Bioprocess Engineering and Fermentation Technology

Bioprocess Engineering and Fermentation Technology

  • 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!

    Justin Kaffenberger · University of Minnesota Twin Cities

    Perfect!  I'lll go with Accellerase 1500.  Thanks for your responses!

  • Michele Rigon Spier 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?

    Michele Rigon Spier · Universidade Federal do Paraná, UFPR

    May you also use peracetic acid , because it is a colorless, or light yellow solution. Its odor is similar to vinegar. Its chemical formula is CH3CO3H. Widely used specially for disinfection in food processing, due to its sterilizer, fungicide, viricide, bactericide an sporicide properties. You should dilute to 0.2%.

    Compatibility for laboratoy use: peracetic acid is compatible with: glass, porcelain, poliethylene, polipropylene and PTFE (Teflon®)

    Compatibility for industrial use: peracetic acid is compatible to: PVC, poliethylene, stainless steel (ex: AISI 304L, 316, 316L).

  • 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. 

    Ajijolakewu Kamoldeen · University of Ilorin

    @ 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 agin 

  • 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
    -Brand: Shidmazu
    -Model: RID-10A
    Size of column: -Product name: APS-2 Hypersil
    Diameter(mm): 250 x 4.6
    -Particle size: (micron-N-)

    Ajijolakewu Kamoldeen · University of Ilorin

    @ Dr Henrik Romar, in case of further works and a possible column blocking, which alternative mobile phase would you recommend ?

  • 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.

    Sivananth Murugesan · National Institute of Technology Karnataka

    Thanks a lot Artun Sukan I will make sure to follow that 

  • Jai Ghosh added an answer:
    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.

    Regards

    Ashish

    Jai Ghosh · Shivaji University, Kolhapur

    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.

  • 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?
    Ghazanfar Sultan · University of Karachi

    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.

  • Bor-Yann Chen 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. 

    Bor-Yann Chen · National I-Lan University

    At least 3 suggestions are provided as follows: determine optimal compositions of nutrient media via Box's experimental design first,  then use minimal principle to have feeding strategy (i.e., exponential feeding strategy for high cell density culture by fed-batch mode of operation).  In addition, for large scale operation, you have to consider removal of significant heat generation.  BTW, OD 50-100 is not considered to be sufficiently high for E. coli culture (ca. < 30 g DCW/L).

  • 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.

    R. Sangeetha · Vels University

    We tried phenol red plates and got surprisingly good results. Zones were visible.

  • 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.

    Philippa Ojimelukwe · Michael Okpara University of Agriculture, Umudike

    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 morphology

  • Sudhir Kumar Pal added an answer:
    Is there a tool available to analyze the AT-content of all the genes from a database?

    Dear all,

    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?

    Sudhir Kumar Pal · Dr. B.R. Ambedkar Center for Biomedical Research

    try generunner, i think it can work. or online expasy repository might help you.

  • Zhongping Shi added an answer:
    What are the process control strategies used to control acetate accumulation in Ecoli BL21 strains when grown in glucose?

    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)

    Zhongping Shi · Jiangnan University

    This is a common fermentation process control problem.

    1) Glucose and acetate generally can not be on-line measured, therefore feedback control of glucose or acetate would not be available.

    2) Feeding methods of DO-Stat or pH-Stat could control acetate concentration at low level and avoid the Crabtree effect, but the cells growth would be very low. High density cell concentration may not be reached.

    3) The DO/pH on-line measurements based artificial neutral network (ANN) substrate feeding strategy is highly recommended. Please refer to the following our papers: 1) An on-line adaptive control based on DO/pH measurements and ANN pattern recognition model for fed-batch cultivation, Biochem. Eng. J, 30(1), 88-96, 2006; 2) Effective induction of phytase in Pichia pastoris fed-batch culture using an ANN pattern recognition model based on-line adaptive control strategy, Biochem. Eng. J, 37, 26-33, 2007. They are available in ResearchGate.

  • Jai Ghosh added an answer:
    Does anaerobic condition of fermentation cause consumption of fewer amino acids or vice versa?

    the pattern of consumption of amino acid is depend on yeast strain. 

    but is there any one knows the availability of oxygen also cause to consumed more amino acid or not?

    Jai Ghosh · Shivaji University, Kolhapur

    It all depends on what do you mean by consumption.  I would like to know what are the amino acids that you are looking at.  See consumption may mean anything, like it may be used as a carbon source, energy source, nitrogen source, growth factor or even it may be getting conjugated to form some secondary or tertiary compounds.

  • Vladimir Dusevich added an answer:
    How can I prepare a smear of chemical pretreated sawdust for SEM Analysis?

    Hello Every one,

    I am working on pretreatment of lignocellulosic biomass, I am facing a problem while preparing a smear for SEM analysis of pretreated sawdust.  please let me know the  smear preparation in-detail and suggest me one of the best methods.

    Thanking you All.

    Vladimir Dusevich · University of Missouri - Kansas City

    For specimens like wood conventional (not environmental) SEM will provide better images of better quality.

  • Revathi Dhanashekar added an answer:
    What are the possible reasons for the decline in O.D value in a batch fermentor?

    I deal with production of Lipase from Candida sps. (CALB). The fermentation has been recently scaled up to a 3L fermentor. The problem that I face now is the fluctuating O.D values of the broth measured through a period of 48 hours. I cannot seem to point out the actual cause of the decline in O.D. 

    Revathi Dhanashekar · Indian Institute of Chemical Technology

    Szymon Strnad- Thank you for offering to share your thesis. Unfortunately I don't know polish. I will be performing the same experiment soon keeping the above mentioned points in mind. Hoping for a smooth run.

  • Anatoliy Zhivotchenko added an answer:
    When exactly should the 100% calibration of a DO probe be done during fermentation? When the media is saturated in normal conditions or at the highest condition?
    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.
    Anatoliy Zhivotchenko · Biovectra

    The calibration technique is a standard and typically is done after sterilization before inoculation at 1 vvm of air supply and at the maximum rpm used in the fermentation (or 400-500rpm is enough),after DO is stable, around 15 min, the stable value is 100%, what important for this procedure is back pressure that typically is set-up at the value of the fermentation process, typically 0.5 bar.

    The DO,% is relative value and 100% is equal to the soluble concentration, mg/L, that can be found according to Henry Law, and it depends on temperature, oxygen concentration in the supply gas (in air it is constant, around 21%) and back pressure.

    Therefore if your air supply fluctuates then most likely your back pressure will fluctuates too and at the same value of DO,% the actual soluble concentration of oxygen in mg/L will be different that you should keep in mind, also when you compare two or more batches you should compare mg/L not DO,%.

    One more tip, if your process is going to be scaled-up then no sense to use agitation higher than 400rpm in a lab fermenter because at this rpm you have KLa =300-400 that is typical for large fermenters, if you use higher than 400rpm then you have to re-develop the process in the scale-up fermenter.again that will be very costly.

  • Reeta Rani Singhania added an answer:
    Does anybody have experience of using oxygen vector in the fermentation medium?

    After several trials I am unable to maintain DO during fermentation; increase of gas supply, addition of oxygen is not working as it increases pressure also. RPM increase is not a solution for filamentous fungi that too when the process have to be replicated at industrial level. I would like to know suitable oxygen vectors if anybody can suggest and kind of experience one can share.

    Reeta Rani Singhania · Indian Oil Corporation Limited

    Dear Biryukov, I am so much thankful to you for your extended help.

    My email id is reetasinghania@gmail.com

  • Mahmoud Abouseoud added an answer:
    Can we use crude extract to determine Km or Vmax enzyme?
    If we want to learn about metabolic flux, can we use crude extract of enzyme to know the Km or Vmax value? Or do we have to purify that enzyme first?
    Mahmoud Abouseoud · Dr. Yahia Fares University of Médéa

    You do not have to purify enzyme in order to calculate Vmax and Km...unless you may know that some imprities taht may exist in the crude extract could interfere or inhibits your enzyme .....

  • Anatoliy Zhivotchenko added an answer:
    What is the logic behind assigning high and low values to N no. of variables for an N+1 no. of experiment in placket barman design matrix?
    Placket-barman design matrix
    Anatoliy Zhivotchenko · Biovectra

    typically it is 20% (10-30%) from the original value

  • Anatoliy Zhivotchenko added an answer:
    How can we maintain the homogeneous condition in the chemostat feed medium while long chain fatty acids are used as a sole carbon source?

    it always forms two layers ,due to polar and non polar properties, so fatty acids are not properly transfer in to the medium ,some one would suggest me to solve the problem

    Anatoliy Zhivotchenko · Biovectra

    1 - any usage of emulsifier will result in foaming issue

    2 - the simplest way is homonization that is used in food industry (milk, sauces,...), and the main idea is to create the small size of fat, in the lab scale you can use a microfludizer that can create nanoparticles below 100 microns of your fat, but still the process requires any kind of a stabilizer of this emulsion - and in the milk it is proteins and phospholipids, even if you do this way you should keep in mind that the emulsion most likely cannot be stable in the flask scale because of absence of the impellers, for the same reason the fermenter agitation should be kept at the highest rpm as possible.

  • Anatoliy Zhivotchenko added an answer:
    What direction do you think is of greater potential in biofuel-related studies?
    Intensive studies have been focusing on the science and technology in the conversion of biomass to energy. At least tens of thousands of papers have been published in this field every year. Those studies cover from biomass pretreatment, microbial consortia, enzyme cocktail synergism, to enzymatic degradation mechanisms, biosynthesis pathway design, cellulase engineering, bioreactor design, plant genetic engineering, and etc. In order to avoid blind following, what do you think is the most critical issue that we need to consider in biofuel-related study? What is the future of this type of study? What kind of studies is of greater value to our society?
    Anatoliy Zhivotchenko · Biovectra

    The best technology is the Brazil one because it is the cheapest one and profitable one, all other technologies are not profitable and subsidized by government, the Brazil technology is based on sugar cane that harvested 3 times per year and sugar cane does not need to be treated as the corn technology to convert starch into glucose, therefore the Brazil technology can be done only in Brazil and at present almost all Brazil cars are driven on ethanol.

    the 2nd most profitable technology is the sugar produced from the wood not from the straw or similar light weight material (any kind of waste), and the main problem here is that the raw material should be heavy enough (wood) to compensate expenses for transportation and even this technology should be built in the area with heavy forest around, how to get sugar from wood is question, at present more profitable the acid hydrolysis not enzyme one.

    the 3d one should be considered that involved conversion of any waste into synthetic gas and so on, because the plant can be built in any area where the waste raw material is enough.

    all other technologies that involve biodiesel-starch from grains, or similar ones that depend on oil price (growing-harvesting-treatment = oil expenses) are subsidized by government and will be subsidized logically forever, but advantage of all these technologies that based on them that other more expensive materials can be produced in future and actually all new small companies that used the government support is used the biofuel money to develop something better. 

    In general, the main disadvantage of all technologies is that they cannot be large enough to be profitable because of transportation expenses, and at present all ethanol plants is relatively small ones and any small company has much higher portion of expenses on labor therefore another direction is engineering one to decrease the labor expenses by using automation. 

    For future generation the most perspective technologies are ones related to the sun because so far it is the only source that will last for long period of time, but any kind of fermentation technologies (algae or similar ones) are not profitable and cannot be profitable most likely never, therefore it should be developed the cheap technologies (solar, wind, hydro) and the best one would be creation of a small sun that will work in similar way as the sun, any of these technologies have another important advantage - they save our food, do not use oil and do not pollute the earth - therefore governments must subsidize mostly these type technologies including ones that related to them (electrical transformation).

  • Anatoliy Zhivotchenko 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.
    Anatoliy Zhivotchenko · Biovectra

    Diastix strips for fast but not so accurate measurement (1 min test better with the supernatant broth sample), blood glucose analyzer - small device like a computer mouse size with strips (enzyme reaction, 1 min test better with the supernatant sample) and it is very accurate up to 30 mM and can bought in a regular drug store

  • Ravi Kant Upadhyay added an answer:
    What could be the explanation of the accelerated conversion of maltotriose to maltose?

    In the application of Maltogenic amylase in maltose production, a site-directed mutagenesis was performed to accelerate the conversion of maltotriose to maltose.

    It is confirmed that the mutants truly did better that of the wild-type enzyme in the conversion efficiency without conversion rate increase. However, the Km value of the mutants is larger than that of the wild-type.

    The mutation is designed based on the assumption that reduced transglycosylation activity is better for accelerated maltose production, and the mutants did have reduced transglycosylation activity. I wonder if the reduced transglycosylation activity is enough to support the accelerated conversion regarding the increased Km value. If not, what could be the explanation otherwise to support my result?

    Thank you.

    Ravi Kant Upadhyay · Deen Dayal Upadhyaya Gorakhpur University

    Nonionic surfactant, polyoxyethylene mono-N-dodecyl-ether effects  number of units of ethylene oxide moieties, on the behavior of Bacillus amyloliquefaciens α-amylase in the hydrolysis of maltoheptaose . The addition of the surfactant to the enzyme-substrate system increased the amount of reducing sugars during the hydrolysis.  During the hydrolysis, the concentration of maltose get  increase after addition of the surfactant, whereas the concentrations of other products possible remain unchanged. Therefore, the increase in the amount of reducing sugars is thought to be due to the increase of the maltose fragment. Because the transglycosylation of hydrolyzed maltose takes place during the hydrolysis, the suppression of transglycosylation accelerates the production of total reducing sugars. The interaction between the maltose fragment and micelle surface might induce the release of this fragment from the enzyme active site and then suppress the transglycosylation. The bindings of the enzyme, the substrate, and/or the products to the micelle surface may affect the hydrolytic behavior including both the hydrolysis and transglycosylation catalytic conversions.

  • Ghasem Najafpour D. added an answer:
    How can continuous fermented gas production be determined?

    This is with respect to evaluate the quality of feed.

    Ghasem Najafpour D. · Babol Noshirvani University of Technology

    In a system of gaseous substrate mass transfer coefficient plays important role. The gas must be soluble in liquid phase in order to be transported into the cell. Since we culture the organism pure or mixed culture does not matter; we have inlet and outlet streams for the bioreactor then gas samples are drawn for GC analysis. Changes in mole fractions or molar composition of inlet and outlet would let us to know exact gas analysis. Please search for my published papers for detail information about hydrogen sulfide and carbon monoxide.  

  • Dan Minchin added an answer:
    Does anyone know what is a ferromanganese nodule?
    I'm working with manganese-iron oxidizing bacteria
    Dan Minchin · Klaipeda University

    Francisco,  I am not a geologist and I have come across what I believe are ferromanganese flat nodules  on the river bed in an Irish river..  I am hoping to examine the distribution of these next year.  I have heard that bacteria may form these and some might consider these to be stromatolites. I have found these up to 12cm in diameter.  I have been unable to find any information on these elsewhere in Ireland.  I am particularly interested to learn more about these and I would be interested to know how these form, how old these might be and what the composition is.  I can send pictures should you be interested further.  Dan Minchin moiireland@yahoo.ie

  • Ravi Mohan added an answer:
    How do you compare the results of enzyme activity estimated by different methods?
    Lipase activity is estimated by spectrometric, colorometric, and titrimetric methods. In each case the activity is expressed differently. In some case if estimated by a single method the calculation and enzyme activity differ drastically from one method to another. How can you compare or corelate the results in such cases?
    Ravi Mohan · Gulbarga University

    Thank u all for the answer .

  • Ning Zhang added an answer:
    Why does my algae culture smell like E.coli?

    Hi, I ran a batch in 4 flasks starting 6 days ago, 2 heterotrophically and 2 mixotrophically. The 2 heterotrophic ones smell bad when I take samples everyday, and for some time, I think I smell the same thing in one mixotrophic culture. The smell reminds me of the E.coli cultures I used to grow, not in the current lab I am in but from a few years ago. I just remember that stinky smell.

    To check for contamination, I examined the culture under microscope at 400X resolution, and I found nothing other than healthy algae cells. And yesterday I took one proteose agar plate that I use for the algae, and streaked onto it with one heterotrophic culture that I suspected of E.coli contamination. After incubating at 37C overnight, nothing showed up in the plate. But the smells are still there. I am kind of confused. I used glycerol and yeast to feed my algae. Can certain metabolites from algae cause the same stinking smells as the E.coli? 

    Some one please enlighten me on this. Thanks!

    Ning Zhang · Clemson University

    Thank you all for the advices. 

  • Yossef Shabtai added an answer:
    Can someone provide me the list different PHA monomers?

    In my gc ms experiment I have got long chain monomers  c15 to c19 while using linoleic acid as a carbon source in pseudomonas putida .Can p.putida capable synthesize long chain polymers?

    Yossef Shabtai · YSFERM

    Ahil,

    The answer is YES. P. putida can metabolize and degrade long chain hydrocarbons and fatty acids. During the beta-oxidation hydroxyl fatty acid are formed at various chain length and can be incorporated into a poly-hydroxy-alkanoate polymer. In most cases C8, C10 and C12 are the major constituents of such PHA, but it all depends on conditions especially on the affinity and selectivity of the synthase enzyme responsible for the polymerization.

  • AbHaS KuMaR MaHaRaNa added an answer:
    Why do we need to check the protein concentration (mg/ml or mg/l) in withdraw fermentation broth?
    I am interested in this issue, regarding the checking of protein concentration in fermentation sample.
  • Selvaraju Sivamani added an answer:
    What concentration of NAD and Tris Cl should I use for ADH enzyme assay?

    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. 

    Selvaraju Sivamani · Kumaraguru College of Technology

    Please refer the link below:

    http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-alcohol-dehydrogenase.html

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