- Veronica Martinez added an answer:Is 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 ?
I recently asked a similar question (about sucrose). I have two articles with several colorimetric methods that I find convenient, since a spectrophotometer is better to work with (at least for me where I work). Hope they help!Following
- How 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:How 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:Can 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:Can 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:Why 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
- Fatma Allam added an answer:What 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:Why 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:Can 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:Any 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
- How 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.
You won't get this from any place from any catalogue. I suggest that you synthesize it yourself. It is easy and then purify it, which is also not that difficult.Following
- Carlos Enrique Perez Heredia added an answer:What 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:Would 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:Which 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:Is 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:What 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:Does 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:Novozyme 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:How 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:How 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:Can 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:How 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:Can 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:What 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
- Sivananth Murugesan added an answer:How can I quantify the PHA with the Crotonic acid assay ?
I have been quantifying PHA produced by the microbes via crotonic acid assay. I had centrifuged 15 ml of medium to obtain a considerable amount of biomass , and I had treated the biomass alone with acetone , methanol and water to remove the cell debris and then I had dissoved the left out pellet in chloroform and boiled it at around 90 degrees and after which 20 microlitre of the chloroform extract was used for crotonic acid analysis by standard protocol.
When I compare the spectral readings with the standard graph and quantify I get a few micrograms in 20 microlitre and when I back calculate there is only a few micrograms in a liter of fermentation medium.
where as when I used thermogravimetric analysis of PHA in biomass I obtain micrograms of PHA / micrograms of biomass. can someone help me out with the back calculation.
Thanks a lot Artun Sukan I will make sure to follow thatFollowing
- Recombinant protein Expression in E.coli: Which gene's expression levels can be used to monitor the developed metabolic/cellular stress inside E.coli?
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.
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.Following
- Ghazanfar Sultan added an answer:Can someone advise on microbial catalysis and biotransforamtion?I am working on the microbial transformation of triterpenes using A. niger
the microbe was incubated for 24 hrs in 30 c , then 10 mg of the substrate have fed to the microbe.
As a screening experiment I incubated the fungi with the substrate for 2, 4 and 6 days in 3 different conical flasks.
I harvest the and check on TLC, noted that the substrate very slightly or has not consumed at all.
And there was no any marks for any metabolite or transformed product?
Dear, if you are not look for specific products then why do you bother on depending only one fungi. Try other available fungi and bacteria too.
Although I am agree with Salman Zafar's proposal, but remember that most of the transformations (especially oxidation reduction reactions) need whole-cell procedures because they need mitochondrial or microsomal machinery.
So I suggests you to follow the Salman's strategy as well as try some other cultures.Following
- R. Sangeetha added an answer:Does anyone have advice on lipase activity with tributyrin agar?
Ttributyrin agar has been dissolved 20 g in 1 litre distilled water and sterilized by autoclaving at 121°C for 15 minutes. It has been cooled to 80°C and added 10 g neutral tributyrin. But we couldn't see a clear zone, even by Rhodococcus spp. and Pseudomonas spp.
We tried phenol red plates and got surprisingly good results. Zones were visible.Following
- Philippa Ojimelukwe added an answer:How can I differentiate bacterial contamination in fungal wet mount microscopically?
Basically I work on solid state fermentation of fungus and find it really difficult to confirm if my seed cultures are pure.
You could stain with lactophenol cotton blue stain which helps detect septa special mycelia and spore structures. On the other hand, the gram stain reaction will help detect bacteria which could be either gram positive or gram negative. This is in addition to colony and cellular morphologyFollowing
- Sudhir Kumar Pal added an answer:Is there a tool available to analyze the AT-content of all the genes from a database?
I am interested in finding the AT-content of all the genes of Saccharomyces cerevisiae(SC). Is there a tool available that can analyze the entire genome database of SC and can tabulate the genes in the order of their AT-content? Moreover, it would be great if the tool can analyze any fixed sequence length of each gene in the database, say, the first 500bp of all the genes and then tabulate them in the order of their AT-content?
try generunner, i think it can work. or online expasy repository might help you.Following