA system without previous conduct were adjusted to the analysis circumstances within the system developed. The potential differences among the indicating and reference electrode were calculated by way of a Crison digital voltmeter.
All chemicals were of analytical reagent grade and deionised water was used during the preparation of the solutions.
Results and discussion: A schematic demonstration of the FIA manifold used for the determination of citrate in soft drinks is illustrated above. The system was developed optimising every parameter individually and considering the best negotiation between sensitivity, reproductibility, sampling rate and low utilization of reagents.
Conclusions: The citrate determination in soft drinks by means of potentiometric detection presents a superior alternative to spectrophotometry, allowing the direct analysis with no interference of sample colour or turbidity.
5.0 Executive Summary:
The Optimisation of citric acid production can be achieved with gene manipulation of the yeast Saccharomyces Cerevisiae. The metabolic flux investigation throughout the metabolic pathway was conceded out using MATLAB. The methods comprise gene deletion, down regulation and up-regulation and the dimension techniques can be utilised to monitor product and transitional formation and overall efficiency.