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Hello, I am currently doing an assignment that focuses on designing CSTR. I am currently trying to figure out how to choose an agitator and its specifications such as the material used, type of impeller, speed of rotation, direction of rotation (if it affects the reaction) etc.
Below are the specification of the process:
Approximate volume: 60 L (Lab size reactor)
Process: Transesterification of Glycerol and Dimethyl Carbonate to produce Glycerol Carbonate (All Liquid) + Methanol as side product (gas)
Catalyst: CaO (solid)
Reaction temperature: 75 degree C
Reaction duration: 90 minutes
I would be truly grateful if anyone would share useful info, equations or anything that can help me in this. Thank you in advance and good day. :)
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If we consider, for example, heterologous insulin biosynthesis in E. coli, we often find that a strategy based on synthesizing the target protein by expression plasmids has simply been adopted. But wouldn't that be a problem in terms of plasmid stability during continuous production on an industrial scale? Shouldn't such operations be performed with modifications at the chromosome level? There are definitely different nuances in other hormones, enzymes and proteins, thus I would appreciate it if you could explain by giving some examples.
Thanks in advance..
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Dear Pierre, thank you very much..
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After charging media in Bioreactor when we Sparger air through the media its pH increases
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As previous scientists said before me, CO2 stripping must induce such pH increase.
Looking at the dissolved CO2 equilibrium in water:
CO2 + H2O <==> H2CO3 <==> 2H+ + CO32-
Sparging air engender a decrease of dissolved CO2 then reduction of free proton H+ and pH increase.
Best regards,
Paul R. Jr BROU
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My current research is in the carbon flux distribution of L. rhamnosus in a biofilm reactor and I am struggling to find research pertaining to the continuous metabolic analysis in literature. Any help would be highly appreciated.
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Dear Hendrik,
maybe these recent publications on 13C metabolic flux analysis in biofilm-forming P. aeruginosa and agar plate-grown E. coli might be of help:
Best,
Michael
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I am working to find a bioactive compound from a bacterial source which can show antifungal activity. So I have established the bacterial culture in a synthetic medium. What we found that the enzyme/proteinous compound is mainly extracellular in nature. Now I want to go for isolation, extraction and purification of the compound from the liquid culture of bacteria at laboratory scale. So I am asking to share your knowledge about the sort of suitable methods which will be helpful for getting the desired compound.
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Try this website protocols
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in general batch anaerobic digestion process which is the right optimum substrate/ inoculum ratio 0.5 or 1.0
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The Italian norm on BMP test (UNI/TS 11703:2018) states that the inoculum/substrate ratio must be comprised between 2 and 3 for general biomass digestion (i.e. 0.5 to 0.33 if you define as substrate/inoculum). Nevertheless, it also states that some substrates (glycerol, fats, industrial effluents containing inhibitors like olive mill wastewater, etc.) may require teh test to be performmed with I/S >= 5. This must be established by the lab operator and stated in the report of the test. For a discussion on the pros and cons of the Italian , German and IWA draft on BMP assay, please see chapter 6 of my book https://www.crcpress.com/Managing-Biogas-Plants-A-Practical-Guide/Rosato/p/book/9781138626614
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I am conducting a reaction with a highly viscous chemical, which is the reactant as well as the solvent, with the other reactant being less viscous and immiscible. I was able to achieve the mixing in a 2L flask by vigorous stirring using magnetic bead, which facilitated the mixing as well as the reaction. Reaction temperature is 120 deg C.
However, moving to a 10L vessel (glass, round bttom, 4 neck) with an overhead stirrer, there are issues with the mixing of the two chemicals, as the two reactants remain immiscible. Button stirrer with a PTFE blade was used.
What kind of agitator/impeller would be best for use in this condition?
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A propeller type that promotes vertical movement downward is best for different densities. Baffles are almost always needed even if they are removable through nozzles in the top.
For larger reactor a pump around loop with mixing jet inductor is helpful.
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As my aim is to study the anti-fungal activity by the bacteria, Is there any expertise member who can explain how to know a bio-active compound(may be protein, enzyme or other molecules) isolated from a bacteria is extracellular or intracellular in nature ?
The literature search could not helped me to find out the appropriate solution for this particular microorganism so I want to go for various experimental methods. So is there any proper step by step procedure for better confirmatory tests ????
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Hello, see attached info.
Good luck!
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I am struggling to calculate the columbic efficiency for acetate and glucose used as substrate in batch mode double chamber MFC. i haven't find a single paper for batch mode which had used formula for columbic efficiency with all those constants and other parameters units .
 with thanks
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Should I add some preservatives or other chemicals to this solution?
What is the correct temperature to store this stock?
How long can it be stored in these conditions?
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Dear Dr. Pourkhaloee
I stored cellulase and pectinase stock at -20 centigrade for a month without any problem. I think it could be right.
Best wishes
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I'm producing lactic acid via anaerobic fermentation. I'm going to the equations in Michael L. Shuler Textbook "Bioprocess Engineering." He stated that there are two types of substrate inhibition for bacteria: competitive and noncompetitive. I need to figure out which one I have and how to find Ki, which he does not explain how to do in the textbook.
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Dear Kaila,
In order to figure out which model should be used, it necessary to investigate which one fits experimental results better. I could find the following paper, which concerns determination of kinetic parameters of phenol biodegradation (competitive inhibition by the substrate itself):
Hill, Gordon A., and Campbell W. Robinson. "Substrate inhibition kinetics: phenol degradation by Pseudomonas putida." Biotechnology and Bioengineering 17.11 (1975): 1599-1615.
In this paper:
Kumar, Arinjay, Shashi Kumar, and Surendra Kumar. "Biodegradation kinetics of phenol and catechol using Pseudomonas putida MTCC 1194." Biochemical Engineering Journal 22.2 (2005): 151-159.
the authors used a linearized Haldane model for the determination of inhibition constant.
I hope the above-mentioned papers will help.
Best regards
Szymon
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I converted salmon oil into FAME using lipase. I used BSFTA and tetrahydrofuran to derivatize the sample and injected in GC for separation and quanitification. I now have peak area for individual fatty acid methyl esters. Now how to calculate the FAME conversion yield?
For example: researchers say that they got 75% biodiesel yield at 1:3 oil:methanol molar ratio after 24 h of reaction time. I would also like to calculate in that manner. Some of the formulas are confusing and doesn't make any sense. Can someone help me out? 
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1. Yield of biodiesel(%) =mass of biodiesel obtained / mass of oil used
Normally we get an yield of 95-100%. Mass of biodiesel  ( mass of oil + mass of alcohol - mass of glycerol - mass of unreacted chemicals  ) it could be greater than the mass of oil. Theoretically it possible to have an yield slightly higher than 100%.
Method : measure weight of row oil before the reaction and weigh biodiesel after reaction and post treatment.
2. Ester Content (%)= cumulative mass of methyl esters/ mass of biodiesel.
It is very important to understand that biodiesel does not means that its 100% methyl ester (or pure) it may even contain un-reacted triglyceride and partially reacted mono-glyceride and di-glycerides. Fatty acid methyl ester /ester content could calculate using EN-14103 method. According to EN-14103 minimum ester content should be 96.5%. we should use an internal standard like C17. In GC area of a peak is proportional to the concentration of that component.
method
a. take a known amount of internal standard (x gram) and mix with known amount of biodiesel (y gram).
b. perform Gc test
c. find out area corresponding to C17 ( area A)
d. find total area (Area B)
Please try to understand that y gram biodiesel does not contain y gram of methyl ester it contain 'U' gram of methyl ester and 'V' gram of contaminants ( we dont know value of U and V , test doing to find these values)
Area A proportional to x gram
total area corresponds to x+U gram not for x+y/ x+U+V
Area B proportional to x+U gram
Area (B-A) proportional to U gram
ester content = U/y
U= (B-A)*x/A
ester content = (B-A)*x/(A*y)
Ester content(%) = ((Area total/Area C17:0)-1)*(mass of C17:0/mass of biodiesel)
3. Conversion Efficiency(%).
It denotes amount of raw oil converted to methyl esters.
Conversion Efficiency(%)=Ester content(%)* Yield (%)
Note:
1. If the ester content is above the limit (96.5%) then other physical properties like density and viscosity will be within the limit.
2. if you loose biodiesel during washing or though any other way , it reduces the yield and there by conversion efficiency but not ester content value.
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I am working on the reaction kinetics of biodiesel synthesis from rubber seed oil. I am proposing a pseudo-first order rate equation since the methanol will be in excess. I needed to test the proposed rate equation with available published data from literature
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Hi Dr.horiguchi
Do you use  a bioreactor? I work on optimization of  hydrodynamic  forces in stirred bioreactors for mass production so, I wanted to talk more about this.
Best Regards
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A few questions would give me a better idea of what you are trying to accomplish.
  1. What is the biomass in this process?
  2. What is the objective of the process?
  3. What are the conditions for this process; temperature, pH, and pressure?
  4. What is/are the microbes involved?
  5. Is the inbound material homogenous or non-homogenous,  sterile or non-sterile?
  6. Does the process have to be sterile?
  7. Is there a financial objective attached to the process?
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i have found the method for Electroporation of C. necator.
one of the steps says:-
The cells were then harvested, washed twice with 50 mL cold H2O, and concentrated in H2O to a final optical density of 25.
May i know how to do achieve the final OD of 25?
·        
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Simply put, you can't measure OD bigger then ~1.0
Once the density of bacteria (think of them as particles) is increasing beyond ~1.0 you start having optical side effects such as shading and biological once such clamping. This is true for any measurement of suspended particles.
So measure OD of your cells re-suspended in water, calculate how much more concentrate they need to be, and adjust your final re-suspension volume accordingly.
Yoram
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I'm using 1 g of bagasse for my solid state fermentation. How to calculate the pigment yield obtained and express them as g/kg dry weight?
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You can measure the absorbance of your extract from solid state fermentation. With a standard curve of abs x pigment concentration, you will find the concentration of pigment on your solution. Finally, you have to multiply the result of concentration of pigment on the extract by the volume of liquid you have used per gram of bagasse for extracting the pigment from the solid fermented material.
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In a continuous system using immobilized Saccharomyces cerevisiae, how does one achieve steady-state since the rate of cells flowing out of the system will not be consistent given their trapped nature in the alginate matrix? Are there any papers that describe this scenario?
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I am trying to determine the reducing sugar yield after enzymatic hydrolysis. If, after DNS, the reducing sugar concentration is 115 g/L, what is the yield?
1 g of feedstock was added to 10 ml buffer for the enzymatic hydrolysis section.
If I am correct, I am getting 1.15 g/g, which seems implausible?
Please assist.
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1 g of 100% pure and dry cellulose should only give 1.11 g/g glucose (extra 0.11 g in weight from the H and OH added during hydrolysis), so it is possible you are getting 100% glucose out (plus a bit error to bring it up to 1.15 g).
But if your feedstock is not 100% pure and dry cellulose, then it could possibly be due to an interference, e.g. Discoloured solution that is affecting your colorimetric measurement. If this is not the case, you are measuring how many equivalents of DNS is converted, and converting this to mass using glucose equivalents. If the molecular weight of your reducing molecules are less than that of glucose, then you end up with an overly large mass.
If you care about what's in there, likely you will have to set up a HPLC process to investigate. If you only care about glucose then you will need a glucose-selective quantification method. For example, a colorimetric process using glucose oxidase. Many conventional blood-glucose meters will work well in a buffered solution, and typically selectively quantify glucose between 2 and 10 mM (but not every model works selectively or works quantitatively in an enzymatic broth, so a calibration curve would be required). 
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Understanding algae responses to stress can help to improve our knowledge about algae adaptation strategies which can be used to engineer tolerant strains. Also, it can be used to manipulate culturing condition to yield the higher amount of secondary metabolites like carotenoid. 
In most of the articles that I am reading it is not mentioned why is important understanding adaptation mechanisms. Are there any other applications other than what I stated?
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Thank you for your time and guidance, it is very useful paper. 
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I read an article saying  that 66.07 g of NH4+ will be available in 1 litre of water if  1 mole of ammonium sulfate is introduced to water
But i presume that 36 g/lit of ammonium should be present but the above mentioned reference says that 66 g/lit. kindly clarify me 
1mole of ammonium sulfate = 132 g/lit ( N= 14,H=1,S=32,O=16) 
Ammonium sulfate when dissolved in aqueous solution produces two NH4+ ion i,e 2*18 = 36 gm.
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Hi, Ahamed!
You are correct. I think it is obvious that the "66.07" is a typographical error, as 6 and 3 are next to one another on a computer keypad.
Also, you mean "... if 1 mol of ammonium sulphate ...", not "1 M", just to be clear.
And if I remember correctly, IUPAC switched to spelling sulfur with an "f" in 1990. :D
regards,
Daniel
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Kindly elaborate on pros and cons of both, if possible.  
I'm interested in understanding the differences in levels of recombinant protein produced, ease of production and ease of genetic manipulation.  
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1. plant-based platform for commercial production of recombinant protein is usually considered as more cost-effective platform (ex. no need of a constant controlled environment), and a scalable-production platform to allow large-volume manufacturing, when compared to other systems. Grow yeast also have contamination worry.
2. Post-translational modification:
(1) For yeast, one major concern for producing therapeutic glycoprotein for human application is that yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and hyper–immunogenicity and thus render the therapeutic glycoprotein less effective. (attached paper)
3. If you want to isolate those recombinant proteins, I think yeast should be much easy to do that
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I want to know if could be degradation of the components of the medium.
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I think it is not necessary to  autoclave media twice because single time autoclaving is much enough to sterilize it. If you will do it second time then important nutrients can denature, as well as salt concentration will  be increased and change in pH can effect the growth of microbe. I have experience with this media and never had the problem to for autoclaving it twice. 
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It is known that nitrogen and phosphorous both are vital for the growth of cells, then why in limiting condition does it enhance PHA production?
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PHA granules are actually food reserves in the organisms and therefore nitrogen and phosphorous values increase.
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In ABE fermentation by clostridia, does continuous immobilization culture solves problems associated with continuous fermentation using suspended cells.
- Mixture of acidogenesis and solventogenesis in continuous system
- Degeneration due to solvent toxicity
- Difficult to achieve steady state
What are the other problems rise for continuous system.
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I once speculated too about immobilized cells as a solution to avoid solvent toxicity and to overcome sporulation/programmed cell death. But I'm not an expert in the field of fermentation, so I cannot technically answer your question. I know that removing solvent from the medium alleviates reaching toxic solvent concentrations and that controlling pH just before entering the acid shift allows to propagate the exponential growth phase and to induce some retaking of acids and direct solvent formation from substrate. Yet I don't know if there's a way to reach a continuously efficient solvent production without triggering sporulation, unless you're working with a sporulation mutant, which is in turn probably not the most efficient solvent producer because of poor solventogenic enzyme induction. So I guess you need a flexible time schedule in your continuous fermenter (with some device for solvent removal from the medium if you intend to recirculate) to do a lot of trial and error with the best strains you have... In the end, assuming you have an outlet filter to avoid lost of biomass, probably cells won't care whether they are immobilized or not as long as they are not compelled to sporulate... :-)
Best regards,
José
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I want to measure certain intracellular metabolites, NAD and NADH. Thanks.
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Dear Miss/Mrs Mandep,
I guess you should mixed up the air environment with nitrogen gas so that the air redox potential < 7 (microaerophilic condition).
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Is there any solution to get rid of precipitation or is there any salt which doesn't get precipitated when added with CMC (Carboxy Methyl Cellulose)?
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cmc what concentration you r adding to which salt..... and what is the purpose?
cmc is a surfactant ...need to dissolve in water to get the foam
wheather the salt is added to cmc solution or direct to cmc?
pl make clear.... toanswer
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recently i have studied articles about biodiesel wastewater. we are producing large amount of waste waster in water washing of biodiesel. we can reduced waste water by adding some percentage of distilled water 10 or 5 min before ending transesterification process as prewash.
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Dear colleague,
You are using WCO (high FFA%) as feedstock oil and you chosen base homogeneous catalyst (KOH) for this reaction. In my opinion, you were not right in this  case because the saponification reaction is very easy to occur and lead to form soap. The big amount of soap make the transeserification stop soon.
So you need do esterification reaction first by using acid catalyst (H2SO4). After finished this step (FFA% will significant decrease), you can use KOH for next step (transesterification).
About washing process, your idea is not good. Because the hydrolysis reaction can occur if you use distilled water soon. For this reason, the amount of ester (ester content) will unexpected decrease.   
For the better idea, after finished reaction, glycerol phase should be removed from biodiesel phase. After that, you can wash biodiesel phase by distillated water. This process should be repeated 6 times (3 times without stirring and 3 times for stirring or slow shaking).
I hope this comment will be useful for you.
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Hello! We did a small biodigester at home, it had two units: a recipient with 0.25L and a another variable volume recipient for gas storage. The biodigester starts up with 1.5kg of food waste, 1 kg of cow manure and 5L of water. The system seems to be good during adaptation time (we planned 15d for this and then to operate it in semicontinuous mode), pH measurements every three days was nearly to 7. Neverthless, 9 days after the start up we have to burn  the biogas the because the storage recipient was too small, but... it didn't burnt.
We have an hypothesis about what was bad in our design: the (inoculum/substrate ratio) was to small (0.6 aprox.) and for food waste the recommended ISR must be over 2 for preventing the inhibition (food waste are easily biodegradable matter)
We are not sure about adaptation time or if we can burnt the biogas daily, but in this case the pressure maybe won´t be for flow through the tube. Maybe we must change the design or we are doing something bad during the start up?
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Good morning, Lenis, I have just seen your question. Your assertion was correct, in my opinion. However, for start-up and acclimatisation at the same time, even an inoculum/substrate ratio of 2 would be far too high. For example, just to carry out biomethane potential tests using already acclimatised biomass we use a ratio >6 to minimise potential stress. Now, we operate full-scale food waste AD plants in the UK. We have done it for over 10 years now and the volumetric loading to our 16000m3 plant is around 300m3/day => 50 days HRT.  I see that your are probably starting in semi-batch mode, but at that initial loading I am surprised the pH has not dropped further. If it was me, I would start at an inoculum/substrate >25 and monitor biogas composition more regularly, if possible. It CH4>50% increase OLR gradually. I normally control start-up on the basis of biogas CH4 content and flow. Since you are looking at start-up and acclimatisation (i.e., you are not using an inoculum from a food waste digester), I would expect start-up to take a long time, as Rowayda indicated (or longer!). Can you not get some UASB solids to start-up. I hope this is useful.  
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I need commercial grade Cellobiose dehydrogenase (CDH). Does anyone have the information about any company/organization/supplier who can supply commercial grade CDH?
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At the moment there is no commercial provider of CDH. There are only about 20 research groups that produce it and use it.
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Firstly, we tried preparing feather culture media 1 & 2 using NaCl 0.5g/L, KH2PO4 0.4g/L, K2HPO4 0.3g/L, feather 10g/L, pH 7.5 (Media 1) and Media 1 + NH4Cl 0.5g/L, MgCl2.6H2O 0.1gµl, yeast extract 0.1g/L.
We optimized the temperatures for both media to 40 and 50 Celsius, but did not get satisfactory results. Thanks.
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The keratin  can be dissolved in alkaline buffer at pH 9 and above and used as substrate for keratinase assay. 
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Now I need to design a transfer function for my bioreactor. The controllable parameters pH, DO, Speed, Temperature available in the system. I need to control all the parameters.
Is it possible to add more parameters within a single transfer function?
If yes, Then how i control the parameters separately?
More over i have a plan to design a MRAC for that system.
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Yes. The Stirrer Speed in RPM
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My article's reviewer wants me to determine the pore size of the membrane by gas (liquid)/liquid method, but I don't know this method. What is it? How does it work? If I don't determine the pore size by this particular method, which method is the best replacement for it?
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Liquid/liquid displacement method and gas/liquid displacement method, the pore is filled by liquid (says liquid 1), where certain pressure is applied to gas or liquid 2 to displace liquid 1. It uses Laplace equation to determine the pore radius and its distribution. In laplace equation, pore radius is inversely According to the Laplace equation the required pressure is proportional to the interfacial tension and inversely proportional to the pore radius.
delta P = 2*gamma*cos(theta)/r
Where gamma is surface tension, r is radius, and theta is contact angle. For more detailed procedure, please check in this paper :
L. Germic, K. Ebert, R.H.B. Bouma, Z. Borneman, M.H.V. Mulder, H. Strathmann (1997). Characterization of polyacrylonitrile ultrafiltration membranes. Journal of Membrane Science 132 131-145
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I'm designing a bioprocess using Nicotiana Tabacum BY-2 plant cells, I need it's kinetic parameters, thank you.
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You can find the contents of "Biochemical Engineering & Biotechnology" from added file here.
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It is assumed that to be a two stage system, the HRT should be greater in the acidogenic reactor because the hydrolysis step is which required most time.
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HRT should be designed considering the growth kinetics of the microbial consortia. Fermentative consortia have faster growth kinetics compared to methanogens in the second stage, thus the HRT in the methanogenic stage i.e. second stage is higher than the first stage. 
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The ungassed power number (Np) of a single impeller is 5.2 and for the two impeller system the power number can be assumed to be twice that value. Power consumption of agitation is reduced when gas is introduced into the vessel. Np,g/Np can be assumed to be 0.7 if Flg≥0.01.
height (H) to tank diameter (T) ratio H/T = 2. The fully baffled reactor is agitated with two Rushton turbines with a diameter D = 0.4T. The actual production  takes place in a 50 m3.
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The stirrer speed depends on the power number according to the definition: P = Np rho N^3 D^5. To calculate the speed you need the net power input. This can be measured inaccurately via the electric power (correction for system friction) or using strain gauges. Apparently this is no option at that size of reactor or if you are dealing with design. So you have to choose a power input (depending on your system between 1 to 3 W/kg. (5 W/kg is very heavy and not very useful when using the impellers primarily for gas dispersion) ). In general you want to operate above flooding, keeping reduced power input of the lower impeller (somewhat) higher than the power introduced by the gas g×vGs (power per mass equals gravity field strength times superficial gas velocity). All this provided that you are using aqueous fluid.
Note that there are serious inaccuracies in the assumptions you use: Np = 5.2 (+/- 0.8 (?) for (standard) Rushton); The reduction in power draw by gas of 0.7 (can easily be 0.4 for larger gas flows) for both impellers (the upper one is less loaded as a result of shortcut flow around the upper impeller). So it is not possible to calculate the stirrer speed exactly (as function of power), only to estimate it.
Success
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Hello everybody,
I am interested about to design differents reactors to treat wastewater so as knowing which calculus I must do. 
Thanks for advance.
Ysabel
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Dear Ysabel Huaccallo
The waste converted with biological approach to biodegradable hazardous materials to harmless and stabilized by products with help of microorganisms under elevated temperature. In-vessel composting reactors are of two general types: plug flow reactors (vertical and horizontal) and agitated-bed reactors. The increased temperature, typically 50 to 700C  results from heat released by microorganisms during the degradation of the organic materials. Aerobic composting is used to degrade sewage sludge whereas anaerobic processes are more suitable for hazardous waste treatment. The in-vessel composting of contaminated soil utilizes a bulking agent, such as saw dust or animal waste, to increase the porosity of the media. Conveyors must be used to transport materials in the compost-based reactor system.
And also in rural pit are created underground about dimension L,B,D(4X5X6) mtr.dump waste material and cow dung or bacteria generated waste to be added. And covered air tight so that oxygen cannot be introduced. After some time anaerobic bacteria convert all waste in compost fertilizers.
Composting technology can be applied to municipal sludge, soils, and lagoon sediments contaminated with biodegradable organic compounds. It has been demonstrated that composting is suitable for pentachlorophenol (PCP), refinery sledges, insecticides contained in cannery wastes, explosive-contaminated soil, ethylene glycol contained in landfill sludges, and polycyclic aromatic hydrocarbons (PAHs). High rate composting is conducted in an enclosed reactor and the curing may be conducted in a reactor.
Please find attached herewith suitable literature.
Regards
Prem Baboo
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I have been using mason's trichrome kit by sigma, can acetic acid play an important role in staining? I leave the tissue in bouin's for overnight and then hematoxylin for half n hour and then phospho tungstic and phospho molybdic acid for 5 mins and then aniline blue for 5 mins followed by 0.1% acetic acid.please suggest me as to what can be my mistake.
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Masson trichrome staining procedure contains three solutions 1) sol A- Acid fuschin- 5 min, 2) Sol B phosphomolybdic acid - 5 min ,3)Sol C- Methyl blue - 2-5 min. Before enter into Sol A,B and C. The tissue sections were subjected to celestine blue staining for 5 min , followed by Hematoxylin for 5 min and then blueing using tap water and then differentiate it with  1% Acid alcohol then go for Sol A, B and C. 
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Hi
I wanna make an alginate scaffold. I have a sigma batch of alginate. In some papers, alginate is purified before further use. I wanna know how much it is necessary to do the purification?
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Thank you for your responds. The protocol I use for purification is just simply using activated carbon. Is it enough or I need more complicated methods?
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I am doing a biological methane potential test and want to determine the chemical oxygen demand of the substrate amended with maize straw before / after the fermentation. The standard method I am using is the macherey nagel nanocolor CSB 1500. However, this method is only accounting for the soluble, but not the solid phase. Does anyone know a reliable method to determine the total COD of such samples?
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Use TOC instead
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Hi, everybody
I am working on phytase production by a B. subtilis strain. We are going to scale up the production using a NBS BioFlo 310 Bioreactor. We are going to use NBS EX-2000 Gas Analyzer for measuring O2/CO2 from the bioreactor exhaust, but unfortunately, we are having difficulty working with this equipment. Although, the air enters and exits from the machine, but its flow-meter doesn't work and the results aren't satisfactory! Also, how can I make sure that the machine is working properly?
Thank you in advance!
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Hello Karim.
I would contact the manufacturer (Eppendorf) and ask for advice.
Perhaps your exhaust gas flowrate is out of range (0.5 to 1.0 L/min) or the pressure is too high (0.5psig max).
I would also calibrate it before each use, you can buy specific gas mixtures from compressed gas suppliers.
Good luck.
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How to optimize the poor viable cell dansities (VCD) in 5L Bioreactor after 10 days of seeding?
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Dear Smit,
What kind of cells are you growing, i.e., bacteria, yeast, animal cells? Is the medium OK., i.e., have all the necessary ingredients? If it is a commercial medium, check the expiration date as certain ingredients, e.g., vitamins, can lose potency over time. If it is "home-made", you may have left something out. Is the pH correct? Sometimes the pH can change during sterilization (autoclaving) and may require use of a buffer, or adjustment w/ sterile acid or base after autoclaving. Did you use a large enough inoculum? Do the cells require that the Bioreactor be sparged w/ sterile air or oxygen, and agitated w/ an impeller? If you have animal cells, the Bioreactor's impeller may be causing the problem. Animal cells lack cell walls, and are less robust vs. bacteria. Impellers used for bacteria, yeast, etc. are not appropriate for animal cells, as they can cause shear, i.e., they can tear the cells open. For animal cells, a pitched-blade impeller is recommended, as it provides gentle, non-shearing agitation (See R. Mirro & K. Voll, BioProcess International, Jan. 2009, P. 52-57.). Pitched-blade impellers are, unfortunately, expen$ive. If you can't get one, try lowering the agitation rate of the conventional impeller. Also, if you have animal cells, it's a good idea to add an anti-shear ingredient to the medium, e.g., Pluronic F-68.
I hope this helps you. If you need more information, I am available @ wcolonna@iastate.edu.
Bill Colonna, Iowa State University, Ames, Iowa, USA
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I am providing oxygen through sparging to my system (fermented broth) and it increases drastically the DO level in the broth. I am doing also agitation  in the system for breaking of the large molecules of air bubbles, formed due to sparging, hence for proper mixing. But without sparging, the agitator system individually  does not help in increasing the DO level in the broth.
I have checked it for lower to higher agitation speed maintaining the time of oxidation constant and no aeration,but still the DO is not increasing.
What does it mean?
Is that my limitation of agitator system?
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 Dear Zakaria Al-Qodah,
Maybe I misunderstand what you mean to say, but I do have some deviating opinion.
Ionic strength is not likely to change much during the experiment. But it does have a lowering effect on solubility. As such it may lower DO, but ionic strength may also increase mass transfer (smaller bubbles), resulting in a higher DO.
Broth viscosity as such does not reduce solubility unless it increases the ionic strength. Of course the presence of compounds in larger quantities may influence the measured solubility of the broth. But, indeed, higher viscosity (above 20 mPas) leads to strong coalesence, reduced mass transfer and hence lower DO. But again it is not likely to occur during the experiment. However, it is often the reason for not being able to control DO any more at a certain stage of the fermentation.
Two kinds of vortex formation may result from stirring. Vortices behind the blades result in turbulence and better dispersion of the gas, leading to higher mass transfer and higher DO. Also a vortex from the top surface may arise, possibly leading to the entrainment of gas from the headspace. This may result in a higher DO, depending on the composition of the gas.
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I am protecting Dopamine with Acetyl groups and a protocol suggests running Argon through the solution to remove the HBr, which in the reaction is a 33% HBr solution. The link to the patent is here.. 
Im also wondering if I could replace the HBr with Acetic Acid, and if that will do the same job. That way I can evaporate the Acetic Acid through reduced pressure. 
Anyone tried this before? 
Thanks
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No, because you are protecting the acetyl group of dopamine. Any excess of acetyl radicals will first remove this protection (chemical kinetics)  Remove HBr with Argon bubbling only. 
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We are interested to use the Aber Futura Biomass probe (Dielectric spectroscopy) to measure the capacitance data of E.Coli and also scanning data of E.coli cell parameters (Cole-Cole alpha,Cell Diameter,Biovolume, Conductivity etc...). We are using bacterial mode(Default: 1.1MHz) with Polarisation OFF but we couldn't able to measure the capacitance data using default mode. Our offline data suggested that cells are growing well. Anyone can help us to solve this issue.
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Hi,
Aber Futura, work with two types of on-line biomass measurements, optical and capacitance. The second one, takes a measurement of available cells in the medium. You should ask them, the range of capacitance depending of your type of cells and the type of medium (sometimes there a lot of nutrients that can affect the capacitance measurements).
In the attached file, you can have an idea of this.
Best Regards.
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i have chitosan:polyethelene oxide combination nanofiber is availabe in that i want to remove polyethelene oxide alone?
chitosan is non soluble in water & PEO is soluble in water but i have nanofiber how i will remove?
any method is available or give me some suggestion?
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Gurumoorthi,
     Water will remove the PEO.  However the real questions are:1. Are the Chitosan and the PEO phase separated in your fibers and 2. If they are, what do the phase domains look like?  If your Chitosan phase domain is continuous through out the fiber, then you will have a continuous(although likely void filled) structure.  If the Chitosan domains are discrete, then you will not maintain your fiber/textile structure.  You are more likely to maintain a continuous network with lower PEO content.  Either way, you are very likely to get an irregular morphology for the material that is left.  If your application requires  high surface area structure(like filters or sensors) this could be a good thing.  If you application is structural, then perhaps not so much.  Hope this helps.
Regards,
JMD
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We are interested to use the Aber Futura Biomass probe to measure the capacitance data of E.Coli and also scanning data of E.coli cell parameters (Cole-Cole alpha,Cell Diameter,Biovolume, Conductivity etc...). We are using bacterial mode(Default: 1.1MHz) with Polarisation OFF but we couldn't able to measure the capacitance data using default mode. Our offline data suggested that cells are growing well. Anyone can help us to solve this issue. 
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I have used at 0.5MHz and couldn't get the capacitance values over period of time. 
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i am currently fermenting pichia and I am having issues with contamination. A red film keeps appearing in my solution after i inoculate it. instead of going acidic overtime the solution becomes basic. I have made new seed stocks which were grown in zeocion. Using the new seed stocks still leads to contamination.
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 I would suggest the following strategies: 1. You may want to streak plate your culture and inoculate a single colony for seed culture to rule out contamination in the seed culture itself. 2. Increase the inoculum volume 3. Increase the amount of methanol in the culture media. This apart, you may like to re-ensure that all the steps are being carried out asceptically as well as check for any leak in the fermentation vessel as well as its proper sterilization. Hope this helps....All the best!
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I want to understand how much % of the water soluble extractives in switchgrass or corn stover is sugar which can be fermented or further utilised.
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The commercial objective is more related to the supply chain and logistics costs of these crops. Both, switchgrass and corn stover are the substrate for production of biofuels. So, basically the broken down sugars from these biomass will be fermented to ethanol if we go by biochemical route of conversion. If sugars are lost during a rainfall event during field drying, they have to be compensated by harvesting more biomass which increases cost, or minimize the loss of sugars by drying in high density windrows if rainfall is expected. So we are trying to quantify the loss of sugars during rainfall events. 
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I would like to know the investment needed into a single-use process 2,000 liter monoclonal antibody facility, incl. labs, warehouse, offices etc. ?
what are the typical operating costs, especially HVAC ?
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I propose you use de "SuperPro 9" software (Intelligent Inc.) for process simulation. This software is very strong for simulation process and economics evaluation (raw materials, materials, human resources, single use technology, clasified areas, etc). Into software help existing an example for antibody production with single use technology. Regards
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I like to study the barotolerance of few bacterial strains by applying high pressure. In the Bioreactor pneumatic pressure is applied. Can anyone suggest me high pressure resistive materials to inoculate cultures and incubate in the reactor? Similar studies has used sterile plastic bags, hungate tubes, blood pouches were they have applied hydraulic pressure. The material should withstand high pneumatic pressure.
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I have used saline pouches similar to blood bag for inoculation and the pouches were tested till 50 bar pressure. There is no leakage and cross contamination..hope it could be tested at more pressure condition. Thanks everyone.
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I need to know any further consideration beside the actual area of the biopharma plant.
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You can use the book "Piping and Pipeline Calculations Manual" to me has helped me. In conclusion I think you should consider the following:
- Theoretical needs of the productive system.
- Type of fluid: related to auxiliary systems and process lines.
- Type of material: to define friction losses, pressure drops.
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I have tried to increase molecular weight of poly lactic acid of lower molecular weight by chain extension with di dissociates . It give me weight average molecular weight of 200000 Da. I would like to know does there is any other method to get ultra high molecular weight PLA.
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Helena is completely right with her answer. In order to obtain high Mw PLA, you should use ROP from lactides.
cheers,
Miguel
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When I use diluted seed oil with ethanol to react with DPPH in ethanol, I found that it couldn't dissolve it well and could see some precipitate that interferes with the measuring of absorbance. How can I solve this problem?
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what seed? Maybe you have impurities in your oil and need it a filtration. You can dissolve your oil in DMSO
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Nicotiana Tabacum BY-2
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I have no information on this. Sorry.
Ben
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I am currently conducting a research into fungal enzymes involved in glycosyl hydrolysis. I am to select one of three organisms for enzymes production. The activity of the enzyme produced by the best of these three organisms is twice the second best and three times the least. However, when I used the same enzyme units (30FPU) for the three organism in a target hydrolysis, enzymes from the other two organisms (those with lower activities) had  higher sugar yields (10%  total sugar yield more than the best organism). Of course, I thought that the other two organisms might posses some other complementary enzymes which act in synergy to produce a higher sugar yield but I am concerned that they however require a higher volume of crude enzymes or some expensive enzyme concentration mechanisms that will rather make the hydrolytic process non cost effective. I urgently need a decisive guide about this. 
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Enzyme activity by set protocol and applying these enzymes for particular target reaction are totally different things. Factors influencing the target reaction might have significant effect on the enzyme from your three organisms. So I suggest choose your organism based on the actual performance in your target reaction and then further investigate the parameters influencing the enzyme activity such as temperature, pH, solvents, buffer etc. 
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I have been quantifying PHA produced by the microbes via crotonic acid assay. I had centrifuged 15 ml of medium to obtain a considerable amount of biomass , and I had treated the biomass alone with acetone , methanol and water to remove the cell debris and then I had dissoved the left out pellet in chloroform and boiled it at around 90 degrees and after which 20 microlitre of the chloroform extract was used for crotonic acid analysis by standard protocol.
When I compare the spectral readings with the standard graph and quantify I get a few micrograms in 20 microlitre and when I back calculate there is only a few micrograms in a liter of fermentation medium.
where as when I used thermogravimetric analysis of PHA in biomass I obtain micrograms of PHA / micrograms of biomass. can someone help me out with the back calculation.
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Make sure you follow the exact same procedure for your standard curve. You might be having problems with your extraction step. I dont know what protocol you are using though. Depending on what organism you are using you might need some harsher methods to break open the cells. I would recommend 30% warm NaOH solution treatment and then chloroform extraction at a milder temperature like 37C. Ratio of chloroform phase to sulphuric acid is important as well. I use crotonic acid method all the time and time or reaction is critical. Treat sulphuric acid mixture at 80C for at least an hour. Crotonic acid is usually a very reliable method TGA might need special expertise.  Hope this is useful.
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Cell Viability is critical for achieving high cell density and produce recombinant proteins in Ecoli/Pichia/Chinese Hamster Ovary cells. I appreciate any useful input.
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Many bioreactors are having a different set up for measuring the cell viability,.since the probe of the bioreactors is  not much accurate best way to do is you take 1-5 ml of sample asceptically and measure the cell count by using trypan ble exclusion method. One more advantage is that you can also see if any contamination occured.
all the best
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If we tend to consider optimization of the degree of freedom and maximal information of a chemical reacting system to be obtained, how and what can we conduct?
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Dear Chen,
I suggest you to go for Plackett-Burman design to screen the major factors and exclude the minor factors. After screening, Box-Behnken design or Box-Wilson design may be used to determine optimal settings.
Please refer the link below for clear idea:
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the pattern of consumption of amino acid is depend on yeast strain. 
but is there any one knows the availability of oxygen also cause to consumed more amino acid or not?
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Yes, you are correct. The alcoholic fermentation is conducted by yeast of the genus Saccharomyces. The two common species involved are S. cerevisiae and S. bayanus. These two species are closely related, and the subject of a continuing debate among taxonomists as to whether they constitute separate species or races of the same species. Saccharomyces converts the glucose, fructose and sucrose found in grape must and juice into ethanol via the process of fermentation. In fermentation, an organic
compound, in this case acetaldehyde, serves as terminal electron acceptor. This leads
to the production of ethanol. In aerobic respiration an exhaustive consumption of amino acids occur, but in anaerobic respiration fewer amino acids are consumed. 
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I want to use temporary immersion for differentiation phase in somatic embryogenesis.
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Also available at: Application of bioreactor systems for large scale productio...
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i have a pichia pastoris clones (3) with my gene of interest which was confirmed by PCR and sequencing. My protein of interest is extracellular protein and when i am trying to express the protein with standard protocol as mentioned in the invitrogen pichia pastoris manual. After 96hrs of induction phase with methanol i am unable to see my protein on SDS-PAGE either by loading supernatant directly or after concentrating..what could be the possible reason for this?
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Have you checked the intracellular fraction? The protein might not make it all the way through the secretory pathway for a number of reasons, so it is important to find out if this is a case of no expression at all, or failure to be secreted.
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Placket-barman design matrix
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typically it is 20% (10-30%) from the original value
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After several trials I am unable to maintain DO during fermentation; increase of gas supply, addition of oxygen is not working as it increases pressure also. RPM increase is not a solution for filamentous fungi that too when the process have to be replicated at industrial level. I would like to know suitable oxygen vectors if anybody can suggest and kind of experience one can share.
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DO can go low i.e. greater 20% saturation is normally acceptable.
The problem can be approached from two angles.
On the supply side where increased mass transfer can be improved by
1.increases pressure (i.e depth) in the reactor, raising partial pressure of oxygen
2. changing aeration scheme where airlift reactors may be a better approach to agitate non-newtonian fluids like fungal biomass
3. changing impeller arrangement
On the demand side
1. reduce metabolic activity, (lower temp, restricted feed of nutrient, i.e. fed batch approach)
regards
bob
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In my gc ms experiment I have got long chain monomers  c15 to c19 while using linoleic acid as a carbon source in pseudomonas putida .Can p.putida capable synthesize long chain polymers?
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Dear  Dr. Ahil;
I hope it is not too late. The following attached book is an extremely important one in the area you are working in. Regards
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it always forms two layers ,due to polar and non polar properties, so fatty acids are not properly transfer in to the medium ,some one would suggest me to solve the problem
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Dear Ahil,
Apart from the solutions menthioned above, you could from oil emulsions.  I would suggest forming an emultion and adding this using an acurate second feed pump.  
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I am trying to determine accurate ethanol yields on glucose of different yeast strains in batch aerobic fermentations using bioreactors. I am using media containing around 240 g/L sugars which, using S. cerevisiae in anaerobic conditions, could result in up to 13-14% ethanol (v/v).
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you use 240 g/l, it is known that at high concentration of the carbon source the yield is lower therefore to get higher yield you can use the glucose fed-batch, I would use the initial concentration not more than 50 g/L, the glucose feed concentration can be 70%-solution that does not increase the volume so much.
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I need to extract this compounds from my biodiesel sample in order to characterize them in HPLC tests. Any suggestions are welcome.
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Thank you Lorena
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The rapid decrement in C/N ratio indicated active microbial degradation on the carbon source.
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Hello Mohd. By nature lignified structure are more resistant to microbial / enzymatic attack; influence of LC content on biodegradability is quite well established. (see for instance "Towards new indicators for the prediction of solid waste anaerobic digestion properties"  in Water Science Technology 2006 53(8) pp 233) ;
Nitrogen loss is usually not associated to lignocellulosic compounds, but with proteins degradation.
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Can any change in substrates characteristic influence or contribute to microbial diversities? Need explanation on this
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Temperature is the one which controls the microbial community since there three types of microbes psychrophiles, mesophiles and thermophiles based on temperature. mesophiles are the organisms which could live in temperature above 20 degrees to 37 degrees. during the starting stages of compost the material in the com[post will degrade once degradation is in process the heat increase in the material will automatically increase the temperature to thermophilic which supports the organisms which grows above 40 degrees and again when the degradation of the material completes the temperature will automatically down to mesophilic which supports the spores of thermophiles and streptomycetes and actinomycetes. 
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I would like to produce gel filament of cell entrapped in polymer solution. But when I mix the suspension with the polymer solution, a pre-gel is formed and air bubbles are also entrapped in the mixture. I've already tried to removed the air bubbles by vacuuming but it takes very long time and isn't good for cell viability. Any idea how to remove the air bubbles without causing cell damaging?
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Hi, maybe you can use slower stirring speed while mixing the solution and add it gradually, i had same problem with prepare hydrocolloid solution at high concentration and i added the particles gradually to the solution at lower stirring speed.
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Which is more efficient in the case of bacterial system?
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Most of the time 70-80 % ethanol (chilled) works, i think it should work
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As I know, lactose serves as both carbon source and inducer. Thus it is consumed as a substrate, which may decrease the capability as inducer. The good part is that excessive lactose is not harmful to cell growth. In contrast, IPTG serves only as inducer and cannot be metabolized, so it is more stable. However, I found that excessive IPTG will be "toxic" to cells and the cells aggregate and finally will be lysed if incubated further. Why is it like this?
In terms of protein or metabolite production:
How will the transcription change when induced with lactose instead of IPTG? Will it drop significantly over time?
Is it better to induce with excessive lactose than moderate IPTG?
In a bioreactor, is it useful to induce continuously but with relatively low concentration of IPTG?
Some more general questions:
Is it useful to optimize the amount of inducer dosage?
Is it better to induce at early log phase than at mid log phase?
Thanks
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Dear Zhang, Lactose is a naturally occurring sugar (diasaccharide of glucose and galactose) which is metabolized by a number of prokaryotes and Eukaryotes. However IPTG is a synthetic and structural analogue of allolactose which can be used as a substrate but cannot be metabolized. The beauty of IPTG is that since it cannot be metabolized by the organism its concentration remains constant during the entire course of experiment. Moreover it is used for induction of lac operon in in the concentration range of 100 μM to 1.5 mM.
The amount of Lactose or IPTG used for induction depends on the binding and dissociation constant of the IPTG and lactose to the lac repressor and I believe that that two have different binding constants for sure. Hope I could partially answer to your question
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I am looking for a protocol for ion pair reverse phase hplc using Luna C18(2) column for estimation of intracellular nucleotide precursors (e.g. UTP, UDP, ATP, AMP etc.).
The major concern is that the salt concentration in the protocol should be as low as possible.
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Vlado, we are using Tetrabutylammonium.. the problem that we are encountering is of salt precipitation.. that increases column pressure with time...
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I did the experiment for the determination of reducing power by oyaizu 1986 method and found absorbance values but how to do the calculation to determine reducing power or the extract? do we have any formula? Or the absorbance values corresponds to reducing power? What unit should I use?
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Please refer eplantscience.com for clear-cut calculations
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I am going to check the activity of an enzyme on a substrate which gives acetic acid as one of the products.
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The choice of the detector will depend on the concentration of acetic acid that you are trying to analyze. For lower concentrations, PDA works better as suggested above.
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The problem is that the container of 20l which I will be using cannot be autoclaved so I will be using 500 ml conical flasks. However, that will require a large number of flasks and it will take lot of time to do the autoclave. Is there an easier way to do this? My lab has a vertical autoclave and a horizontal autoclave.
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You may use filtration.
Algae for biodiesel requires extremely large production. Sterilze water cannot be used at very large scale. Keep this in mind before you make any technology claims. You can only use sterile water at small scale, may be in the initial seed preparation stages.
In our lab, we do perpetual algae consortia with high lipid content using seawater with no treatment.
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Natural carbon source.
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Natural carbon source is generally referred to glucose, starch and sucrose. Many crop products or crops itself contains large amount of natural carbon sources. Also the extracts from the crop residues acts a cheap carbon source for PHB production.
As PHB is produced under nutritional stress conditions, so instead of using crops/ natural products... I suggest you to go for distillery waste effluent (also called as Molasses). It contains sugar content in abundant quantity. Also, the discarded and degraded food grains from godowns can be used as rich natural carbon source.
Thanks,
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I need detailed procedures on how to measure color (that is, L, a & b) using a spectrophotometer.
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It is the simplest method and even easier than spectrophotometer. You just calibrate with black and white tiles available with the instrument and start to measure your sample by placing at a certain distance. Details are available with the instrument manual. You will receive L stands for lightness, a has two values (-a stands for green and +a for red) and also b (+ for yellow and - for blue). For greeny vegetables you will get -a and for your sample you will get L, +a and +b values.
If you have the instrument strat today and enjoy the tristimulas color values.
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Does anybody have any ideas about water binding capacity and water holding capacity? Are there any differences between water binding capacity and water holding capacity? And how to measure them?
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Water binding capacity is the tendency of water to associate with hydrophilic substances whereas Water holding capacity is the ability of a matrix of molecules to entrap large amounts of water in a manner such that exudation is prevented. Bound water exists in the vicinity of solutes and other non-aqueous constituents, exhibit reduced mobility and properties differing significantly from “bulk water” in the same system and does not freeze at -40°C.