- Gurunathan Selvakumar added an answer:99+A quicker method to to quantify glucose in a fermentation sample?I am working on a bioethanol project and our HPLC is always in use. Is there another, quicker method to quantify the amounts of glucose in a sample.
I am using GODPOD reagent for determining the D-Glucose. I am doing this determination with food stuffs. Food stuffs are giving high amount of glucose. For that, how to increase the linearity level of the GODPOD reagent to determine the high concentration of glucose without more dilutions of sample solution...???Following
- Charles Rashama added an answer:4Does anyone know about wood-based sugars fermentation?
Anyone with links to publications on fermentation of wood based sugars? Specifically I am looking at sugars generated by autohydrolysis during medium density fibreboard manufacturing?
Thank you guysFollowing
- Aparna Gunjal added an answer:10Is anybody working on value addition of Protein by-products interested to collaborate in this Edited Book Chapter Invitation (Elsevier)?
I would like to invite you to contribute a book chapters
1. Value addition of waste derived proteins to biofuels and biochemicals
2. Rendering industry wastes- transformation to high value products
3. Application of waste-derived proteins in animal Feed Industry
In: Book entitled “Protein by-products: Transformation from environmental burden into value-added products” 1st edition By ELSEVIER publishers, Editor- GS Dhilon, PhD., P. Biol. (ASPB).
=> The preliminary acceptance is only upon submission of the draft abstract.
=> The book will be possibly published in second quarter of 2016.
=> There is no processing/publication fee
=> There are no page charges for black and white figures and illustrations submissions provided they are sent to Elsevier with the source files. Color illustrations are billed at the current rate.
=> An author guidelines will be sent to all authors after acceptance of the abstracts
=> In consideration of your contribution to this book, Elsevier will provide to all authors an electronic edition of the book, besides giving a 30% discount on all Elsevier Science books for life.
Please text me within 2 weeks if you are interested.
GS Dhillon, PhD., P.Biol (ASPB)
I would like to contribute chapter in the above mentioned book chapter of protein by-products. I would like to know till wnen can we likely submit the abstract and what is the word limit of abstract??Following
- Manuel Serrat added an answer:4Can someone suggest a pH 3-4 buffer system for yeast shake flask culture?
In bioreactors, I maintain a low pH via combination of cell metabolism and base addition. In shake flask, base addition is not possible, and I've been looking for a suitable, biologically compatible buffer system for a pH between 3 and 4. Most biological buffers are designed for much higher pH ranges.
I've tried sodium citrate/citric acid, but my cells grow poorly and lyse.
Any suggestions? I plan to try a glycine-HCl system, but I'm not too confident about it, the HCl doesn't sound cell-culture friendly.
I have had very good results with 25-50 mM citrate buffer. Urea as nitrogen source is a good option too.Following
- 7How can I calculate specific consumption/production rates for a yeast cultivation?
I'm having some difficulties in calculating specific consumption rates from the data I generate from my cultivations with the yeast Pichia stipitis.
Let's consider the specific substrate consumption rate with these data:
TIME of inoculation (0 h)
- 9.66g of substrate present in the medium
- inoculum corresponds to around 0.25 gDW
TIME 13.25 h
- 0.94 g of substrate left in the medium
- biomass concentration is 5.3 gDW
First of all I would like to know if the formula I'm using is correct:
qs = g of substrate consumed / (time * g DryWeight)
Here's how I do it:
qs = (9.66 g - 0.94 g) / (13.25 h * 5.3gDW) = 8.72 / 70.2 = 0.124 g g-1 h-1
Now, what I would also like is to express the result on a C-mmole base. To do that I convert the grams of substrate in mmoles and then I multiply by 6 (substrate is mannitol, C6H14O6, MW 182.2 g/mol). So:
- 8.72 g of mannitol = 47.85 mmol * 6 = 287.2 C-mmol,
- qs = 287.2 / (13.25 h * 5.3gDW) = 4.08 C-mmol g-1 h-1
I'm not so sure these is all correct since these numbers seem to be on a totally different order of magnitude from the data I find in literature.
Thanks a lot,
It is very well answered by Habibollah.Following
- Nizar Matar added an answer:5How can I get Poly lactic acid of high molecular weight ?
I have tried to increase molecular weight of poly lactic acid of lower molecular weight by chain extension with di dissociates . It give me weight average molecular weight of 200000 Da. I would like to know does there is any other method to get ultra high molecular weight PLA.
In the following paper, high molecular weight PLA was prepared by ring opening polymerization using a chain extender (which is hexamethylene diisocyanate). My regards.Following
- Greg Cote added an answer:4How can I explain different calculation substrate in the method of DNS, when calculation DE value?
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
I have not, but John Robyt and his coworkers have done so. Please see the attached paper.Following
- 6Is there any quick and inexpensive way of estimating residual glucose and xylose concentration during fermentation apart from DNS ?
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 ?
Immobilized enzyme like the respective oxidase in the form of a suitable electrode.Following
- 1How can I determine adsorption capacity or saturation point of a bio-filter medium (such as wood shavings) used for syngas cleaning?
i am using biomass materials to clean syngas (tar removal) generated from downdraft gasifier and filter medium is connected at the outside.i also want to predict and figure out how long such mediums can be used for the purpose. longer the saturation point, better the filter medium.
Do not try to design yourself such biofilters. These are readily available in the market and has been designed with lots of care and repetition. They come with the adsorption capacity mentioned on the labels.Following
- Timothy L. Turner added an answer:8How can I buffer a YNB medium for growing yeasts?
when I culture my yeast for more than 40 h it stops growing even if there is stil a lot of sugar left in the medium; when I measured the pH I noticed that it was 2.7.
So, in the hypothesis that growth stops only due to excessive acidification I would like to buffer my medium. I'm using: YNB (1.7 g/L), (NH4)2SO4 (5 g/L), CSM (0.8 g/L) and I have 20 g/L of sugar as only carbon source.
My questions are:
- Can I add a buffer to my medium preparation? Can I autoclave it together with the base medium or should I make a stock buffer separately?
- And if so, what buffer should I use? I don't want to use citrate or succinate because they could be use by the yeast as carbon/energy source and therefore alter the experiment. Would PES be a good choice?
- How do I choose the concentration of my buffer?
Thank you all in advance,
It seems that several others have suggested good buffers for you to use. I wanted to mention, as an alternative to using a buffer, you could try adding a neutralizing agent such as calcium carbonate or calcium hydroxide. Neither of these will fully dissolve in the media, so you will not be able to easily separate your cells from the neutralizing agent - this inhibits your ability to measure cell concentration. But, in general, addition of either of these neutralizing agents should work quite well for maintaining the pH above 2.7 in addition to neutralizing any produced acids. You can use a non-limiting amount in both cases - how much depends on your specific fermentation, but I can say that 35 g/L final concentration of either neutralizing agent should be more than enough for a 20 g/L sugar fermentation - you can probably reduce the concentration of the neutralizing agents quite a bit below the 35 g/L amount, but it is a good starting point to ensure an excess amount.
Using these powders may not be ideal and I would probably consider the buffers suggested above first, but I wanted to bring this to your attention in case you have some issues with the buffers.
Best of luck,
- Aldo Amaro Reyes added an answer:8Can anybody help me with my pullulase activity assay from fermented broth of fungi using DNS method ?
I performed pullulase activity from fermented broth of fungi, by using DNS method, but each time I got the reverse result. Can any one suggest me why?
0.5 ml filterate+ 0.5ml Pullulan incubated for 15 min at 300C. 1 ml DNS Added, then heated in water bath for 10 min . volume made up to 5 ml by distilled water. taken OD at 570 nm or 540 nm.
Substrate is pullullan and product is glucose
Measuring product conc. using DNS
Result obtained each time
0 hrs 0.150
24 hrs 0.127
48 hrs 0.106
72 hrs 0.112
96 hrs 0.114
Try raising the temperature of the assay to 50 °C. Most fungal hydrolytic enzymes act around 50 °C.Following
- Ghada Abd-Elmonsef Mahmoud added an answer:4Can we consider certain vitamins such as Riboflavin, vitamin B12 etc. as secondary metabolites?
I learned that primary metabolites are produced by Microbes in log phase and secondary metabolites are produced in stationary phase. Riboflavin is produced by Ashbya gossipii in stationary phase (i.e., when organism ceases to grow in the absence of glucose). In this case, riboflavin can be considered as a secondary metabolite. But, in many text book, it has been written that vitamins are primary metabolites. Is it context dependent? ...Is it secondary or primary?
riboflavin called pseudo secondary metabolite as it needed in small amounts in the for microbial growth and its produced in stationary phaseFollowing
- Nuresh Manukonda added an answer:10Why does the pH rise during fermentation with Pichia pastoris MutS?
I have a P. pastoris MutS phenotype which express very well in shaking flasks. I was trying to scale up the culture in a fermentor.
The culture was behaving normally for most of the fermentation process, but as soon as I started the methanol feeding (after a DO spike to make sure all the glycerol was consumed) the pH started to rise and the acid pump worked for the whole methanol feeding process.
I took samples at different time intervals and check them for protein expression. What I noticed is that the protein started to express as soon as 3 hours after the induction, then the expression increased in the next hours to a peak expression at 9 hours of induction. After that the cells were expressing less and less until the end of the fermentation.
Comparing the expression level of the peak in the fermentor (9 hours) and the expression in flasks for 48h, in the fermentor I anyway got 5x less protein than flasks.
Do you have any idea how to improve the expression?
Why the pH of the culture was increasing during methanol feeding?
Decrease in protein expression, could possibly due to no more nitrogen source in the medium?
The medium I used was the "Basal salt medium" as described by the invitrogen guidelines and the only nitrogen source was the ammonium hydroxide also used to keep the pH at 5.0
Thank you very much for your help,
pH will decrease normally in fermentation while feeding a carbon source (glycerol/methanol), the catabolic activity leads to acid production. But in this case the pH is increasing i.e, the cells were not able to use the methanol and it's toxic effect kills the cells and pH will raise.
I can make some points to solve this problem.
All fermenter designs are not same so find out the air flow and rpm that is being used are sufficient or not. Using excess air & rpm also not good.
Glycerol feed must be controled to create carbon limiting condetions, try to keep 40% above DO by feeding the glycerol.
glycerol fedbatch is also show it's impact on induction so the feed rate(50%glycerol) should be around 5mL/L/hr.
After reaching the desire OD wait for DO spike.
Methanol must be supplied with PTM salts, make sure the concentration of PTM salts, methanol alon can not be used by the cell.
Check the OD for every 3hrs during adaptaion time 3-5hrs it will reduced and after that the OD should increase.
Check the DO it takes time to respond, after 5hrs you can see the DO response.
It is better to add 1% final concentration methanol and wait till DO spike. Then u can add it based on DO. It should be above 40%.
Improving expression is possible only if it consumes methanol and DO response.
You can try improving expression by doing the follwing
Optimize the OD for induction 300-400 is good, you need to increase glycerol fedbatch to reach this OD.
Make sure you product stability some are stable at low pH and some are stable at neutral pH. The expression will improve with the stability of your product.
Reducing temperature is also another option to control host cell proteins in case of extracellular protein.
Adding nitrogen sources like ammonium hydroxide, ammonium chloride, ammonium sulphate, arginen and cassamino acids.
If you are using ammonium hydroxide you need some additional nitrogen source or add additional ammonium hydroxide and neutralise it with acid during induction.
All the best.Following
- Fatemah Allam added an answer:5What is the mechanism of DYE REMOVAL from a sample by a microbe?Is it by activity of enzyme or degradation or uptake of the dye molecule by the microbe as a food source ?
Is there any publication?Following
- Cesar García added an answer:11Why does pH increase with increase in redox and DO in a plant biomass fermented broth?
I am oxidizing my plant fermented broth by air.I found increase in DO and redox because of more solubility of oxygen in the broth. The dissolved oxygen concentration increases and because the reaction in the broth is an oxidation process, the redox is also increasing.
Oxidation also increases with pH decreasing but why?
When I am supplying oxygen for a long time, redox decreases as well as the pH also behaves the same way.
Why this is happening?
I think what what you mean is that increasing in the DO consumption, it means a related productions in CO2. This can affect the pH of the medium.
- Dirk Müller added an answer:5Can I measure intracellular metabolites in non-steady state conditions?
I want to extract and measure some intracellular metabolites as well as redox cofactors (NAD(P)H and NAD(P)+) from the yeast Pichia stipitis.
I'm afraid I won't have the possibility to set up a continuous culture in a chemostat, so my question is: if I just take samples from a batch culture in bioreactor will I still get a meaningful quantification of cofactors and metabolites?
Also, if I perform a series of batch in identical conditions except for OTR, will the NAD(H) measurements be comparable between the different batches? I'd say that samples must be taken always during exponential phase, but how can I make sure of that since the growth profile will be inevitably different between the cultivations?
Finally, should I express the results just as concentrations (mM) or as "mM per gram of biomass" (mM x gDW)?
(My reference for the cofactors extraction protocol is the attached paper)
you can certainly measure intracellular metabolites under non-steady state conditions, provided you employ a suitable rapid sampling/quenching strategy. This being said, NAD(H) and NADP(H) are notoriously difficult to quantify even with appropriate quenching. So usually, you can obtain a quite reliable figure of the total pool concentrations (NADP+NADH and NADP+NADPH, respectively), whereas the oxidation state is not (cg. eg. Mailinger et al. (1998) J Biotechnol 63(2):155-66).
Regarding the chemostat: growth regulation is very different in batch vs chemostat and so can be metabolite levels. Even if the NAD/NADP levels may be not too dissimilar you may opt for a process strategy that better mimicks your chemostat. Maybe you can more easily realize a glucose-limited fed batch at a fixed growth rate? That can much better approximate your chemostat conditions physiology-wise and does not take that long.
As for the measurement units: you should report intracellular concentrations mmol per L cell volume or L cytosolic volume if possible (e.g. in yeast 70% cytosolic volume is a typical assumption). Omitting the volume reference will invite ambiguity. However, the single-cell denstiy (gDW/L cell volume) is quite variable with growth rate: about 250-400 gDW/L cell vol for baker's yeast (the lower for fast batch growth, the higher value typical of slow growth (e.g. 0.1 1/h in a glucose-limited chemostat). So maybe mmol/gDW would be the more robust measure in your case.Following
- Uma Kranthi added an answer:9Any advice on the purification of polygalacturonase and amylase enzymes by ammonium sulphate fractionation?
Hie can any one help me out with the step by step protocol for fractionating the polygalacturonase and amylase enzymes using ammonium sulphate present in my fermentation broth. My fermenting organism is a yeast belonging to Geotrichum species. I tried it once I found good amount of protein present at 80% fraction but it did not show any enzyme activity. My control OD value was more than my test enzyme value.
ok thank you all for your suggestionsFollowing
- Carlos Enrique Perez Heredia added an answer:2What should I take into consideration to determine the length of the pipes in a Biopharma Plant?
I need to know any further consideration beside the actual area of the biopharma plant.
You can use the book "Piping and Pipeline Calculations Manual" to me has helped me. In conclusion I think you should consider the following:
- Theoretical needs of the productive system.
- Type of fluid: related to auxiliary systems and process lines.
- Type of material: to define friction losses, pressure drops.Following
- Gideon Balakrishnan added an answer:7Would it be okay if I use sodium acetate buffer to extract protease enzyme from fermented solid substrate (SSF)?
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?
The choice of buffer depends upon the optimal pH of the enzyme to be extracted, which again lies with the source of the enzyme. Of-course you can use sodium acetate buffer if it is acidic pH range.Following
- Hesham El Enshasy added an answer:6Which kind of glass bioreactor/fermenter is more suitable for growing aerobic bacteria? heat-blanket or water-jacket bioreactor?
I would also appreciate any advise on temperature control for optimal growth at 37ºC.
I think both are fine if you cultivate cells under low cell density (even I usually prefer double jacket one as the culture will be more visible). The problem start for choosing which is better in case of:
1- High cell density cultivation for yeast and bacteria (as cells will produce heat, and increase temperature accordingly).
2- Cultivation of recombinant E. coli with thermoinduction system (in this case you need very good design to support high heat transfer and also good control system).
In these two above cases, if not using stainless steel double jacketed bioreactor, I recommend to use the glass single walled reactor with stainless steel bottom for high heat transfer from the company New Brunswick-Eppendort of Bioflo 310 and above. This to support high heat transfer and also minimize any high heat oscillation during thermo-induction (rapid increase of heat from 37 to 42) during recombinant E. coli cultivation especially under high cell density.Following
- Solange I Mussatto added an answer:4Is there any method to quantify lignin derivatives without the interference of carbohydrates after biosolubilization of lignocellulose by fungus?
I am getting a thick liquid distillate after fungal biosolubilization of sugarcane bagasse in Solid State fermentation conditions. I want to quantify lignin derivatives (if any) in the liquid distillate. Can anyone help me out?Following
- Muhammad Zaid added an answer:7What is the difference between anaerobic and anoxic conditions?Are both same? When an organism is in anoxic condition, does it shift to anaerobic mode of metabolism?
anoxic environment have only bound oxygen but anaerobic environment do not have any type of oxygen neither bound nor freeFollowing
- A. V. Frolkova added an answer:8Does anyone know what you can use, such as an extractor agent, to separate the mixture butanol-ethanol-water?
We need to separate a mixture in a column distillation using an extractive distillation system.
It depends on the composition of your mixture. You can use for example a complex of distillation columns with a splitting vessel (limited mutual solubility of water and butanol). If you consider extractive distillation the only way for separation try as an extractive agent DMSO or DMFA. Perhaps water can be used as autoextractive agent. But you need to check it.Following
- Justin Kaffenberger added an answer:6Novozyme 188 is discontinued.Is there any alternative?
Novozyme 188 has been discontinued by Sigma Aldrich. Is there an alternative supplier? If not, what is a reasonable replacement for standard enzymatic hydrolysis testing (i.e. NREL protocol, Selig et al. 2008)? Any help is greatly appreciated!
I considered the 49291 product. To get the same level of activity as a 50 mL bottle of Novozyme 188, you need 13 bottles of the glucosidase from A. niger. This is equivalent to a 40 fold mark up in price, and doesn't seem feasible. I think I'll go with the accellerase, though it's not available through Sigma yet. Thanks.Following
- Florian Glauche added an answer:8How can I achieve high cell density for E.coli (OD 50-100) in shake flasks?
Its useful to study the nutrients behavior (Carbon ,Nitrogen,Phosphate,Magnesium and Inducer effects,Byproducts ), recombinant protein profile, process parameters (Temperature,pH, OD,Viability, Plasmid stability,Biomass growth profile) in high cell density cultivation to understand the process at large scale.
For recombinant protein production, we have achieved optical densities of 30 - 60 using baffled shake flasks covered with oxygen permeable membranes (Thompson UltraYield Flasks + AirOTop Seals) and fed-batch medium (EnPresso B, BioSilta Ltd.). Have a look at publications from Kaisa Ukkonen and Jian Li for further details.Following
- Philippa Ojimelukwe added an answer:15How to measure cell density absorbance with many particles?
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?
Yes, but use a standard material for the filtration process. A filter paper is better than muslin cloth. This will ensure uniform treatment of your samples. You may also consider diluting the samples after centrifugation with buffer before taking readingsFollowing
- Szymon Strnad added an answer:3Can anyone suggest the optimal growth conditions and the favourable medium for Rhodotorula rubra ?
I have an isolate which grows well at 19C. but i have come across literature reporting a growth temperature of 30C.
The medium I used for Rhodotorula rubra was Sabouraud agar with chloramphenicol and the temperature was 20C. But I isolated my R. rubra strain when carrying out cabbage fermentation. I think the most optimal temperature would be the one of the environment your strains originate from. But making a growth curve would be advisable anyway.Following
- David Mandepudi added an answer:5How can I sterilize a fermentor with plastic tank?
I want to know if I have a fermentor with plastic tank, can I sterilize it with alcohol(ethanol 70%) instead of autoclave in general fermentors?
In general, plastic fermentors are recommended for single use as they come in sterile condition for ready use. If they are to be used repeatedly then one has to compromise on certain parameters and deal with the complications that may arise while its usage in the previous runs like damaged surfaces that pose complications in terms of reactivity, deposition of residues, microbes and so on. So one has to rationalize and ensure its intended usage. In such case, the surface sterilization may be recommended with 70% alcohol in water, in the presence of sterile ambient like HEPA filtered laminar hood, followed by UV sterilization for the better results.Following
- Ajijolakewu Kamoldeen added an answer:9Can anyone please assist me with a decisive guide on which organism to choose for my enzyme production?
I am currently conducting a research into fungal enzymes involved in glycosyl hydrolysis. I am to select one of three organisms for enzymes production. The activity of the enzyme produced by the best of these three organisms is twice the second best and three times the least. However, when I used the same enzyme units (30FPU) for the three organism in a target hydrolysis, enzymes from the other two organisms (those with lower activities) had higher sugar yields (10% total sugar yield more than the best organism). Of course, I thought that the other two organisms might posses some other complementary enzymes which act in synergy to produce a higher sugar yield but I am concerned that they however require a higher volume of crude enzymes or some expensive enzyme concentration mechanisms that will rather make the hydrolytic process non cost effective. I urgently need a decisive guide about this.
@ Dr Ghosh, my concerns had been expressed in my question. I am actually not isolating enzyme but selecting the best organisms by a set of protocols for their enzyme activity and a reaction using their enzyme products for target hydrolysis.
Suggestions, especially by @ Guldhe and others, have actually been very helpful.
@ all, Thanks aginFollowing
- Ajijolakewu Kamoldeen added an answer:10What are the possible causes of tailing peaks in my HPLC chromatogram?
I am unable to have a definite peak in the chromatogram of the five monomeric sugar and cellobiose analysis using HPLC. All five monomeric sugars, except Glucose, forms tailing peaks while Cellobiose is not detected. This tailing peaks caused overlapping when all sugar standards were run. My sugars and cellobiose were either from Merck or Sigma.
HPLC conditions are well stated. Other details are:
The brand and model of HPLC used: -HPLC Type: Fisher Scientific Thermofisher
Size of column: -Product name: APS-2 Hypersil
Diameter(mm): 250 x 4.6
-Particle size: (micron-N-)
@ Dr Henrik Romar, in case of further works and a possible column blocking, which alternative mobile phase would you recommend ?Following