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Microbial Fermentation - Science topic

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I am doing normal batch fermentation calculations (for the first time) and am confused about how to carry out some of the calculations. Specifically, I would like to know:
  • The equation for substrate uptake rate (g/L/h)
  • The equation for specific substrate uptake rate (g/g/h)
  • Whether it makes sense to do these calculations (in addition to yield and growth rate) for the overall batch or between each of the sample points
The calculation of specific/substrate uptake rate seems pretty easy from a dimensional analysis stand point. However, I am not sure whether to use the specific growth rate, μ (1/h), to account for the time dimension in calculating specific/substrate uptake rate or to use Δt instead (h). Each of them leads to different results it seems. I have seen answers that use either of them and now I am confused.
Any help would be highly appreciated.
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How to Identify and Estimate the Lactic acid from the Microbial fermented broth other than HPLC using Simple methods?
if possible such as
1. Calorimetric or Spectroscopic methods
and much more
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8/31/20
Dear Vignesh,
Another way to quantitate lactic acid in a fermentation is by measuring the amount of NH4OH required to maintain the fermentation at a pre-determined pH value. See the attached document. We did this when we conducted a fermentation workshop several years ago. We fermented glucose w/ Lactobacillus casei (homo-fermentative) and monitored glucose consumption and lactic acid production over time. Lactic acid was determined using an automated enzyme-based method (YSI....very expen$ive instrument!!) and by titrating the culture fluid w/ NH4OH to maintain the pH (either 4.5 or 5.0) to allow the cells to continue growing. We compared moles Lactic Acid (LA) determined enzymatically (YSI) vs. NH4OH titration. The attached graph shows that the correspondence of LA production by the 2 methods was very good. The NH4OH measurement method is not very elegant, but it works. If may work for you assuming that the microbe you are using produces ONLY lactic acid, and not other acids. We could do this w/ L. casei as it produces only LA. It won't work if your microbe is a MIXED ACID fermenter.
I hope this information helps you.
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu
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Is there any medium or visual method to determine the acid production of bacteria? if so, kindly send the literature and references.
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Agree with Rudy Situmeang
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Hello,
Can someone tell me how to increase the shelf life of pickles to 6 months to 1 year..?
Below are details
- Should not have artificial preservatives
- Packaging should be glass bottles
- Quality of the product should not deteriorate (Taste and authenticity should remain same)
- Cannot use Retort because it is deteriorating the taste of the product.
Thanks,
Balasubrahmanyam
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The Indian pickle already contains salts, sugar, oil and spices as basic ingredients. These ingredients act as preservatives itself, there is no need for the extra preservatives. Salts and sugar works on the principle of osmotic pressure on microbial cell wall result in disruption and spices acts as antimicrobial agents. The oil layer creates anaerobic conditions in pickles by reducing oxygen supply which may inhibit microbial growth. In addition, the shelf life of the pickles can be enhanced via refrigeration in airtight glass/plastic jar.
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Hi,
I want to analyse some fermentation samples for detection of Butanol, Ethanol, Acetone, Butyric acid, Acetic acid etc. using a TR-FAME (fatty acid methyl esters) column and GC-FID method. In this column no water based samples should be introduced as it may harm the column matrix. Commercially purchased analytical grade Acetone and Butanol gave fine peaks when analysed in this column. In this situation what is the procedure for preparing non-aqueous samples from the aqueous fermentation broth that will contain the produced acids and solvents to be analysed in GC?
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Thank you for your response.
I understand that HPLC-RID method is so far most widely used technique for this kind of analysis. However, in some columns injecting aqueous samples are not allowed due to the moisture sensitivity of the column matrix. For direct injection, can you name a suitable column that will allow aqueous fermentation sample and will analyse Volatile fatty acids and alcoholic solvents simultaneously?
Regards,
Shiladitya
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I have a protein of interest that is expressing in Pichia. It is expressing well in the shaker flask, but when I apply the same expression methodology but now in a bioreactor I get minimal expression.
Any logical reason for this? Is the density inhibiting the expression somehow. I would think that that more cell mass = more protein expression but could there be some kind of quorum inhibition by pichia?
There is room for more biomass production in the bioreactor, but I keep the feeding rate such that it does not go to the maximal limit to allow for protein synthesis.
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I understand how frustrating it is to grow yeasts and I hope that you have had some success by now. If not, perhaps my comments below may be helpful. I also do not know how much fermentation experience you have already, so my apologies if I am stating what may be obvious to you.
From my own experience growing yeasts and getting them to grow and express something, it might help if you focus on the following:
a) The pH profile.
The pH in a shake flask is uncontrolled - perhaps you can set up some flasks that express well and then take offline pH readings and product concentration readings periodically to have a better idea of what is happening?
b) The temperature profile.
I have found that yeasts are very sensitive to temperature. It appears that you have been able to get them to grow well in the bioreactor already which is great, but I would keep that in mind anyway.
c) The DO (dissolved oxygen) profile.
The DO is a shake flask is very low - perhaps that is what you need in the bioreactor?
Have you tried DO-stat feeding? That is where you have a DO setpoint and when the DO increases suddenly (a DO spike) then the feed pump turns on, delivers some feed and then turns off until the next DO spike.
d) You may need to have a growth phase DO, T and pH setpoint and then something completely different for the production phase. I have found it best when the cells remain yeast shaped and not filamentous.
e) The composition of your growth medium and your feed. Especially if you use yeast extract since there are many commercial yeast extract compositions. You may need to supplement your feed with something (ie not just a carbon source).
This involves a lot of patience and much trial & error. A design of experiments approach can be helpful - for example an eight run partial factorial design with 5 factors. But you should figure out which factors are important to your yeast and some idea of what the upper and lower settings should be first.
I wish you all the best and I am sorry that I did not see your question until now.
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Please researcher shear few research paper with Fermentation optimization techniques. Both In Silico such as predictive modeling and In Vitro such as substrate standardization.
I'm working with Bioflavour producing Microorganism thus need this help!!
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Dear Roy, please follow the article link about the
Microbial Fermentation and its optimization
technologyhttps://link.springer.com/article/10.1007/s002530000403
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I know about silicone and alcohol waxes.
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Hello Allan, your question was asked 5 years ago and I am surprised to see that it has not been addressed till now. I am just seeing it. It so happens we share similar research interest. Non-toxic antifoam reagents are mostly glycol-based. Antifoam and defoamer for fermentation is a non-toxic, non-silicone defoamer specially designed for fermentation process in distilleries.It is a combination of polyalkylene glycols and fatty acid esters.It is water dispersible and can be applied in natural state by aspersion or by automatic dosing pump. It shows excellent knock-down performance in the control of foam, improves the flow and reduces viscosity of the medium even at very low dosage without leaving residue. The shelf-life is long and therefore recommended.
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An oil rich substrate is not producing the expected result of reduction in ANFs after microbial fermentation. Could it mean that defatting or solvent extraction using hexane needs to be done first prior to solid state fermentation? Pls kindly add a published reference
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High oil or fat content can sometimes be detrimental to fermentation. But first of all, what do you mean by high fat or oil content? In some cases, certain microorganisms grow at high lipid contents because they are adapted to produce lipases, thereby allowing access to fatty acids. On the other hand, as mentioned by Mr Calt, a high content of oil in a fermenter can increase the viscosity of the system and consequently reduce the KLa negatively impacting the fermentation.
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What affects the protein quantity from an inclusion body during microbial fermentation? Are there any available resources to quantify the exact protein yield expected from inclusion bodies in E.coli?
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concentration of inducer, pH, time of induction, temp., all play a role. you have to optimized all parameter for getting high quantity of protein. thanks.
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I want to use MES buffer in my microbial fermentation media, The content of buffer will be also used by microbes or it is not usable?
It will needs to care about the buffer contents in media also or it cannot affect the media composition?
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It depends upon the microbe. Suppose the buffer is also action as carbon source then it will be utilised by microbe but quantity of buffer added will be low (volume, drop wise addition) which will be utilised in maintaining the pH rather than microbial growth. Also there is preferential growth in which microbes favours one source of nutrient over another like we choose our food over other being both are present in medium.
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is there any counting chamber with a grid so small to allow for bacteria counting with a 100x immersion objective? Burker and similars have 0,0025 sq. mm grid which can only be entirely visualized with a 40x objective. However, some cocci are hard to spot with 40x magnification, I need to go with a 100x.
Thank you all in advance
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I have used the petroff hauser countng chamber as well. 
They work well with oil immersion at 100x
Also Hawksely makes a nice bactera counting chamber too with a Thoma style grid. 
Best of luck!
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Pichia pastoris is fermented for recombination protein.
I set fermentation condition that DO value 50%.
I want reducing the pure oxygen rate, but maintain or increase DO value.
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In addition to all the answers above...
The DO in your fermentor will depend on the balance between oxygen input (gas-liquid transfer rate) and oxygen consumption by your yeast. If input is high and consumption is low, DO will increase and vise versa. Be aware that oxygen transfer rate is not the same as the volumetric oxygen input, most of the oxygen you will bubble into the fermenter will just espace to the headspace without dissolving into the liquid. Reducing the oxygen gas flow rate might even improve the dissolution of oxygen, if the reduced gas flow rate results in smaller bubbles.... So indeed, like proposed in the notes above, the trick is to improve the oxygen transfer rate QO2 which is a function of kla and the oxygen transfer driving force (=difference in oxygen concentration at liquid/gas surface and your intended 50% DO).
Options are (with increasing order of complexity):
- Reduce bubble size, mechanically by using a adapted gas injector (sintered metal or glass), increasing stirrrer speed, increase residence time of your bubbles in the fermentyer by using baffles, etc.
- Increasing oxygen pressure (this will not improve DO partial pressure in %, but will increase the dissolved oxygen concentration in mg/l)
- Change medium composition to improve specific oxygen transfer rate (kl) and/or bubble size a (e.g. by adding surfactants)
-Add oxygen in another way, e.g. by membrane units (either inserted in the fermentor or as an external unit)
Hope this helps. ;-)
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Someone has information on fermentation with abnormal development of volatile acids in the rehydration of stockfish?
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Nice
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I intend to evaluate the influence of the parameters inoculum ratio / must, fermentation time and temperature on the wine parameters, mainly ethanol. For this I need a same amount of sugars in the must to perform the experiments.
Could I do this by monitoring only soluble solids content (sucrose) in a refractometer, and occasionally adjusting with sugar, or would it be more convenient to monitor glucose, fructose and sucrose and try to fix values of these sugars for each sample?
can anybody help me?
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You need to do a lot more reading in terms of which sugars a preferentially fermented for as has already been mentioned sucrose is a disaccharide and will be fermented last because the yeast needs to excrete invertase in order to break the disaccharide into monosacharide unites of glucose and fructose.  These can then be fermented.  Also there is the need to understand the influence of the total sugar level for this affects the yeast's ability to ferment the must and hence the individual sugars. Refractometers are just too simplistic.  Don't equate the refractometer reading with sugars for it is sensitive to the effect of all soluble solids on the refractive index as well as the alsohol as it is fermented. Fine for industrial use but for research go with the HPLC.  Of course the level of sucrose in grape must is quite low anyway.  
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What might be the reasons for lesser yield of recombinant protein from Inclusion bodies during microbial fermentation? No change in process. 
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Something must have changed. 
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It would be helpful if I get an answer for calculating the exact amount of a oxidized compound after the reaction. For example if 5 mg of substance is mixed with an electrolyte. I need to know how much amount of substance oxidized during different cycles and different scan rate. Is there any protocol or methodology to find the quantity rather than getting the current values.
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I'm assuming that you have run CV and you know the reaction mechanism.  Then perhaps you can get the amount of material oxidzed from the integrated charge under the anodic curve, as #moles oxidzed = (Charge)/[(Farady const)x(# e)].  If you did not run CV then you'll have different way of obtaining the charge but the rest is the same! 
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I need to use 6M sulphuric acid as an electrolyte solution for cyclic voltammetry studies. I know 0.5 M or 1 M sulphuric acid can be used. What will happen if higher concentration of sulphuric acid used. Also during the course of reaction, will it affect the counter platinum electrode or reference silver electrode?please clarify the doubt or send me some related reference  papers
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Is to high concentration- May cause erosion. Also the high concentration of H+ has negative effect on cv. Hydrogen evolution is the main consequence
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Hi all,
I would like to study the effects of human intestinal bacterial on flavonoid, and some one suggested I use sheep's gut as the animal experimental model. Any one have some experiences about this, for example what's kind of medium do you use? Thanks a lot for your help.  
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Test rats
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I am trying to isolate fungal mutants with enhanced xylanase activity. What is the most appropriate inducer for the same which can be part of the medium.
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Hello.
There is a  important source of  xilose in nature. It´s   the plant topinambur ( Helianthus tuberosus), this plan contaín  inuline, a xilose polisacaride that  you can hidrolise whit enzymes (xilanase) and obtain xilose.
Best regards
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Could you suggest a method to give the effect of pressure on microbial growth? Kindly give the procedure or kindly give some reference article to follow?
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Hi Ramadoss,
This type reaction can be done in high pressure reactors. See the procedure followed in this journal http://amb-express.springeropen.com/articles/10.1186/s13568-014-0077-0
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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 want to produce a Bacteria that belongs to Bacillus species at large scale so can i use LB medium or is there any other suitable medium that can be used with ease. I need your valuable suggestions. Thanks 
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It depends upon for what purpose you are going to multiply the bacteria. If you are interested in only multiplying the bacteria then you can use LB media or any suitable growth media. But if your purpose is to obtain the products like extracellular enzymes ( you have mentioned fermentation so0 then you have to use specific media.
I hope this will help
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Hi everyone, I have a question regarding oxygen transfer in stirred tank bioreactor. I'm currently doing research regarding rocking-motion bag bioreactors. As I did my literature review, I noticed that stirred tank bioreactor is more widely used for microbial fermentation compared to rocking-motion bioreactor.
Many attribute such common application to its superior oxygen transfer performance which is critical for aerobic microbes cultivation. May I ask usually what kind of transfer coefficient (kLa) could a stirred tank achieve in laboratory scale, say 10-20 L? And if possible, can anyone tell me what is common oxygen transfer coefficient (kLa) in industrial scale, say 1000-2000 L? Thanks
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As indicated by mr Osadolar the actual value of kLa depends on many factors. The given value of 0.15 s-1 is realistic for water. In large scale reactors the value will be smaller as a result of reduced specific power input. A possible relation for kLa in water is 0.13esG0.4eG0.5, where esG is the gassed specific power draw of the impeller and eG is the specific power input by the gas. Both in W/kg and valid for small and large scale. During fermentations the value is in general higher, but may easily be lower e.g. when the viscosity increases during the process to above 20 mPas as a result of increasing biomass concentration. A rule of thumb for oxygen transfer at large scale is 1 g/m3s.
Your bag reactor will show also a dependency on power input, but the power draw is not easily determined. Mass transfer may be higher due to the formation of droplets. Droplets are very efficient for mass transfer.
Success!
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I have put up a fermentation experiment using yeast cultures. I am using synthetic sugars for the process and for detection of bioethanol produced at the end of the process, I would like to prefer Gas Chromatography. But DNS test of my culture supernatants suggest that there is some sugar left in them. Can I use these supernatants directly for GC analysis or do I need to pretreat them to precipitate out the sugars?
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Hi Shalley,
The best, to my opinion, is to measure ethanol with an enzymatic assay based on ethanol oxidase that produces H2O2 from ethanol, then hydrogen peroxide is further oxidized to dioxygen by horseradish peroxidase and amplex red substrates (i.e. N-caetyl-rsorufin) is stoechiametrically transformed in resorufin (both a chomophore absorbing at 570 nm), and a fluorophore excited at 535 nm elmitting at 585 nm).
SIGMA-ALDRICH sells this kit under the reference MAK076 (400 euros for 100 assays).
Best regards,
Philippe
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During my storage study with a particular type of fermented food product samples, the count of lactic acid bacteria was found to increase with storage time, which would reduce the proliferation of pathogen. However at the same time in one of the samples, after six weeks of storage Staphylococcus spp was observed to be growing and reached to a maximum at the end of storage time. What might be the possible reason?
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Staphylococci can never grow in media with a low pH sich (no growth bellow pH 4.4). But it would depend on the nature of your samlpe. In meat products staphylococci can grow especially S. xylosus  S. simulans and S. carnosus. In liquid dairy products staphylococci cannot grow because of the acidity and bacteriocins production but in cheeses some staphylococci can grow or survive at the suraface. In cereals based foods rice, soya beans etc...) Staphylocci can grow easily even if the pH is low. In olives and vegetables fermentations Staphylococci cannot grow. Please tell us the nature of your fermented food for we can help you more. All the answers from the colleagues given here are true and you might get the idea about your problem.
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Where can I find good readings on production of PHAs from Gram positive bacteria?specially actinomycetes
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I grew C.butyricum in flask RCM medium, and it grew up to OD620=2 in 24h.
I transferred 100ml culture into 2L glycerol medium containing no yeast extract (start OD=0.2 after innoculation), and it can grow up to OD620=0.7.
However, the problem is that glycerol concentration was not decreased at all for several days (pH6.5, 37C, 250rpm).
I guess they grew using RCM medium (100ml) and dont use glycerol.
According to several papers, they can consume glycerol rapidly.
What do you think about this problem? and what can I do?
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This is difficult to predict, the OD value depends on the morphology of your organism, the wavelength used for measurement, the cell concentration an other factors. As your strain ferments, it likely makes only low biomass amounts, but rather forms acids as fermentation products, so OD could be low, despite glycerol is completely gone. you might check the pH, as medium acidification could also lead to incomplete growth and the phosphate buffer does not efficiently buffer below pH 6 anymore. check, if glycerol is left so see, if grwoth was complete.
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I am performing co-fermentation experiment with glucose and xylose and to determine the residual sugar concentration I generally prefer HPLC method but HPLC is not in work for a few days. Can anyone suggest me a way to estimate glucose and xylose concentration separately because I want to see how much xylose has been consumed ?
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A alternative way will be mesuare the reducing sugars by DNS method (for exemple), and the glucose by enzymatic kit (GOD-POD). By difference you will know the quantity of xylose. 
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I would appreciate if you could share your ideas on this.
Regards
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peptone fermenting anaerobic organisms often produce H2S. P aeruginosa is a facultative anaerobe that may produce silfide from cysteine. Then conc of sulfide should in the same range as cysteine. If it is much lower - then sulfide may originate from S-compounds in yest extract.
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One day I visited the scale up section of a fermentation plant. They were talking about the DOI. What I think is productivity may increase to some extent but there may be chances of oxygen toxicity. Is there any chances of oxygen toxicity?
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This will depend on the relative amount of the oxygen added to the medium in addition to the mixing efficiency. It is not recommended to increase oxygen tension (concentration) beyond 80 to 100 % of saturation in order to prevent oxidation reactions or toxicity 
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I'm currently conducting research on the production of xylanase, pectinase and protease via solid state fermentation. The crude enzyme I harvested from each set of SSF is my primary source of each enzyme I mentioned earlier.
From my literature review it is okay to use sodium acetate buffer for the extraction of xylanase and pectinase. How about protease? does sodium acetate buffer suitable for the leaching out of protease from the SSF?
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Since most of the pectinase work in acidic pH therefore the extraction buffer used is generally citrate or acetate. Protease can be neutral, acidic or alkaline. Based on type of protease secreted by your fungi during SSF, you need to choose the buffer system. When I was doing my Ph.D. even physiological saline or 1% Tween-80/20 used to be sufficient for leaching out protease from the SSF... 
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is it possible to perform a mixed culture between Pseudomonas aeruginosa or Acinetobacter baumannii  and a yeast?  how it is possible to monitor mixed culture and to shift the favorable conditions to a yeast without causing the death for Pseudomonas aeruginosa in the same culture?
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You may wish to have a look at these two publication. You won't find a recipe for your intentions, but a lot of helpful hints.
1. Prokaryote–eukaryote interactions identified by using Caenorhabditis elegans
Anton Y. Peleg, Emmanouil Tampakakis, Beth Burgwyn Fuchs, George M. Eliopoulos, Robert C. Moellering, Jr., Eleftherios Mylonakis
PNAS September 23, 2008 vol. 105 no. 38 14585–14590
*2. INFECTION AND IMMUNITY, Aug. 2009, p. 3150–3160 Vol. 77, No. 8
The Acinetobacter baumannii 19606 OmpA Protein Plays a Role in Biofilm Formation on Abiotic Surfaces and in the Interaction of This Pathogen with Eukaryotic Cells
Jennifer A. Gaddy, Andrew P. Tomaras, Luis A. Actis
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Would you please help me with a laboratory protocol for pectin determination in fermentation broth?
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Dear Eloane,
The content of galacturonic acid could determine photometrically. One actual publications is attached. I haven´t checked this protocol at this time, but I´m going to do it in autumn 2015.
Kindest regards
Telse
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During recombinant protein expression (RPE) cellular/metabolic stress is developed inside E.coli, which then depresses the RPE over time.
Over the past few decades, several genes have been identified which can be used as a biomarker or probe to monitor this stress e.g. ppGpp, whose expression level increases with increased oxidative stress developed inside cell. Like this there are various stresses e.g. heat stress, osmotic stress etc. which are well correlated with the RPE.
I am trying to catalogue all those genes which can be used to monitor different type of stresses developed during RPE in E.coli.
Your valuable suggestions, ideas or Paper references will be well appreciated.
Regards
Ashish
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In E. coli infact this is a challenge for RPE gene. Note normally heat stress or osmotic stress do not give satisfactory results. You will get good results if you use oxidative stress.
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The innoculum sizes for starter culture (controlled fermentation) seem to vary. How is it dependent on the organism being used or is there a minimum quantity that will ensure that the desired organism is responsible for most metabolic transformations.
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Since you have not mentioned the fermentaion system and there is no generalize rule but its logic goes in the following way.
Inoculum % depends on time at which reaches log/exponential phase and shaking
1) Aerobic microbes uses oxygen for growth/final electron acceptor producing high amount of ATP hence growth rate is high. Hence you need a lower number of cell at start point (inoculum) to reached a desired number at a particular time (remember time at which you want desired growth is also imp,) .
   So around 1% to 2% of inoculum is generally good enough for aerobic microbes. for e.g E.coli  for 16 hrs in LB broth for 16 hr gives O.D of 5-7
2) Obligate Anerobic fermentative microbes produces less ATP, so require higher number of microbes in starting inoculum to reached a particular number in particular time,
      So around 10% to 15% used. E.g Clostridium acetobutylicum for 24-36 hr in TYA medium give O.D 5-7.
3) Facultative anerobic microbes can grow at intermediate oxygen level and mid % of inoculum is needed
       So around 5% to 10% is good enough. E.g yeast for 24 hrs at  70 rpm in MGYP medium gives OD of 7-8
4) mixed consorita of anerobes requires more inoculum % because of different growth of different microbes present.
        So around 10 to 15%. E.g Methanogenic consortia takes 20 days in Msp medium to reach O.D of 2-3.
5) Anerobic respiration. Depending on final electron exceptor number of ATP produce varies. I have no persional experience so cant help you with that but initial inoculum % should be around 5 to 10%.
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Solaris Biotechnology srl is a sme located in Porto Mantovano (Italy), available for collaboration in EU research projects (i.e. development of microbial fermentation processes at lab and industrial scale, downstream processes, photobioreactors development for algae cultivation).
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Dear Radhouane Kammon,
thank you for the message. Are you praparing some EU proposals about LCE-12-2015?
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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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When a microaerophilic microbe is used for the production of PHA and glycerol is used as the sole carbon source what would be the risk of contamination in doing an open batch fermentation process, Mineral medium is the base media where in trace element solution is added . can there be contamination with the addition of trace elements
I just tried an unsterile fermentation mode and I could produce PHA but the production is comparatively very less than that of sterile fermentation conditions.
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The controlled fermentation requires sterile conditions including the medium. In all these fermentations, when working under non sterile conditions the yield is low, to escape this situation you have to work in sterile conditions. This is not a proof that your low yield is due to the contamination, it could be due to other factors.
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Solaris Biotechnology srl is a sme located in Porto Mantovano (Italy), available for collaboration in EU research projects (i.e. development of microbial fermentation processes at lab and industrial scale and downstream processes).
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Dear Daniele Spinelli.,
We are looking for this type of collaboration, and we are interesting, Presently We have one Research foundation, Peptides , Proteins and bio products, we are doing.,
I Will Contact Soonest..,
Regards R.Selva....
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Dear all,
I would like to produce lactic acid via the fermentation route, mostly simultaneous saccharification and fermentation (SSF).
I am looking bacterial strain which will give the highest lactic acid yield at optimal SSF conditions.
Any suggestions?
Buy from ATCC? 
Who is willing to share his/her lactic acid bacteria strain?
We may set up a research collaboration.
Thank you.
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Lactobacillus spp.:
L. acidophilus
L. bulgaricus,
L. plantarum,
L. caret,
L. pentoaceticus,
L brevis and
L. thermophilus
The above lactic acid bacteria (LAB) can be used for lactic acid production. I suggest you to perform extensive literature review on lactic acid production for a bacterial strain yielding highest lactic acid.
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Bio-transformations of natural and synthetic compounds are performed by using a large range of bacteria and fungi.
Can I use a magnetic stirrer for water insoluble compounds; or should I use emulsifying agents for this purpose?
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I have worked with bioconversion of Progesterone to 11-alpha-hydroxyprogesterone using fungal strains. I used a low concentration of solvent that was tolerated by fungal strains to improve substrate solubility and used a magnetic stirrer to keep the substrate dispersed (I used a 5 Lt Schott bottle as a mini bioreactor in absence of actual bioreactor). Using micronized substrate powder also helped. If you are using a small bioreactor, there should be no problem of keeping substrate dispersed. Uptake will definitely be an issue as others have already indicated, but dispersing or emulsifying agents can help. The challenges could be also in analytical measurements to obtain representative samples. Type of microbial strain you need to use for bioconversion will also be an important issue. I would prefer to use a small bioreactor than shake flasks, if you can avail the facility though. Give it a go, and I am sure you will work it out as I did.
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I am interested in this issue, regarding the checking of protein concentration in fermentation sample.
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Protein concentration check is the necessary part because if you are working on an enzyme and you are chking it for maximal production then u have to optimize both the protein and activity simultaneously by calculating the specific activity of ur fermentation broth sample. It is needed coz if u have thinking of getting a purify product from that broth then u must go to do all purification steps which means there will be loss of proteins during ur purification processes. So to minimize heavy loss of protein you have to chk the first fermentation broth for higher specific activity so as at the end u will also get a good protein having good activity and potentiality. Hope I answered ur question. All d best.
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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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A. niger can produce large number of organic acids. Certain time i would like A. niger to prduce Specific acid more. Example, if my interest is citric acid, then what parameter will help me to get more citric acid than other organic acid like gluconic acid, oxalic acid, etc.
(Under submerge Condition)
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for production purpose it is better to keep pH around 3 that decreases risk of contamination
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How can we estimate the acetic and formic acid content in a solution using a UV-Vis spectrophotometer? If anyone has the protocol, kindly provide it as some protocols I have found on the internet are not working. Thanks.
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If you decided to determine acetic and formic acids via UV, you have to measure the absorbtion of the total of acids at 220nm, in the first step. Then submit an aliquot of the sample to oxidation of formic acid by HgO in a boiling water bath in a closed vial. Cool the oxidized sample and filter /or centrifuge/ the suspension. Measure UV spectrum of the oxidized sample. The absorption maximum of the oxidized sample should be proportionally reduced due to removal of HCOOH and would corespond to content of CH3COOH. From the difference between the both spectra /total - CH3COOH/ you can detrmine formic acid.
I would prefer to detrmine both acids by GLC on a Chromosorb 102 column, detection with TCD. Temperature 140 - 150 oC, H2 or He as a carrier gas.
Or I would use conductometric alkalimetric titration.
Note: if your sample represents H2O extract from some biologic sample, the UV spectra might not give you correct data due to acids interferrence /overlapping// with some other watersoluble extractives.....
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I'm seeking for a gene that can be used to differentiate between C. ljungdahlii, C. autoethanogenum and C. ragsdalei. As far as I know from whole genome sequencing data, they have identical 16S and 23S genes. ITS regions also vary within the multiple rRNA operons those cells have so it is not a good choice.
I checked complete genome sequences for autoethanogenum and ljungdahlii to find candidate genes to differentiate among them, but could not find acces to ragsdalei data apart from the 16S rRNA gene.
I'm also having a similar problem with C. carboxidivorans and C. scatologenes.
Any help will be welcome.
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C. ljungdahlii and C. autoethanogenum are basically the same organisms the second carries multiple deletions in the chromosome. I don't know about C. ragsdalei but you may want to check the chaperonin 60 (cpn60) sequences
Hill et al 2004 cpnDB: a chaperonin sequence database. Genome Res 14: 1669–1675.
Jian et al 2001. Int J Syst Evol Microbiol 51:1633–1638.
Verbeke et al 2011 Syst Appl Microbiol 34: 171–179.
Goh 1997 J Clin Microbiol 35: 3116–3121.
Suerte
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I am writing to ask about a problem that has been happened in cultivation of a microbial strain, lactobacillus Bulgaricus.
I used to culture this microorganism in a 10litere bioreactor and reach a certain optical density but after my previous working banks finished, I made another series of working bank from the same mother stock. These new working banks are not as efficient as my previous working bank. Although the inoculums which are prepared by culturing the working bank growth well as usual, the final optical density in bioreactor has decrease to a quarter. For finding the reason of the problem I repeated this procedure twice and the result was the same. Even using direct inoculums from another mother bank that had not been thawed before, could not solve this problem and the final optical density was not as high as before. The fermentation conditions and medium ingredients were the same throughout all the experiments. I am looking for the reason of this problem and want to find out what was happened to these cells.
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You have to go back to the mother culture and streak it on MRS plates to obtain many colonies. Then try randomly to pick around 20 separated colonies and start prepare different inoculations. Most probably that some cells within the mother culture mutate for some reason which resulted in two types of cells which can ferment different carbon types. In lactic acid bacteria this can happen. Another suggestion is maybe the glucose or lactose fermentation is controlled by some genes located on plasmid. The bacteria may loss this plasmid during storage or when transferred from medium to another
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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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I want to study the effect of temperature on growth of pseudomonas aeruginosa in solid state fermentation, so I want to determine the growth of biomass at the end of fermentation period at each temperature. The production of CO2, during the fermentation, is not enough to be sensed by sensor. How can I determine the biomass?
The bio reactor is a vessel like a Graduated cylinder with volume 50 cc. I put an air distributor at the bottom of the Graduated cylinder and an effluent on the head that is connected to the sensor of CO2. By the way, because of temperature gradient that occur in greater bio reactors, I can not use greater one to reach much CO2 Concentration.
I put about 10 grams mixture of Corn bran+Corn germ in the bioreactor,
this substrate mixture have protein and it is not possible to use protein test to estimate biomass.
who knows what else substrates can I use that is tested before for pseudomonas aeruginosa to produce rhamnolipid?
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Given the fact that the the CO2 amount is too low to be measured, it is very unlikely that methods such as C or protein measurements could give significant result : if there is "no" CO2, there is "no" biomass, and the background "noise" due to the substrate will probably be much higher than the biomass value.
I would stay with CO2 determination : instead of feeding the fermenter continuously with fresh air, use a small pump to recycle the gaz, after making sure that the whole setup (for which you gave no clue...) is airtight. The CO2 will accumulate and be of course easier to measure. The drop of the O2 concentration should be also measured and the respiratory quotient calculated.
I have use this trick to detect the tiny CO2 production during vinegar fermentation.
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I need the procedure to standardize it in the laboratory for practice.
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The attached article indicated the use of tertiary amine for recovery of another organic acid, lactic acid.
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I would like to know how to calculate KLa in a flask and in baffled flask?
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I am using the PreSens system having a pH and a DO probe inside my shaker flasks, that way you can get all data needed. Maybe they can give you a test system for you to figure out if it suites you Link: http://www.presens.de/products/brochures/category/sensor-probes/brochure/non-invasive-oxygen-sensors.html
One more articel:
Hope this helps you CU Jan
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Microorganisms involve usually in spontaneous fermentation belong mainly lactic acid bacteria and yeast. Production of African traditional cereal alcoholic beverages need two obligatory spontaneous fermentations steps: a lactic acid fermentation which is carried out by a complex population of environmental microorganisms and an alcoholic fermentation.
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Microorganisms involve usually in African traditonal beverages processing belong mainly lactic acid bacteria and yeast which govern spontaneous fermentations.
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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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For calculation of the generations in broth medium, I determine the initial number of bacteria and then the final number of them in a given time. Afterwards, by using the formula, I can determine the generations.
Now I would like to know the method to determine the number of generation in a solid culture medium.
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I think it is not possible to do this determination in solid medium. For achieved this you should count the number of bacteria every hour on the plate for example. Maybe taking a picture, but you would have much error accompanied, even if to make a densitometry analysis of the photo, and you would have to do a calibration curve to know how many cells correspond to a pixel for example. I work with yeast and never saw that parameter determined in plates. Also, the generation time is generally measured in the exponential growth phase, in this phase the cell growth is constant, as well as the duplication of cells. In solid medium cells rapidly deplete their immediate resources and one of the criteria in the exponential phase growth is that none of the nutrients are limited.
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Please let me know your opinions on which approach is better (pro and con), if one would like to investigate microbial populations (bacterial, and/or fungal if possible) in plant leaves fermentation processes over a time period (some months).
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Thanks Steve, I will look closely at the link you posted...
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Antifoam concentration and it´s effects on growth of E.coli.
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harvest might go up to 100g/L after centrifugation. And we use 0.5 % antifoam 204
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I am growing e.coli in fermenter with glucose in minimal media at pH 7.4 (50mM SPB). Technically, first D.O. should decrease (to nearly zero) due to cell growth, and then it should increase, due to optimum growth. But, in my case, D.O. is decreasing up to 6-7% and remaining unchanged till end. Although growth and other vital parameters are normal, pH is decreasing and O.D. at 600 is increasing properly. I can't understand what is happening?
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You mentioned growing e.coli in fermentor but did not describe how you operated the fermentor system while growing this e.coli.
Did you just run the fermentor at constant airflow and agitation?.
Did you notice a change in these two parameters toward the end of the run?.
Did you have a chance to check the function of your D.O electrode to see if the electrode is still responding properly to air/O2?.
Other than that, unless the culture pH dropped to growth inhibitory point, as you mentioned; cell OD still going up, this indicated the culture is still in a O2 demanding mode and not in a stationary or late stationary stage of growth where you will see oxygen level goes back up due to the supply oxygen not being consumed by the culture.
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Usually, detection of hydrogen after fermentation process is use GC, HPLC, gas meter, etc. Do you think is there any methods which can detect microbial community which can produce hydrogen only base on the using of medium?
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I suggest using a pair of redox electrodes - platinum and titanium-silicate. The difference between these electrode's reading is indicating for hydrogen production by microbial community under fermentation conditions. There are many papers on this topic.
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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.