Hi Rasoul,

Basically there are two kinds of lag phases, as defined by Pirt (1975).

1- A true lag which is defined as a lag caused by factors such as change in physiology, change in nutrients, presence of inhibitors, spore germination, state of inoculum culture.

2- An apparent lag, where a fraction of the inoculated cells are deviding normally while another fraction is dying or simply not deviding.

The result in both cases is that your cell count is constant. therefore, in the 2nd case, increasing your inoculum size will lead to an increase of the deviding (active) cell fraction. In other words:

growth rate is defined as:

dX/dt = µX

if only a fraction of the cells are deviding, then:

dX/dt = µX*F; where F is the fraction of the active cell population and where µ*F in the apparent growth rate.

If you increase your X (inoculum size), then you F will tend to 1 and µ*F will tend to µ.

In microbiogical processes, inoculum size can be high, especially in the case of yeasts. In industrial processes for yeast production, where performances are required, the inoculum size can represent 40% in terms of v/v. In these cases, Inoculum has to be prepared and concentrated before adding it in the reactor.

You will find more details in Principles of microbe and cell cultivation (Pirt, 1975).

Hope it will help.

Amine

Hello amine!

Thanks a lot. It is a helpful explanation.

Hello Rasoul,

While it is true that increasing the amount of inoculum can decrease the lag phase of the culture, you have to take in mind that some strains are affected by the cell density and they wont grow after a threshold has been acquired, one of the the best ways to decrease the lag phase is to have your preinoculum adapted to the conditions of your medium, such is the case for heavy metals and mediums with high osmolarity or toxins, in this cases working with low concentrations of the toxins or inhibitors will lead to an increased tolerance of the cells towards this element, then you can increase the amount of the toxin until you reach the desired concentration and the lag phase will decrease significantly, this principle also applies to cells growing for industrial purposes, like yeasts were you can use ethanol to increase the cell tolerance towards this element.

Hope it helps.

Best regards

It isn´t true. The lag phase depens on the metabolic activity of the microorgansims. If you want to reduce the lag phase you must to know the behave of the culture. You must know when the culture is in exponecial phase of growth. Almost at the end of exponencial phase you put your inoculum in fresh media culture (5-10% of inoculum). If your culture is growing activilly the exponecial phase of your culture continues and probably your culture will not show lag phase.

The inoculoum dosen´t affect the lag phase (between 5-10%), if you microorganism is allready adaptep on the media culture and if it is growing exponentially, the exponencial phase continues without lag phase

The amount of inoculum affects the lag phase because many species of bacteria use quorum sensing to orchestrate gene expression. Bacterial populations behave as a group and not only as a collection of isolated individuals. The minimum amount of inoculum needed depends on the bacterial strain.

If you are curious, I recommend this publication:

QUORUM SENSING IN BACTERIA

Annual Review of Microbiology

Vol. 55: 165-199 (Volume publication date October 2001)

DOI: 10.1146/annurev.micro.55.1.165

Melissa B. Miller and Bonnie L. Bassler

Hi Rasoul, 5% inoculum is rarely used in industry, more like 0.5 to 2%, unless particular species are used. The reason why it is sometimes beneficial to have a fairly large inoculum does have to do with the environment, especially when dealing with oxygen sensitive organisms, i.e organisms lacking catalase or superoxide dismutate. In a vigorously stirring bioreactor, there will always be some level of hydrogen peroxide and O2-. If the ratio bacteria vs. H2O2 / O2- is high, i.e. a high percentage inoculum, the net damage per bacterium is low and it won't interfere with growth. The opposite will retard growth or inhibit it altogether. The other effect is that metabolic routes change due to higher substrate levels in the fresh medium, but that does not take more than a couple of minutes. Hope this helps, cheers Marcel

Hello!

I think, the size of inoculum has influence on the lag phase length. First because some well-known books has mentioned that and secondly, according to my experience. By increasing the volume of inoculum the cells rapidly go into Log phase

@Marcel Yes, it is correct. 5-10 percent is too much for industrial applications.

About quorum sensing, I also agree with Esteban.

So according to the presented comments, by increasing the incoulum size, the cells need less time to be adaptaed to the new medium.

but

How can increase the volume of inoculum cause decrease the time necessary for growth of each cell?

in other words, how does the size of inoculum accelerate the metabolism of each cell?

Are you comparing inoculum cells in the same growth phase Rasoul?

Yes.

different volume from the stationary phase to a new medium.

Can it be explained on the basis of qourum sensing?

I do like this:

1) Growth of the bacteria in a broth culture medium until it reaches to stationary phase

2) Different volumes are transfered into new culture media

3)The number of bacteria (by direct counting under microscope) is determined at time 0 and 1, 2 3 h va ....

4) The first significant increase in the number of cells in considered as the initial of Log phase

As far as I understand you are using different volumes but the same concentration of inoculum. Is that right? Is the concentration after inoculation the same?

for example:

10 ml inoculum +90 ml culture medium

5 ml inoculum +95

2.5 ml +97.5

1.25 ml+98.75

So finally we have different cell numbers in the new culture media

Dear sir, when u increase the concentration of inoculum 5%-10% it will not change the time of lag phase due to adaptation of bacteria is always same. but it increase the population at log phase.

I think all the comments are correct but the condition of inoculum defines the lag phase.

If the inoculum is not adapted well, then the quantity of inoculum is important; because normally the population is strongly heterogeneous, and it takes time for all the sub-populations to be adapted to new conditions. If the new medium contains some components which cause stress for the bacteria, some sub-populations may be entered in death phase because of the inability of cells to cope with new stressors. Amine's comments will be helpful to explain this situation.

However, if the inoculum is previously adapted to new conditions, then the lag phase is almost independent of the quantity of inoculum.

Please let me know if this conclusion is correct?

Hi everyone,

Rasoul,

If I am getting it right: you are harvesting cells in stationnary phase from medium 1 to medium 2 then calculate which of the added volumes takes you to exponential phase rapidely.

1- Medium 1 and medium 2 are same ?

2- normally, during inoculum production, cells should not be cultivated until stationnary phase. They have to be harvested and inoculated to the new fresh medium while they are in late exponential phase, right before complete depletion of your limiting substrate(s). In this case, you will make sure that cells are still in the desired physiological state. Harvesting them in stationnary phase induces several changes in metabolism leading to a change in physiology that require therefore more time to be reactivated.

3- I would suggest the same experiment but by harvesting earlier (mid or late exponential phase); you would eliminate the bias due to physiologial difference between log and stationnary phases.

4- If medium 1 and 2 are different (different C source, N source, physical and chemical medium properties etc), then probably you'd make the following experiment:

Make a two steps inoculum preparation :

Step 1 Growth in medium 1 until mid log phase

Step 2 harvest and transfer a fraction of cells (say 2%) in medium 2

Step 3 use whatever you produced in Step 2 to inoculate your may fermentor/flask where you want to conduct your experiment. this would help cells to adapt to the new substrate and reduce adaptation time.

Awaiting your comments

Best regards

Amine

Hello everyone,

I guess the size of the inoculum will have the biggest influence if:

a. Cells interact with each other (quorum sensing).

b. Cells need enzymes to break down a substrate in the (complex)-media.

In the first case this can be crucial for growth of some cells which require certain signals from their buddies. On the other hand growth might be restricted if too many cells share the same volume.

In the second case an insufficient inoculum will possibly result in cell-death since the required amount of enzyme to deliver enough nutrients cannot be produced till the organism starts to starve.

In theory I would say if the substrate is directly consumable for the organism (like glucose), calculations like:

dX/dt = µX

will fit the result very well. But what if glucose is converted into acetate and acetate affects µ but not the conversion of glucose into acetate itself.

I think overflow-metabolism and other forms of regulation (substrate inhibition e. g.) will make such systems very complex and you will get a different result with a different inoculum even in a defined media. But this has been never tested by myself would be very interesting to hear if somebody here has real data.

Stephan

Hello,

I see that there are many relevant answers. I think it is also preferable, especially for microorganisms that have a long generation time, to have a preculture in exponential phase and produced, if possible, on the same culture medium than the main culture.

In my experince 5% inculum is right quantity but depend on your bacteria and media if bacteria get suitable media then you can decrease the time

Again, to reduce lag time you should develop an inoculum in the same medium and when the culture is in late exponential phase you can use it to inoculate the final culture, and the lag phase almost desappears

If you add inoculum at different concentration and then observe the lag, I don't think it would make much of the difference as far as duration of lag phase is concerned. But you can achieve stationary phase much earlier with higher concentration of inoculum.

Hello everyone,

From my humble opinion, and above all bearing in mind my experience, I think that Rasoul has mentioned above an interesting as well as key parameter: the heterogeneity of seed cultures. Physiological heterogeneity plays a key role in the adaptation and further growth of inocula. The underlying physiology of inocula is underestimated by traditional methods. Currently, we know that high-throughput techniques such as flow cytometry allow us to know the underlying physiology. If inoculum is mostly composed by damaged and dead cells, both lag phases and poor adaptation to culture media would appear. Conversely, if inocula are composed majority by metabolically active/healthy cells, microorganism would start to growth at normal rates. Dead and damaged may be the largest subpopulations if the inoculum is harvested at stationary growth phase, so it is important to make sure that inocula is harvested during exponential growth phase in order to avoid the appearance of lag phases. Lag phase undoubtedly affects fermentation performance and metabolite production profile, appearing as a signal of poor culture optimization.

Another influence ma be the Anaplerotic carbon dioxide fixation in the TCA. If the inoculum is chosen to low the partial pressure in the medium may to low to allow these reactions. If you do not have the quorum sensing or an enzyme involved for the availability of nutrient (As Stephan wrote), the quality of the inoculum is the key parameter for the lag phase. Maybe of interest:

T.Palmen, M. Scheidle, R. Huber, C. Kamerke, A. Wiesner, B. Dittrich, D. Klee, J. Büchs: Influence of Initial pH Values on the Lag Phase of Escherichia coli and Bacillus licheniformis Batch Cultures. Chemie Ingenieur Technik, 2013, 85(6), 863–871

another paper that my be of interest:

Robert Huber, Marco Scheidle, Barbara Dittrich, Doris Klee, Jochen Büchs: Equalizing growth in high-throughput small scale cultivations via precultures operated in fed-batch mode. Biotechnology and Bioengineering, 2009, 103(6), 1095-1102

innocula need a minimal size of innoculum which is a critical size .A "golden conce ntration" for some industrial microorganisms is 0.2G/L dw. This "golden concentration" have to be confirmed for your new strain and of course you must study how to have with reproducibility a constant optimal physiological state of your innoculum.

Beetween 0+ and 0.2G/L(dw) the lag decrease. Beyond this "golden concentration " your growth is directly exponential. If innocula is to big be carefull on linear phase of growyth and effect similar to autocatalics reactions

I agree with most of the answers/ suggestion given before. To extend it further (from my experience in working with Bacterial, Fungal and Actinomycetes fermentation at lab scale and industrial scale) I would say: It makes big difference in deciding lag phase when seed inoculum media is different from production media, To avoid extensive lag we sometimes used 'two stage seed' before inoculating the production vessel from seed vessels. And while working for lab scale fermentations also it differs from 'microbes to microbes' and 'products to products'. For eg. when we are working for biosurfactant production by Bacillus group we are using 2% seed transfer single stage seed, whereas when we are working for biosurfactant production by Yeast we transfer 10-15% seed.

Agree to almost all answers Quality inoculum - free from contaminats - age and size will determine the success of not only the fermentation but also the time taken. As nearly all answers the emphasis is on numbers and age - an adequate amount (5-10% v/v if aseptic conditions ; more if unsterile medium which as its bacterial load) and an actively growing culture in the log phase grown in a near similar seed medium as the production medium (including the pH) will cut lag phase significantly

Yes, the larger the amount of inoculum, there will be shortening of the lag phase. This is only true when the cells are not in synchronized growth. It does not hold true in case of continuous culture, where the quantity of substrate and its affinity plays a very important role.

No this will not shorten the lag phase, but log phase to achieve stationary much earlier

The lag time does not depend on amount of inoculums otherwise it is against any theoretical principles described in text books.

The lag time depends on many factors:

1 -depends on type of culture - the faster growing culture has the shortest time (E.coli)

2 - depends on differences in composition of inoculums and a new fresh medium,

3- depends on activity of the culture - the best time of the inoculums usage is exponential phase,

4 - depends on the composition of the new fresh medium - if the inoculums has protein source but the new fresh medium is a mineral medium without protein source then the lag time will be much longer,

5 - depends on the fermentation parameters used for the new fresh medium - if pH, DO, T is not optimum then the lag time will be much longer.

Very interesting topic! I am wondering if the growth rate during log phase (calculated by exponential curve fitting) can be corrected somehow by the duration of lag phase. I mean, I want to compare the growth rate obtained from several cultures which started from different amount of inoculum and showing different lag phase periods.

Thanks.

There are a lot of good answers above, but one further thing to consider is "apparent lag" which is very much innoculum size dependent. If the number of viable cells is below the detection limit (I presume you are measuring optical density) you will not be able to measure change in OD until the cell density is high enough to be in the measurable range.

I think the explanations given above about effect of innoculum size are correct. his published article by Agbogbo et al., 2006 on the co-fermentation of glucose and ylose for bio-ethanol production is also explanatory on the subject matter:

www.sciencedirect.com/science/article/pii/S1359511306001784‎

The most used proportion is between 5 and 10%...But an optimization could be a better solution....

Probability

Two things are important for mastering minimisation lag phase .

1° try to have the optimal physiological state of inoculum with an accurate knowledge of it . For industrial fermentation this step is very important to minimise the time of adaptation

2° the lag phase is dependant of innoculum size under a critical concentration

-X inoccrit -that we need to determine. In general the lag time follow a law like :

              ( T lag=f(1/Xinoc)       if    Xinoc is inferior of Xinoccrit)

Xinoccrit is generally beetween 0.1 and 0.2 Gdw/L

Xinocrit is the cell concentation (dry weigth ) when the lag phase is zero

There is truth in al the above comments.  What I can bring to the discussion is that it is worth having a look at two topics in this reation:  quorum sensing and  the lonely cell phenomenon.

I appreciate the question? It is a nice observation. I am also agree with Dr. Behnam Taidi '' the quorum sensing and the lonely cell phenomenon''. From this question I also learned a lot. So informative. Thanks....Rasoul Shafiei

Hello all, I have a follow up question related to this:

To get a log phase of Salmonella, I am harvesting the cells after 4 hours. Will it matter if I use initial inoculum of of 7 log (approx 1 colony) or 8 log, or 9 log and inoculate in 10 ml of nutrient broth and incubate for 4 hours? I will be glad if references for further reading are provided. Thank you in anticipation. 

To reduce the lag phase your inoculum should be at the end of the exponential growth phace. At this moment you should inoculated your media fermentation.  If you repeat this when your innocolum is at the end of exponential growth face, the lag phace should desapear. If you increase the inoculum could have problems with the performance of your fermentation 

The inoculum is usually in the log phase, so it usually already passed the lag phase, thus putting the log phase innoculum in the fermentation medium can make the microbe continue to be in the log phase. https://lab.plygenind.com/inoculum-preparation-for-fermentation-a-practical-guide