Bioprocess Engineering and Fermentation Technology

Bioprocess Engineering and Fermentation Technology

  • Anbibie F. added an answer:
    How to purify enzyme: as cellulose or amylase by using minimal chemicals?
    As per the protocols we use no. of chemicals for purification so is there any novel chemical which results in minimal use of other chemicals for enzyme purification? or combination of physical and chemical methods will be beneficial for purification?
    Anbibie F.

    I hope this would help. Some enzyme separation processes are related to size based separation. The one commonly used is ultrafiltration. Ultrafiltration membranes with MW-cutoffs ranging from 5000-100,000 Daltons are useful for concentrating enzymes (proteins). The protein to be filtered is placed on certain chamber and centrifugal force or vacuum is used to create the filtrate which will be accumulating in the other chamber.

  • Muhammad Sohail added an answer:
    Is there any report describing production of cellulase and/or xylanase from Saccharomyces cerevisiae?

    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?

    Muhammad Sohail

    Dear Jonas Nyman!

    Kindly visit following link


  • Vinay Rao added an answer:
    Hi, we are performing purification of keratinase. We are unable to prepare a suitable substrate. How can I prepare azokeratin substrate?

    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.

    Vinay Rao

    Thanks Gopal. I surely will try that.

  • Carlos Araújo Queiroz added an answer:
    Can 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.

    Carlos Araújo Queiroz

    You may want to check answers given to a related question at this forum: «What is the best buffer for pH 3,5?»:

  • Tsepo Lebiletsa Tsekoa 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. 

    Tsepo Lebiletsa Tsekoa

    You may also try FIFTYOD media from Prozomix. See the following link

  • Jai Ghosh added an answer:
    What are the examples of auxotrophic Saccharomyces cerevisiae yeast strains that can be used for ethanol production? What are their selective markers?

    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. 

    Jai Ghosh

    You need not try to clone and try to make such strains. It will be a waste of time and money. There are several such strains with ATCC. You can see their catalogue and then order for one.

  • Jack Rincon added an answer:
    Why in a two-stage system for H2 and CH4 production, the hydraulic retention time in the methanogenic reactor is higher than the acidogenic reactor?

    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.

    Jack Rincon

    Thanks for the responses, has been of great help

  • Ahmed M. Hemdan added an answer:
    What is the optimum additional volume and incubation temperature of AerobicThermophilic bacteria to enhance a bio gas production?

    I want to know The optimum additional volume and incubation temperature of AerobicThermophilic bacteria to enhance a bio gas production?

    Ahmed M. Hemdan

    Thank you

  • Ghanshyam Tandon added an answer:
    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.
    Ghanshyam Tandon

    I n my view GOD POD method will be most suitable fulfilling your requirements. The method is most specific for glucose only, quick and many samples can be analyzed at a time. The reagent is easily available you can follow the method as suggested above by Jurgen Denecke.

  • Jose MARIA Buesa Galiano added an answer:
    What is the proper way to perform DO probe calibration?

    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?

    Jose MARIA Buesa Galiano

    Hi Jirka, you can do the calibration after sterilization because you will filter the gases by a .22nm filter during the culture. I suppose that you want use pure oxigen to mantain a low level of pO2 with a low flow of gas avoiding strong bubbles isn it?. So calibrate zero and calibrate 100% with oxigen or 21% with air unless you use pure oxigen during your process. Good luck

  • Anand Prabhakar added an answer:
    Which is the best online, precise measurement technique for measuring in- and off-gas(CO2, H2 and O2) compositions in fermentation processes ?

    Hi all,

    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  ?

    Thank you

    Anand Prabhakar

    Use Biogas analyzer (BGA) from Siemens

  • Alok Nahata added an answer:
    Can we use Yogurt/Curd as a source of bacteria for inducing fermentation for enzyme production?

    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. 

    Alok Nahata

    Thanks Dr. Jayaprakash Khedkar and Abhas Kumar.... I just wana know which is the bacteria which should couple with the yeast and other ingredients. Little confused because of the variety of bacteria as to which is the best one or which combination should be used. 

  • Dr. Durga Madhab Mahapatra added an answer:
    Is anybody working on value addition of Protein by-products interested to collaborate in this Edited Book Chapter Invitation (Elsevier)?

    Dear Colleagues..

    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)

    Dr. Durga Madhab Mahapatra

    Dr. Dhillon,

    I am interested to contribute a chapter in the 1st theme : 1. Value addition of waste derived proteins to biofuels and biochemicals.

    This chapter involves studies on production of value added proteins from algae grown in domestic wastewater. This also deals with novel way of protein quantification through IR and its future prospects.

     I have tried  contacting you but the mail has bounced back.

    Kindly suggest how to contact you.

    Dr. Durga

  • Charles Rashama added an answer:
    Does 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?

    Charles Rashama

    Thank you guys

  • Jai Ghosh added an answer:
    How can I calculate specific consumption/production rates for a yeast cultivation?

    Hi all,

    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,



    Jai Ghosh

    It is very well answered by Habibollah.

  • Nizar Matar added an answer:
    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.

    Nizar Matar

    In the following paper, high molecular weight PLA was prepared by ring opening polymerization using a chain extender (which is hexamethylene diisocyanate). My regards.

  • Greg Cote added an answer:
    How 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

    Greg Cote

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

  • Jai Ghosh 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 ?

    Jai Ghosh

    Immobilized enzyme like the respective oxidase in the form of a suitable electrode.

  • Jai Ghosh added an answer:
    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.

    Jai Ghosh

    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. 

  • Timothy L. Turner added an answer:
    How can I buffer a YNB medium for growing yeasts?

    Hi all,

    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,



    Timothy L. Turner


    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?

    Methods used

    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

    Time          O.D.

    0 hrs        0.150

    24 hrs      0.127

    48 hrs      0.106

    72 hrs     0.112

    96 hrs     0.114

    Aldo Amaro Reyes

    Try raising the temperature of the assay to 50 °C. Most fungal hydrolytic enzymes act around 50 °C.

  • Chithan C Kandaswami 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?

    Chithan C Kandaswami

    The term, "secondary metabolite", is often employed to denote metabolites (example phenolics) that do not have a primary function in the organism, which generates these molecules. As such vitamins are primary metabolites in terms of metabolic function. Primary metabolites comprise carbohydrates, lipids and proteins. Vitamins are very often precursors to coenzymes. The coenzyme, FAD, is widely distributed in plants. In all living cells, tetrahydrofolate, involved in one-carbon (C1) metabolism, is indispensable for the biosynthesis of purine, thymidylate, formylmethionyl-tRNA, pantothenate, and methionine, and for the interconversion of glycine and serine. The tetrahydrofolate molecule is well distributed present in various compartments of the plant cell. 

  • Nuresh Manukonda added an answer:
    Why does the pH rise during fermentation with Pichia pastoris MutS?

    Hi guys,

    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,


    Nuresh Manukonda

    Hi Marco

    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.

  • Fatemah 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 ?
    Fatemah Allam

    Is there any publication?

  • 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?

    Cesar García


    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.

    Best Regards

  • Dirk Müller added an answer:
    Can I measure intracellular metabolites in non-steady state conditions?

    Hi all,

    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)


    Dirk Müller

    Dear Niccolo,

    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.

  • 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. 

    Uma Kranthi

    ok thank you all for your suggestions 

  • 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.

    Carlos Enrique Perez Heredia

    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.

  • 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?

    Gideon Balakrishnan

    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.

  • 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.

    Hesham El Enshasy

    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. 

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