Fermentation Technology - Science topic

Kuhner AG is the best option for measurement, which provides appropriate results

Thank you so much sir.

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

Dear Sajjeev Jagannathan

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.

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

Your are welcome Priyanka Roy

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.

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.

Please have a look at this useful ResearchGate link.

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.

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!! 

Databases like KEGG or MetaCyc can be useful for metabolic pathways, here is the link to KEGG pathway map for C. butyricum.

 Thank you, Mr. Michael. how about the number of electron and hydrogen produced? any big different between aerobic and anaerobic condition?

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

The method Bill suggests is a good one.  Here is another paper that compares different methods for reducing sugars.

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

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!

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 .

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

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 

I agree with Marius K. Somda answers .

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.

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!

 I think these will be helpful to you

regards

this book will be useful 

Regards

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?

you can write to him, this is email: (kvera@correo.uady.mx), this outhor have many paper about of measure methano 

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

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.

Which clostridium species gives the highest concentration of butanol from the broth? 

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

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

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

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

I have not, but John Robyt and his coworkers have done so. Please see the attached paper.

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!

Thank you Carlos, i sent a msm to Dr Nemecio.

Best Regards

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 ???

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,

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.

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.

Keeping positive pressure inside the bioreactor system may help minimize the contamination issues.

You cannot use the colorimeter method in this case. You can go with plate count method or protein estimation method for bacteria growth.

Hi Jai,

Thank you for the reply, but the point is HB 2151 cannot be used for the large scale expression.

 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.

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. 

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.

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.

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.

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

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.

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.

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. 

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

Cofermentation brings different traits that may be useful in fermentation

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.

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.

 http://www.sielc.com/Application-HPLC-Derivatization-and-Quantitative-Analysis-of-Glycerol.html 

Not required.

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.

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.

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..

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

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...

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

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

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

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

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.

I usually use 15-20% ammonia solution

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.

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.

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.

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

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

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.