Science topics: BiotechnologyBioprocess Engineering and Fermentation Technology
Bioprocess Engineering and Fermentation Technology - Science topic
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Questions related to Bioprocess Engineering and Fermentation Technology
I'm currently growing E. coli in bioreactor and I have experience only with P. pastoris in bioreactor, so I'm not sure, how should it behave. With Pichia, one can try hard to imrove the conditions and it will grow very well to very high cell density (something like OD600 of hundreds), so I had similar approach with E. coli.
I usually grow E. coli (in flasks) in LB medium supplemented with 0.1M K-phosphate pH 7.0; 1% glycerol; 2mM magnesium.
I grew it for the first time in this medium, but the pH shifted up to 8.x after switching the temperature to 18°C (after induction). We thought it was because of the phosphate, although from what I found on the internet, the phosphate buffer shall not be so much sensitive to temp change as other buffers. Anyway, we grew it without the phosphate the second time, but the pH increased anyway (not that much though). Moreover, it was slowly increasing over time, rather than decreasing. It was about 7.2 in the morning (maybe more). What could be the cause of the pH increase?
Second- what is the usual OD600 after overnight growth in bioreactor? The first time we got around 8, but the pH was slowly increasing. At that time I thought it's because of cell lysis, but now in retrospect, it could be related to the changes in pH as mentioned above. The second time we got OD600 around 3, but it was after shorter time (because we thought it's lysing) and the culture grew slower in general in comparison with the first one.
Thank you for your suggestions.
I am looking for a simple spreadsheet for calculating the feeding regime for biomass production in a fed-batch mode.
Basically, I am growing baker's yeast and looking for a simple spreadsheet that I can provide my volumes, substrate concentrations, specific growth rate, starting biomass and target biomass So I can get feeding rate and time of fermentation required without going through the complex calculations and equations that I am not good at. I am using sucrose and 10L steered/controlled bioreactor. Anyone can help with this? I will be very much grateful.
Novozyme 234 is the best enzyme for the isolation of protoplast but in recent days the Novozyme 234 are not commercially available. So is there any other method (enzymatic) for this isolation?
I would like to quantify the OTR and the oxygen demand in different cultures and rpm of shaker by the method of gas in and gas out, but is not very clear which formulas used, and which measurement magnitude are replaces the variables in the formulas.
I am working to find a bioactive compound from a bacterial source which can show antifungal activity. So I have established the bacterial culture in a synthetic medium. What we found that the enzyme/proteinous compound is mainly extracellular in nature. Now I want to go for isolation, extraction and purification of the compound from the liquid culture of bacteria at laboratory scale. So I am asking to share your knowledge about the sort of suitable methods which will be helpful for getting the desired compound.
As C/N is very critical in the optimization of the Exopolysaccharide production by Microorganisms. My concern is that I have used complex media such as tryptic soy broth (TSB) for other optimization experiments. TSB medium contains glucose as carbon and tryptone as the nitrogen source. Should I use different ratio of glucose and tryptone to optimize C/N ratio? Any suitable reference paper please.
Thanking in anticipation
I am performing enzyme assay for ADH. I am confused with what concentration of NAD and Tris Cl can be used.
If any one has a protocol, please provide it for me.
As my aim is to study the anti-fungal activity by the bacteria, Is there any expertise member who can explain how to know a bio-active compound(may be protein, enzyme or other molecules) isolated from a bacteria is extracellular or intracellular in nature ?
The literature search could not helped me to find out the appropriate solution for this particular microorganism so I want to go for various experimental methods. So is there any proper step by step procedure for better confirmatory tests ????
Hello Every one,
I have done saccharification with 50 U/ml crude enzyme extract in 50 ml buffer with 5% solid loading. Now I have a doubt at calculation part. If I get released reducing sugar of 1.23 g/ml what is my % of saccharification? I have taken 1 ml of crude enzyme extract for estimation of sugars released. Now I have to calculate it for 1 ml or 50 ml please let me know in detail in this regard. I am using the following formula:
Saccharificatioon (%) = sugars released x 0.9/ cellulose content x 100
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!
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?
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,
I have seen that both yeasts and filamentous fungi are used for biochemical production in cell factories systems. I am not sure about the differences between the two systems, I mean, are fungi able to produce more complex compounds or are there other reasons? It would be nice is someone could help me to understand it. Thank you so much in advance
Hi, I'm looking to produce a highest biomass production of Saccharomyces cerevisiae in the Laboratory, in a stirred tank bioreactor (with working volume of 3 L), for this purpose, I'm trying to choose the best strain to fulfill my goal.
I appreciate the answers.
We want to use a fermentation volume of 30/40 L and it would be very labour intensive to centrifuge (using bucket centrifuge) the broth before resuspening and lysating it. After lysation it is still necessary to use the centrifuge but then we would no have to use it twice in one process.
During cascading events of a fermentation, agitation and aeration are at a max. Thus the saturation of air with those conditions give a higher DO value compared to the saturation using basic conditions before inoculation.
I need to selectively isolate fungal cellulase/xylanase producers in a selective medium which supports both bacterial and fungal cellulase/xylanase producing organisms. Tetracycline, a broad spectrum antibiotic, has been shown to inhibit some fungal species. Sodium azide and cyclo-heximide can also prevent both bacterial and fungal growth. This condition is also applicable to some other common antibiotics. I need a more restrictive antibiotic with no known antifungal property so that I will not lose any chance of selecting the best fungal strain.
I want to ask about using the mixed culture instead of pure culture for syngas fermentation, as I used the pure culture but they did not produce ethanol. Can I use digested manure as mixed culture after killing methanotrophic bacteria on it and it is enough heating it at 100C for 30min or I need another treatment method? And there are any papers about this topic?
I want to check the interaction between magnetic immobilized enzyme with the nano materials. What are be the reaction parameters should be fixed for this?
Is there any solution to get rid of precipitation or is there any salt which doesn't get precipitated when added with CMC (Carboxy Methyl Cellulose)?
I am studying the effect of surface active agent concentration (1-50 ppm) on the hold-up and mixing time in an internal loop airlift bioreactors. the surface active agent used is "Sodium dodecyl sulfonate".
The problem is we have an overflow of foam even at lower concentrations of SDB.
for fermentation of yogurt culture inside an incubator it needs constant temperature condition for some period of time eg 37 degrees for 4 hours without changing.Hence how can i maintain constant temperature for a period of time inside the incubator ?
To prove the increase in yield and productivity in a better way, instead just with the metabolite profile.
Different kinds of substrates produce different amounts of biogas and have different rates of degradation. They know some method to determine these rates?
I found that for different media different sterilization SOP follows. Some passes the pure steam into the media after reaching temperature 95 or 90 through sparger and close the jacket steam and some not passes the steam inside the media and close the exhaust valve after reaching the temperature 95 and continue the sterilization with jacket steam.
which is best?
I transferred Saccharomyces cerevisiae yeast strain optimized to produce precursor of pathway with beta-carotene producing plasmid (CRTE, YB and I). There is constitutive promoter (TDH3) upstream of all 3 genes. For how many days should I wait to get orange color? Already the colonies are small but white.Or has some one also faced the same problem? I got some recommendation referring to change the temperature to 4 'C.
I am looking forward to your advice.
Final products of Biotech Downstream Separation are commercially relevant (Insulin, Penicillin, Cephalosporin, Enzymes etc). Some are within cells coming out of the fermenter and cell-disruption is needed, some are in the liquid phase coming out of the fermenter and don't need cell disruption.
I need some info about commercially relevant downstream products.
Thanks in advance
I want to design citric acid plant by submerged fermentation process with Aspen HYSYS. But the problems are Aspen HYSYS doesn't provide sorghum properties, many unit operations for submerged fermentation process, many intermediate components, and Aspergillus niger microorganism. Are there any simulation software specifically for fermentation industry?
Shake flasks are predominant platform to culture the microbial cells. Precise measurement helps to add external oxygen to provide oxygen enriched environment that will increase the growth of cells and recombinant protein production.
I wanted to study the effect of 1 % nitrogen source on the enzyme production via Solid State Fermentation.
Which method is correct?
A). The 1% nitrogen source is determine based on the volume of moisture I provide
B). The 1% nitrogen source is determine based on weight of the solid substrate I wanted to use.
Does anyone have experience on this matter? TQ
I have produced lipase by solid state fermentation. I extract the fungal lipase after 5 days of fermentation. The highest activity was achieved at 0.113(U/gSB) on the 5th day. Sugarcane bagasse was used as the substrate along with cooking oil.
I need a supporting evidence on highest lipase recovery on the 5th day. what would be the highest recovery of fungal lipase after 120h? I need to plot a growth curve on this.
Could anyone help me on this? I would really appreciate it.
Thanks in advance
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)
I wonder, if the chromosome XII has an Endoglucanase encoding gene and XIV carries a beta-glucosidase gene, then why there isn't any report describing production of these enzymes from Saccharomyces cerevisiae?
Firstly, we tried preparing feather culture media 1 & 2 using NaCl 0.5g/L, KH2PO4 0.4g/L, K2HPO4 0.3g/L, feather 10g/L, pH 7.5 (Media 1) and Media 1 + NH4Cl 0.5g/L, MgCl2.6H2O 0.1gµl, yeast extract 0.1g/L.
We optimized the temperatures for both media to 40 and 50 Celsius, but did not get satisfactory results. Thanks.
I am going to clone and express a glucoamylase gene in an auxotrophic Saccharomyces cerevisiae using YEp 352 which has a LEU2 and URA3 selective marker, in order to produce a one-step conversion route for ethanol production, therefore a strain which has a strong capability to convert starch to ethanol is much needed. thanks.
It is assumed that to be a two stage system, the HRT should be greater in the acidogenic reactor because the hydrolysis step is which required most time.
We use pure oxygen for fermentation, but ordinary air for DO probe calibration. At this moment we set air as 100 % point of calibration (we use only 1 point calibration). Is this correct? Shouldn't it be 21 % point of calibration, since we use pure oxygen as oxygen supply. Can someone help?
I am trying to figure out which instrument (micro GC/mass spec) is best suitable for monitoring in- and off-gasses during autotrophic fermentation ? Does Mass spec give quantitative data ? which is more reliable/reproducible ?
Process of Fermentation includes the use of microorganisms, like yeast and bacteria for the production of enzymes.. In Submerged fermentation, the production of enzymes is done by microorganisms in a liquid nutrient media. Compounds containing carbon in or on the substrate are busted down by the microorganisms thus producing the enzymes either extracellular or intracellular. The enzymes are isolated by various methods. This approach is used in wine making, brewing, cheese making and baking.
I want to know if the bacteria of yogurt can be used as source for inducing fermentation to produce enzymes.
Thanks in advance.
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)
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?
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.
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 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 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.
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
For example if the maximum tolerable temperature of bacteria is 43 degree Celsius, if we increase the temperature to 45 degree Celsius , how does it affect the bacterial growth?
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?
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.
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.
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?
I would also appreciate any advise on temperature control for optimal growth at 37ºC.
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?
We need to separate a mixture in a column distillation using an extractive distillation system.
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.
I have an isolate which grows well at 19C. but i have come across literature reporting a growth temperature of 30C.
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?
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-)
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.
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.
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.
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.
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?