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I would like to quantify the OTR and the oxygen demand in different cultures and rpm of shaker by the method of gas in and gas out, but is not very clear which formulas used, and which measurement magnitude are replaces the variables in the formulas.
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Kuhner AG is the best option for measurement, which provides appropriate results
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Years back, I have taken notes from that book for my ug assignment, but I missed to note down the author’s name.
Now, I need the book for further reference. I searched a lot but i could not find it.
I could not get the book without knowing the author’s name.
BOOK DETAILS
TITLE: TEXTBOOK OF MICROBIOLOGY
CHAPTER 29: TOOLS OF FERMENTATION TECHNOLOGY ON PAGE 1021
If anyone knows the author details and the edition of the book, please share it.
Thank you in advance.
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Thank you so much sir.
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I am working on bioethanol production from lignocellulosic biomass. However, I require some clarification on the methods for calculating initial sugar concentration, sugar consumption rate, ethanol yield (in g/g and g/l). Could you please recommend simple calculations or protocols?
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Sept. 21, 2023
Dear Merlin,
I will try to provide you with some information that hopefully will help you.
There are many methods for measuring sugar concentration. Some are very sophisticated, e.g., gas chromatography, HPLC, etc. If you don't have access to such instrumentation, you can use colorimetric methods. There are many of these. They use readily available chemicals and require only a bench-top spectrophotometer for analyses. One that I have used in the past was developed by Dubois et al. "Colorimetric method for determination of sugars and related substances". Analytical Chemistry, Vol. 28, p. 350-356. This is an OLD method (from 1956); however, it is still in use. It is very quick and easy to use. Requires phenol and sulfuric acid. Detects many different sugars, e.g., glucose, mannose, galactose, fructose, xylose, maltose, raffinose, etc. It is very sensitive: can detect as little as ~5 micrograms (even less) of carbohydrate (CHO). It can also detect the above sugars in polysaccharides (e.g., starch, cellulose, etc.) and oligosaccharides.
Reagents: 1) 5% phenol in water; 2) concentrated (18 molar) H2SO4.
Procedure: Add your test sample to DI-water to final volume of 1.0 mL. Then add 1 mL 5% phenol, followed by 5 mL H2SO4. (When the acid is added, the resulting solution gets VERY HOT, so hold the test tube by the top, not by the bottom.) Mix the tube contents (vortex mixer). An orange color develops very quickly. Wait until the solution is cool to the touch, then read the absorbance at 490 nm on a spectrophotometer. You will need to run a series of sugar standards for quantitation of the CHO in your samples. CAUTION!! Be sure to wear gloves, lab coat and especially safety goggles when doing the assay. If you're careful, you'll be OK.
For detection of sugar consumption rate, e.g., during a fermentation, you can take samples of the fermentation broth at various times, clarify the broth (centrifugation) and measure the sugar concentration (as above) and see how it decreases with time. You can also measure the corresponding increase in ethanol.
Calculating ethanol (EtOH) yield: Let's assume you are using yeast to ferment glucose to ethanol. Consider the reaction:
C6H12O6 ----------> 2 CH3CH2OH + 2 CO2
Glucose EtOH
If the yeast completely ferment 1 mole (180 g) of glucose, they will produce 2 moles of EtOH (2 x 46 g/mole) or 92 grams, plus 2 moles of CO2
(2 x 44 g/mole) or 88 grams. The EtOH yield is (92 g/180 g) x 100 = 51.1%. If the above fermentation is done in a 1-liter volume, you will have 92 g EtOH/liter or 92 mG/mL. If you divide the weight of ethanol by its specific gravity, that will give you the # grams of EtOH/L. If I recall, the Spec.Gravity of EtOH is ~0.79 g/mL.
From the equation and calculations above, you can see that if you start with 1 mole of hexose (e.g., glucose, galactose, etc.), the EtOH yield will be ~51.1% of the initial weight of the sugar. IMPORTANT: The 51.1% is only a THEORETICAL yield. You will NEVER get the theoretical yield. This is because during the fermentation, the yeast will use some of the sugar as a nutrient on which to grow and multiply in number, so a small amount of the sugar is never converted to EtOH . Of the total sugar, only ~93-94% of it is fermented to EtOH. This % will vary from one fermentation to another.
I hope this information helps you. If you have other questions, let me know. I will try to answer them for you. Good luck w/ your research.
Bill Colonna, Midwest Grape & Wine Industry Institute, Iowa State University, Ames, Iowa, USA. wcolonna@iastate.edu
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Hello all,
I am in the process of setting up an industrial enzyme production facility, initially for producing poultry feed enzymes. Does anyone have a contact of where I can procure the strains for the following enzymes? I am trying not to do R&D initially as we would like to be production ready ASAP.
Thank you!
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There are a bunch of places where you can get microbial strains for making industrial enzymes. Here are a few options:
1. Culture Collections: Many countries have these collections where they keep a bunch of different microbial strains, including ones used in industries. These places usually give out strains to researchers and businesses. Some examples are the American Type Culture Collection (ATCC), the National Collection of Industrial Food and Marine Bacteria (NCIMB) in the UK, and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) in Germany.
2. Biotech Companies: Lots of companies that work in biotechnology focus on developing microbial strains and offer all sorts of strains for industrial purposes. They might have their own unique strains or genetically modified organisms made specifically for making enzymes. Some examples of these companies are Novozymes, Genencor (a division of DuPont), and Codexis.
3. Research Institutions: Universities, research institutes, and government labs often have collections of microbial strains for research. Some of these places might be willing to provide strains to businesses or collaborate on projects involving enzyme production. It could be helpful to get in touch with relevant departments or researchers in this field to access these kinds of strains.
4. Online Strain Marketplaces: There are online marketplaces that focus on selling microbial strains and related stuff. These websites connect people who want to buy and sell strains, so you have a lot of options. One example is the ATCC Microbiology Marketplace, where you can find all kinds of microbial strains.
5. Industrial Enzyme Suppliers: Companies that make and sell industrial enzymes might also offer the matching microbial strains for people who want to make enzymes themselves. If you have a specific enzyme in mind, you can reach out to these enzyme manufacturers and ask if they have the strains you need.
When you're getting microbial strains, it's important to think about things like whether the strain will work well with your production process, if it's genetically stable, and any rules and regulations you need to follow. Also, make sure you're following the safety and intellectual property rules related to working with microbial strains.
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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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Design Expert software used for modelling experiments for optimization of fermentation technology.
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Your are welcome Priyanka Roy
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As C/N is very critical in the optimization of the Exopolysaccharide production by Microorganisms. My concern is that I have used complex media such as tryptic soy broth (TSB) for other optimization experiments. TSB medium contains glucose as carbon and tryptone as the nitrogen source. Should I use different ratio of glucose and tryptone to optimize C/N ratio? Any suitable reference paper please.
Thanking in anticipation
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Dear Ali. To solve your problem, a multivariate analysis is necessary. And as recommended by Sarmad A. Qamar Response surface methodology (RSM). But in view of the complex substrate, it will be difficult to provide a given level of nitrogen.
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What are the new technology available for response surface methodology or other optimization technique. If any one working in optimization of fermentation technology please reply....
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For the fluid aspects, computational fluid dynamics is gaining ground in fermentation. CFD is used to optimize reactor designs (effect of geometry on hold-up, mass transfer, substrate mixing, etc.). CFD has been used to find the optimum between minimal shear rate and proper cell suspension for cell cultures ( Louviere et al.). Siemens STAR-CCM+ offers an optimization add-on where you can automatically vary geometrical parameters to find pareto-fronts for optimization, e.g. energy input vs. mixing time, gassing rate vs. kla,...
In my own work I combined CFD with metabolic models to make in-silico assessments of productivity under non-ideal mixing conditions in fermentors, which can be used in optimizing lab-to-factory transfer and assessing potential yield losses with a model driven approach.
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I encounter Modified Gompertz to calculate production rate in batch fermentation. How about in semi continuous/ ASBR treatment? Thank you
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Currently I am working with a locally made fully manual 5 liter fermenter (Scigenics, chennai). After sterilization, it use to take a lot of time to cool down to RT. Is there any way to decrease the temparature rather quickly?
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You add the medium in the sterlized fermenter and then start the stirrer with chilled water being circulated in the jacket. Note it will depend upon heat transfer coefficient across the material of the fermenter body, and between the medium and the jacket material.
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Dear All,
We are interested to develop a bioethanol miniature plant. Is there anyone who knows any company or institution that can built this type of plant?
Thank you.
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Hello Safri, 
The ethanol production from starch is well studied, if you want to use cassava, try to focus in residual biomass to give a value added to that.
I send you two papers of this.
Good luck!! 
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I want to know range of carbohydrates that metabolised by Clostridium butyricum NCIMB 7423Bergeys manual is helpful in term of describing the metabolism of species, Clostridium butyricum in general. However, is there any other source to check for metabolic pathway of specific bacterial strain. Where can I refer?
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Databases like KEGG or MetaCyc can be useful for metabolic pathways, here is the link to KEGG pathway map for C. butyricum.
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I'm a new researcher who research in Microbial Fuel Cell by adopting yeast (S. cerevisiae) as a biocatalyst. But one of the issue is the reaction mechanism: glucose + H2O --> CO2 + 24H+ + 24 e-  is obtained from anaerobic condition or aerobic condition? some references said from anaerobic condition, but also some references said from aerobic condition. It makes me a little bit confused.
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 Thank you, Mr. Michael. how about the number of electron and hydrogen produced? any big different between aerobic and anaerobic condition?
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Dear all,
I have a similar problem in my anaerobic fermentations (glucose fermentation by C.beijerinckii). I flush the fermenter with N2 (0.5 l/min) entire time and stir.
I prepare my anaerobic media accordingly so that there is no dissolved oxygen in the begining of the fermentation. However, during exponential growth phase, dissolved oxygen level increases drastically, up to 60%. The problem is not the probe nor the equipment. I observed the same pattern in different setups (Aplikon fermenters and microreactor fermentations).
Does anyone experience a similar thing? If yes, how did you overcome the problem?
P.S.I am suspecting oxygen production by the bacteria triggered by a metabolic change, but I haven't found anything about the in the literature.
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Dear Ghasem,
I haven't checked it without flushing. I should do it next time. For the case of contamination or algae, I should do further investigation.
Thank you
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 Dear all , 
 production of probiotics using  fermentor sampling once in 4hrs OD has reduced after 24hrs but microscopy & plate shows no contamination .
kindly post your suggestion .
Thanks in advance 
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The method Bill suggests is a good one.  Here is another paper that compares different methods for reducing sugars.
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I am using clostridium acetobutylicum as an organism and lignocellulosic biomass hydrolysate as carbon source rest components of reinforced clostridial broth are supplied. But it is producing only butyric acid and it is not entering solventogenesis, what could be the possible reason for this?? Can someone give me an explanation or justification of this phenomena.
The lyophilised powder of cells were obtaine 2 years ago but it was kept in that form only. 
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Hi Mriganka
it looks like as you have oxidation power present, if you incubate Butanol long
enough, you have a possibility to get a transformation to butyric aldehyde and acid.
Can you estimate aldehyde in you samples? This might be an indicator.
An Alcohol Oxidase, if present, might not be necessary for your lignocellulosic
degradation, you could knock it out or strip it directly from the reaction to avoid
extensive incubation.
kind regards
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For example, when an inoculum is 5% in terms of v/v, what does the v/v percentage mean?
Also, how is this applied in industry?
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This term is very often used once you have standardized your inoculation procedure. For bacteria o yeasts for example, you may count cells and establish that you are going to use a 10^8cells/mL-inoculum, or xx CFU/mL, or a given O.D. inoculum. But then, you need to standardize how much inoculum of this N° or density you are going to add for starting your next culture. Normally, one uses 5-10% v/v, which means what Dr. Belaouni detailed above. If you are setting a1L-culture you may add 50-100 mL of inoculum (or starter). Being strict, quantitatively, you should discount this volume from the total volume of culture (as Belaouni said). In the practice, at pilot plant scale or industrially, many times you directly add this 5-10% without discounting from the whole culture. The main thing is standardization, which assures you the possibility to reproduce your results EVERY time. Other relevant thing is to keep your inoculum, not only in the same proportion, BUT always in the same growth stage and equally activated. In the case of fungi, sometimes the inoculum processing is slightly different. You may neither count (except for conidia) nor measure OD. But you can add the same number of agar plugs per volume, or standardize by dry weight. Hope it serves. Success!
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How can I get saccharomyces cerevisiae strain?
Dear all,
I would like to know if a regular off-the-shelf baking yeast can be used for laboratory scale and cultured on minimal media without amino acids or vitamins as i want to grow them on 13C-glucose to end up with fully labelled metabolites that could be used as internal standards for quantitative analysis of lipids in other biological samples . and if not, can anyone suggest a suitable strain that will grow on minimal media with one carbon source.
Any pointers given are much appreciated. Thank you!
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Dear Malak
The best way is to procure it from ATCC or UKNCC or NCIMB or PHE-Culture Collection in UK. I mean you will get the right type and authntic  cultures  of the yeast .
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we have Shimadzu LC 2010 HPLC system provided C18 column and UV/Vis detector.
I want to analyze Lactic acid both L & D, Propionic acid, gluconic acid, acetic acid, calcium propionate, calcium lactate, calcium gluconate, sodium lactate, potassium lactate, sodium acetate and other organic acids which obtained from fermentation technology.
please suggest some references and methods for any of the above organic acids. it would be great helpful for me to extend my abilities.
Thanking you.
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We do use the Aminex-HPX 87H column since almost 20 years. It has good  propoerties to separate organics, alcohols and sugars including the compounds you mention. we use it specifically on fermentation samples from all kind of microbes. it uses isocratic diluted sulfuric acid eluent. it will not distingusih l and d lactate. this latter we do by commercial enzymes, which are specific for either D or L lactate.
you wont see differences between the different salts of lactate, as they  dissociate in fermentation broth.
Best,
Christoph
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Hello , I have a question about the concentration of Saccharomyces cerevisiae yeast to ferment a solution f glucose of 50 g / liter , It is clear that I need to grow the yeast in preculture medium for incubation but how much is the concentration of yeast should be used in fermentation medium ??
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A difficult question to truly answer, yeast can tolerate and indeed ferment above 20% total available sugars so 5% is in truth well within their capabilities. Most breweries will pitch as high as possible because they want a quick transit time in the fermenters but most research is carried out at around 10/8 cells per mL but as the other answers say nitrogen, free available amino acids and the fermenter system all change the dynamics of the experiment. You need to sequentially grow S. cerevisiae as Budiatman states, you'll get a quicker fermentation if you keep all media at 5% so the yeast won't encounter osmotic shock as much as possible.
the best method is to get to know how you're yeast works in the system you are running.
The best people to read are Boulton and Quain or Mike Ingledew, these guys literally wrote the book on yeast fermentations.
Hope that helps.
Darren 
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With starting 2% initial concentration of dextrose in fermentation broth. How I maintain minimum dextrose concentration as per OD or biomass gm/l.
There is any thumb rule for that or I have to do some experiments for that. There is consumption of ammonia hydroxide in fermentation broth so feed rate of dextrose how effect the ammonia consumption rate.
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I agree with Marius K. Somda answers .
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I am working on E.coli fermentation process. For maintaining pH I am using ammonium hydroxide 20% solution. During the process ammonia consumption approx 50 ml/l. But I want to know how I calculate the acetate formation during the process for which the ammonia used. E. coli fermentation is mixed acid fermentation so how I can calculate specifically acetate formation during the fermentation process. 
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Simple check the initial acetate (by GC or HPLC) and then check the final acetate by the same method. After all acetate is not so volatile.
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I am working on cold active lipases and having trouble in purifying the enzyme due to very high degree of aggregation. I have tried breaking the aggregates using several detergents such as CHAPS, Tweens, Triton X100, NP40, Brij-35, SDS, etc but no success till now. The high molecular weight aggregates are not able to move in to PAGE and remain stuck at the interface of stacking and resolving gel. Find the attached images of SDS and native PAGE showing protein bands at the top of gel. Any suggestions please..
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What, precisely, is your aim? Do you just want to see the protein in the gel (e.g., for quantification), or do you want to purify the active enzyme (e.g., for kinetic measurements or industrial use)?
In the former case, I'd start with 7 M urea, 2 M thiourea, 10 mM DTT and 1%SDS. 
In the latter case detergents with fat-like structure (lyso-lipids, fatty acids, FOS-choline, LysoFos, neopentenyl glycols) may be able to to solubilise functional protein. In any case, I'd start with a careful literature search. A day in the library can save you months in the lab!
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hi dears,i need help.. i want to estimate lactic acid concentration in fermentation medium by colorimetric method using 4-phenyl phenol. at first i want to draw lactic acid standard curve. can any one tell me an exact and perfect protocol?
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There are many protocols for DNSA where different quantity of reagents is used so its a confusion that which is right method.
if have please provide with reference.
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this book will be useful 
Regards
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10 Years ago we tested a YSI biochemistry analyser on pichia fermentations and the conclusion was that Methanol determinations where not reliable. Things change and technologies advance, so I was wondering if anyone has had any good experiences since.
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We also have an online methanol detector from frings, that has the same working principle, but the signal is not stable in fermentation broth. In water we do get nice reproducible results but that's of little use to us. Despite that, autoclavation of this sensor only works for SIP, the electronic parts cannot go into an autoclave. Could you give me more details about the detector you are using?
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Hello to everyone
I had an instrument "MX6 IBRID" can i use it to measure methane and other gases produced in vitro fermentation of ruminal fluid.
if someone have info about this kindly ans me.
Regards.
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you can write to him, this is email: (kvera@correo.uady.mx), this outhor have many paper about of measure methano 
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does anybody can help me ?
I want the protocol of reverse phase hplc of lactic acid determination .
thank you all
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Actually a  C18 column is more than sufficient. Here is an article I give below:
Ginjupalli Kishore, Armugam Karthik,, Shavi Venkat Gopal,, Averineni Ranjith
Kumar, , Mahalingam Bhat and N. Udupa. (2013),Development of RP-HPLC method for simultaneous estimation of lactic acid and glycolic acid . Der Pharma Chemica,  5(4):335-340
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Crab tree positive effect of Ecoli leads to acetate accumulation. Apart from engineering of Ecoli strains, what are the feeding strategies and process control optimization can be done to avoid acetate (<1g/l)
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Mr Phillip Brumm if you know the correct answer you answer to the question,as per your reply to this question I can quess that you dont have minimum knowledge about fermentation process. Can anybody  in the world maintain DO above  90%  and that too  maintaining DO is no way related to acetate production please mind it.
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I was doing some research on ABE fermentation using bacterium Clostridium acetobutylicum.Before fermentation, deactivated bacteria cultures are stored at -80 °C. The thawed cells are then inoculated into 12 mL synthetic medium containing glucose (30 g/L) and Yeast Extract (5 g/L) in 15 mL Hungate tubes (pre-cultures). Cells are then grown under an aerobic conditions for 48 h at 37 °C, thereafter they are transferred into fermentation bottles.
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Which clostridium species gives the highest concentration of butanol from the broth? 
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We use pure oxygen for fermentation, but ordinary air for DO probe calibration. At this moment we set air as 100 % point of calibration (we use only 1 point calibration). Is this correct? Shouldn't it be 21 % point of calibration, since we use pure oxygen as oxygen supply. Can someone help?
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Hi, Jirka
I am using NBS BioFlo 310 for my experiments and we use Air for setting the span of the probe which is 100%.
In my opinion, it should merely influence the time needed to reach the fixed digits otherwise, it won't affect the the amount of DO in the vessel broth.
Good luck
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I need a bacterial strain that is able to produce Lysozyme enzyme extracellulary in chitinous media. What might do this?
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Dear Sara
You should have some idea on microoganisms producing lysozyme first. I think that the most known for their active lysozyme production are Staphylococci strains. Both coagulase positive and coagulase neagtive are capable of producing Lysozyme but coagulase positive staphylococci are more active because there is a relationship between coagulase and lysozyme. Bacillus strains can also produce lysozyme. You should first isolate pure strains of these microorganisms and after gathering many isolates you should go on a screening test using a bacterial test for detecting the lysozyme activity. Many techniques are described on the net. I cannot write you all the details of these methods.
Add your answer
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What is the most effective way to break down unfermentable sugars to a form that can be fermented?
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Unfermentable sugars may result in a fermentation mash depending on the source , nature of the carbohydrate material and processing conditions. in the case of starch fermentation, alpha amylase will produce fragments of malto dextrins in addition to glucose molecules giving rise to more unfermentable sugars than Beta amylase which produces mainly maltose fragments that are fermentable. glucoamylase breaks down starch molecules to more fermentable sugars. these enzymes and their combinations may be utilized to maximize the concentration of fermentable sugars. other enzymes such as alpha galactosidase, xylanase, cellulase may be added to molasses to convert unfermentable sugars to fermentable ones. prevailing experimental condition will be guided by on what is being fermented
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I want know which types of Probes/Sensor using for fermentation technologies.
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Hi Manoj
I think you mean the types of sensor that can be used in bioreactors for fermentation!
There are different types of sensors for this issue such as pH, DO, Level, OD, Temperature which are online and the offline sensor for measuring glucose, some minerals and so.
They should be very sensitive and autoclave-able. Mettler Toledo is one of the famous companies providing theses sensors for fermentation, you can visit their homepage to  get more information!
Good luck
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DNS method was used to measure reducing sugar as well as DE value , however, this method is under discussion since the standard curve alters when the degree of polymerization of maltooligosaccharide
changes. How to cope with that problem? the used amylase does
not exclusively releases maltose units, it will also release glucose, and so on
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I have not, but John Robyt and his coworkers have done so. Please see the attached paper.
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I isolated the DNA of yeast strains obtained from the Sauerkraut brine and subsequently performed the PCR-RAPD for the strains. Before sitting down to PCR-RFLP, I would like to get busy with PCR-RAPD for the bacteria (hopefully mostly LAB) and here is the thing - what ways of ensuring anaerobic conditions can you suggest, besides using airless chamber or wrapping test tubes with foil?
I haven't experienced big difficulties in cultivating so far, yet I would appreciate it if you could provide me with some solutions in case I'd have to modify something. Thanks a million in advance!
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There are many options, depending on your resources and anaerobic requirements. I work with strict anaerobes, but the concepts apply to facultative anaerobes.
-A rigorous method is given here: Hungate, R. E., & Macy, J. (1973). The roll-tube method for cultivation of strict anaerobes. Bulletins of the Ecological Research Committee, 123-126. 
-A more recent paper for liquid media prep: Wolfe, R. S., & Metcalf, W. W. (2010). A vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes. Anaerobe, 16(3), 216-219.
Sounds like you want simple, however, proper anaerobic culturing requires some work. These papers should give you a start and provide background on what to look out for. Good luck!
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I´d like to know any reference or paper in which the authors deal with the dynamics of pH and Temperature in the modelling of PHB fermentation in Fed-Batch.
I´ll appreciate.
Best Regards.
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Thank you Carlos, i sent a msm to Dr Nemecio.
Best Regards
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Comprehensive study on a two-stage anaerobic digestion
process for the sequential production of hydrogen and
methane from cost-effective molasses 
in the above paper the yield of hydrogen production is 2.8 l/l-reactor/day but the value in L/L-molass/ day is 27 litre. 
**international journal of hydrogen energy 35 (2010) 6194 e6202
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You must bring the volume from which you got the 3.6 L, to perform the calculation. Anyway the standards set for production employs a molasses with certain characteristic, your molasses has the same characteristics ???
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I am looking for media which help in  alcohol production by yeast and gives good yield. Which media composition can be better then GYE (glucose yeast extract).
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carbonic maceration, cane extracts, grape juice, curd, fruits slurry, seeds paste and slurry are best media for alcohol production. A media should contain high levels of ferment able sugars,
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I need better explanation to understand why people often used that terms. Maybe I need more information about the principles and purposes, the microbes as the subject, and other things. 
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Co culture could be developed both by different species or the same species. The main difference is that mixed cultures are usually present in some environments such as activated sludge. they are extracted and bring to the labs, but co cultures are developed synthetically using different microorganisms which are grown in the same medium.
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We need to separate a mixture in a column distillation using an extractive distillation system.
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Hi Joel
Do you want to break the azeotrope? F.E. a separation of water and alcohol inside a destilliation column or do you want to extract something polar from an aqueos Phase..or maybe both?
To break azeotropes you can try to destill under overpressure, this is a suitable method for some difficult mixtures, even if the boiling points are very close, you might have to optimize or enlarge your theoretical destillation bottoms and look om the capacity of your colon.
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I am trying to grow hepatocytes (HepG2 cell line) on a cryogel based perfusion bioreactor. I generally sterilise the scaffold before seeding the cells onto it and the bioreactor design housing this scaffold is completely leak proof. I also autoclave all the spare parts and tubings required in the system and all the handling is done under sterile conditions. Yet, after running for a day, the media reservoir changes to yellow and there is bacterial contamination in it. Is there something I am not doing right? Or something I am missing out? Please help with your suggestions
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Keeping positive pressure inside the bioreactor system may help minimize the contamination issues.
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I am running submerged fermentation of Bacillus with barley husk as carbon source in a bioreactor. When samples are taken, the barley husk particles are also included.However, the amount of barley husk particle that is included in the sampling varies ( not possible to standardise). Problem: The absorbance at OD 610 nm fluctuates too much ( suspected reason: unequal amounts of barley husk particle). I am thinking of filtering the sample with a muslin cloth or a filter paper to remove the barley husk particle before checking of the cell density. Is this a good move?
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You cannot use the colorimeter method in this case. You can go with plate count method or protein estimation method for bacteria growth.
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I want to express the human Fabs in E. coli, the literature shows HB2151 strain is a preferred choice for the same. But I need to make Fabs at levels that should be considered good enough to take forward at production scale. I was wondering if we can use BL hosts, but for them very confusing results in the literature are reported. 
The inputs will be very helpful in taking the experiments ahead.
Regards
Shridhar.
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Hi Jai,
Thank you for the reply, but the point is HB 2151 cannot be used for the large scale expression.
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Fermentation lowers polyphenol in cocoa, I have a question regarding this issue, in fermentation polyphenol are consumed in tanning reaction with protein which cause to insoluble the polyphenol then reduce the polyphenol , when polyphenol is insoluble how it can reduced?
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 Cocoa bean heap fermentations are used to make chocolate in Ghana. Though various metabolites are lower down and break be due to heterogeneity and size of the heaps,due to  microbial activity and its variability. Indeed, differences in microbial activity could be linked with the flavour of chocolates made from the corresponding dried, fermented cocoa beans. Whereas the polyphenol and alkaloid contents of cocoa beans are crop- and heap-dependent, epicatechin and theobromine levels decrease during fermentation due to diffusion out of the bean cotyledons and polyphenol oxidation and condensation. Residual levels are responsible for the degree of bitterness of the final chocolates.The latter encompasses anaerobic yeast fermentation of sugars to ethanol, microaerophilic fermentation of sugars and citric acid to lactic acid, acetic acid and mannitol by lactic acid bacteria (LAB), and aerobic exothermic bioconversion of ethanol into acetic acid by acetic acid bacteria (AAB). These microbial activities result in  the death of the bean due to penetration of mainly ethanol and acetic acid through the husk into the cotyledons, and the creation of an environment, i.e., a
decrease of internal pH from 6.5 to 4.8, an increase bean temperature up to 50 ◦C and a damage internal cocoa bean structure, for development of flavour precursors and pigment degradation by endogenous enzymes, such as invertase, lycosidases, proteases and polyphenol oxidase.
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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 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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Itaconic acid is produced through fermentation process by Aspergillus sp. at pH 3. This species use Kreb cycle intermediate for the production of IA. now the question in my mind is the role of itaconic acid in fungus.. Why the Aspergillus sp is producing IA.. Is IA a primary or secondary metabolite??
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Itaconic acid is reported as a primary metabolite, normally produced by  strains of A.terreus but It is over produced under phosphate limited conditions. There is difference between production and over production.
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I deal with production of Lipase from Candida sps. (CALB). The fermentation has been recently scaled up to a 3L fermentor. The problem that I face now is the fluctuating O.D values of the broth measured through a period of 48 hours. I cannot seem to point out the actual cause of the decline in O.D. 
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It's easy: Bible OLD TESTAMENT Genesis 3:19 You will eat bread by the sweat of your brow until you return to the ground, since you were taken from it. For you are dust, and you will return to dust." Candida makes the same. OD of dust is negligible.
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To evaluate the quality of feed
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The easiest is to invert full (water) measuring cylinder in a water bath and follow gas displacing water with a chronometer (cost 0€). Of course, can also buy a mass flow meter (e.g., Brookfield) but will cost you some 2000 €.
Kindly
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This is with respect to evaluate the quality of feed.
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If it is for evaluation of a ruminant feed then you can use the following technique.
Minson, D.J. and Mcleod, M.N., 1972. The in vitro technique. Its modification for
estimating digestibility of large numbers of tropical pasture samples. Div. Trop. Pastures. Tech. Pap. 8, CSIRO (Aust.)' pp. 1-15.
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How can I extract and quantify the cyanide that has been extracted from cassava tubers into the fermentation medium?
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When I used to work with cassava I applied a test kit as described in the attached paper. I subjected cassava bagasse to fermentation and I quantified the content of cyanides following method. It gives reliable results and it is good for frequent measurements. You may also consider gas chromatography but it is more expensive solution and it requires handling of potassium cyanide which is very toxic.
Best regards.
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I am trying to find the optimum moist heating treatment of fermented food, which is employed by yeast and BAL. I have to maintain the sensory characteristics of the fermented food so they still taste good and look good in color.
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In order to determine the effective F value, you must first define EFFECTIVE. I mean you must know which characteristics you want to maintain and then decide what is the effective value and following this define the shelf life. 
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Using RSM, What is the best for fermentation conditions/medium optimization; make a broad screening of factors by PB design then optimize by Central Composite Design or make preliminary studies for each factor alone using one factor-at-a-time then choose the critical factors and optimize them by Central Composite Design??
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using the available literature directly start with PBD and then go for statistical optimization. is your is a totally new topic then priliminary studies are to be done---> PBD -----> CCD
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My research is about LAB fermentation of shrimp waste for chitin recovery and what seems to be the advantages of cofermentation over that of successive fermentation of shrimp waste to extract chitin? 
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Cofermentation brings different traits that may be useful in fermentation
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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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A quicker method to quantify glycerol in a fermentation sample?
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I have used a few different enzyme kits including the one from sigma. They are not designed for fermentation samples, I have found them highly unreliable for such an application. With HPLC you need to have access to one with the correct column, and detector and if you don't have RI you need to derivative anyway. We have slightly modified the method in this link (below) to use with just a spectrophotometer set to 410 nm and after years of using other methods for glycerol determination this has by far proven the most reliable. If you have other non-glyceril elements in your media that will react with this method you will need HPLC but this should not be too likely.
I should add that this method is much cheaper than any kits.
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When we plot glucose consumption, ethanol production or growth curve in a fermentation process, most of the time we can not get a smooth line connecting the data points. I found some articles e.g.:
1. Alfenore et al. (2002) Appl. Microbiol. Biotechnol. 60:67–72
2. da Cruz et al. (2003) J. Inst. Brew. 109(4): 349–355
These authors seems to use trend line on their graph, since I noticed that the line are not exactly connecting the data points.
Is there anyone know that we may do that? if the answer is yes , can anyone suggest what regression used for those curve and refer me to any reference that mentioned this?
Thank you in advance.
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Not required.
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In this study we evaluated the optimal physical requirements such as the shaking rate and incubation time for applying in liquid media malt extract broth to obtain a high emestrin compound and its toxicity.
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Effect of shaking rate on the production of bio-active components are not fixed, It depends on the environmental factors. You perform a study with and without agitation so that you will conclude whether agitation is required or not. If it is required, then you try with shaking at low and high speeds.
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Representation of results in tables or by graphs? Now a days, graphs have been replaced by tables for representing enzyme activity. Which is the more appropriate method?
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There is no "best" method; it depends completely on what you want the reader to perceive. Do you want your reader to quickly grasp the range of results and the trends, or is it more important for the reader to be able to see the details, the exact values?
Think in terms of what the reader needs to fix in mind in order to understand the text, and equally importantly, to understand what comes next in the paper.
Finally, pay no attention to "nowadays" or to what is trendy. Just think about the reader and what will best help that reader to understand what you have done and why.
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We are conducting a design project where the production of Amevive is our topic. We realise that it was subsequently taken off the market due to it's ill effects on the patient. However, any help on processes developed circa 2004/2005 would be appreciated.
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Well, I do not see any reason for its withdrawl from the market, as it is strictly to be administered under medical supervision. I feel that it has more to do with the relation between Astellas, US and BIOGEN, since Astellas were getting it manufactured by BIOGEN and only marketing it..
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I'm working in production and optimization of pigmented fungi biomass (Gibberella/Monascus) in order to produce an alternative biocolorant. So what should I do to make it grow well?
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You can adjust the C:N ratio ( ( what agrowaste are you using) ; moisture content, add yeast extract for growth factors; inoculum age and size are among the few parameters that you can optimse for pigment production
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When yeast (C. tropicalis ) inoculated to the glucose and xylose medium at 2% rate separately it utilizes the sugars into ethanol and xylatol respectively at 12h but when the sugars are combine in a certain proportion in fermentation broth the yeast was unable to convert the xylose to xylatol but the glucose is conversion to ethanol is efficient one. But in the same fermentation condition the yeast utilize the xylose to xylatol after a long incubation period (more than 144h) but the efficiency was very poor. So can anybody suggest what will be the reason for the yeast behaves like this? What should be done to utilize both sugars in to their respective products efficiently in combined form?
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I never work with this type of yeast. But I assume the enzymes required for xylose metabolism are repressed when glucose present in the media. In other word, the yeast prefer glucose to be metabolized than xylose. Once the glucose exhausted it start to use xylose as their carbon source, but then it is already a long period of incubation, which mean that the yeast already started to loose its viability. Therefore the efficiency that you observed was also poor.
I will follow this discussion to get other answers..
Cheers...
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How do you monitor or quantify residual glycerol concentrations in fermentations? HPLC, enzymatic kits or other? What are we looking for primarily (accuracy, fast, cheap)?
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I am using HPLC with an Aminex HPX-87H column with RI detector. The mobile phase is 5mM with flow rate 0.6 ml /min. The column should maintained at 60C and the rt time for glycerol is 14.4 min. If you are using the auto-sample for HPLC i think it is best method to detect glycerol con up to 10ppm in the fermented samples
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It could be any enhancer or inhibitor or even culture conditions.
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If only focusing on process level did you try a range study of your process parameter? Thinking about DO, pH, Temp, maybe pCO2 in a screening design to see if these parameter alone might have an influence maybe in combination, after that I would be thinking about getting different feeds. Did you try sodium butyrate? Link: http://www.ncbi.nlm.nih.gov/pubmed/16080696
or try:
See you!
Jan
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Can we use a reducing sugar like Glucose or Lactose in a production medium for alpha amylase production where we use DNSA method to check the enzyme activity in which we detect reducing sugar as the final product of the enzyme?
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Glucose is the end-product of alpha-amylase action on glucose polymers. It is therefore a classic repressor of alpha-amylase production. I am not sure why you have chosen glucose as a carbon source for alpha-amylase production. The carbon source for producing high titres of alpha-amylase is usually a polymer of glucose, typically starch. If you use starch then it is acceptable to use the DNS method to assay alpha-amylase activity but you need to be aware that this method measures reducing ends. Dimers (such as maltose) and oligomers of glucose will also show a reaction with DNS. The use of lactose may or may not be a problem because although is not a substrate of alpha-amylase, the microorganism which you are fermenting for alpha-amylase may produce a lactase whose action will result in the release of glucose, thus interfering with the DNS assay. You should read up on catabolite repression to get backgrounding on why glucose is not a good substrate for the production of alpha-amylase.
Clem
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In bacterial physiology, lag phase is defined as the phase which is necessary for adaption of cells to new environment. During this phase the bacteria grow and the size increases; but the population density is almost constant.
In textbook, it is recommended to have at least 5% of inoculum to decrease the lag phase.
Now, my question is:
How the increase of inoculum quantity can decrease the time needed for adaption and growth of each bacterial cells?
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Hi Rasoul,
Basically there are two kinds of lag phases, as defined by Pirt (1975).
1- A true lag which is defined as a lag caused by factors such as change in physiology, change in nutrients, presence of inhibitors, spore germination, state of inoculum culture.
2- An apparent lag, where a fraction of the inoculated cells are deviding normally while another fraction is dying or simply not deviding.
The result in both cases is that your cell count is constant. therefore, in the 2nd case, increasing your inoculum size will lead to an increase of the deviding (active) cell fraction. In other words:
growth rate is defined as:
dX/dt = µX
if only a fraction of the cells are deviding, then:
dX/dt = µX*F; where F is the fraction of the active cell population and where µ*F in the apparent growth rate.
If you increase your X (inoculum size), then you F will tend to 1 and µ*F will tend to µ.
In microbiogical processes, inoculum size can be high, especially in the case of yeasts. In industrial processes for yeast production, where performances are required, the inoculum size can represent 40% in terms of v/v. In these cases, Inoculum has to be prepared and concentrated before adding it in the reactor.
You will find more details in Principles of microbe and cell cultivation (Pirt, 1975).
Hope it will help.
Amine
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If the cellulase determine at 67 FPU/ml, what is the amount of cellulase (?? FPU/ml: 20, 40?) needed to be used in 1% biomass enzymatic hydrolysis?
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Dear All,
Great day. I m really sorry for late respond.
Thank you all the answers provided.
Let take example:
cellulase activity was determined at 70 FPU/ml.
(followed Ghose's Method, 1987)
Q1.
2% substrate in 30ml working volume for 5 days (120 h). 30 FPU would be used,
then how many (ml / ul) of cellulase enzyme should add into above conditions (2% in 30ml)? Could show the way to calculate?
Q2.
If the B-glucosidase activity was determined at 2.8 U/ml (using 1% salicin as substrate).
How many (ml / ul) of B-glucosidase enzyme should add into above conditions (2% in 30 ml)
If cellulase to B-glucosidase ratio is 5:1
If cellulase to B-glucosidase ratio is 1:5
Could show the way to calculate?
I am novice in ligocellulose enzymatic study. Hence, seeking technical advice and your expertise.
Hope can hear from you guys soon.
Thank you in advance.
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I am working on production of acid metabolites in E.coli in which I have to compare the effect of various bases like NaOH, KOH and Ammonia etc. Could anybody please suggest the concentration of ammonia for neutralization? Thanks in advance.
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I usually use 15-20% ammonia solution
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Water and vapor inter conversion inside the filter in between the membranes is unclear to me. Can anybody help me out?
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Im sorry. May be I have understood wrong but a hydrophobic filter always allows little amount of water below its bubble point which can be considered as a paramenter to test. Please clarify what you have been explianing.
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I am going to perform protein determination on an oil palm biomass sample. Among the two methods, Lowry and Bradford, which one is more convenient? Can anyone share their experiences?
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Hi,
I am not sure which method is more convenient. This will I am going to do to down select them.
1. Which one will give you more accurate value
2. Which method is used more frequent in the literature. This will make your data can compare with other's.
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Lactic acid bacteria were isolated from ivorian fermented foods in the view to produce dried starter culture
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HI Amenan,
It was an industrial work , I did it for my previous company not meant for publication as the data was confidential, and we were looking to find a rootcause of a problem, like seaching for a bacteria at various stages of fermentation.
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How can the knowledge of biochemistry be helpful in explaining and propagating 'Bioprocess and Fermentation Technology'?
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In fermentation and in general in a bioprocess you use cells or part of cells (i.e. enzymes) to obtain the desired transformation. Biochemistry can thus give you the necessary knoweledge of how the transformation can work. In a way you change the "black box" to something more obvious
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I've tried incubation-like fermentation to cocoa bean. This method according to Biehl et al (1982) or Biehl et al. (1985). The cocoa bean incubation at medium acetic acid with duration about 24 hours.
How to make it simple?
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Miss Jinda, it'snt use some yeast. the cocoa beans just immersed on medium acid. actually, I'm so Interested with your Publication about cocoa fermentation. may I asked ones of it?? Thanks
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Acetate production in E.coli fermentations
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The best way is to use HPLC or GC (gas chromatography). GC has some advantages because of better selectivity: not many E. coli metabolites are volatile as acetic acid (do not forget to acidify the supernatant to covert acetate to free acid, otherwise acetate at pH 7 is NOT volatile). The protocols for both methods (HPLC and GC) are very well established, I can provide ref. If your lab does not have HPLC/GC, then you can try specialized colorimetric reactions (browse through the Sigma analytical part where numerous kits are listed for the price 100 to 500 USD.