Microcalorimetric Monitoringof Microbial Growth in Solid-    State FermentationsMenert, A., Kazarjan, A., Stulova, I., Lee...
Why calorimetry?                               Calorimetry is an extremely appropriate method for                         ...
Ice calorimeter of Lavoisier-Laplace                                                             The quantities of heat th...
Isothermal                                                         microcalorimeter                                       ...
General advantages of calorimetry                                                low specificity                          ...
Multichannel calorimeters         TAM III SystemXIVth International Society for Biological CalorimetryConference June 2 - ...
Reproducibility of data on TAM III    Lab Assistant Results Report    Ampoule (5-25-06)-lactic acid bacteria.rslt    Summa...
Introduction           Solid-phase fermentations are of great           interest in:                 Cheese ripening      ...
Structure of agar                                  β-(1-3)-D and α-(1-4)-L bonded galactose                               ...
Bacterial growth curve                                                         1 – Lag- phase                             ...
Bacterial growth curve                                                         Lag- phase                                 ...
Bacterial growth in colonies                                             Every colony starts to grow from one             ...
Parameters used to describe the growth of     colonies (Malakar et al, 2002)                                              ...
Every colony starts to grow from one single cell    Morphology of colonies is dependant       on many factors; the simplie...
Determination of bacterial growth in solid state       dependant on the concentration of glucose and agar                 ...
Bacteria studied          Lactobacillus paracasei S1R1 – lactic acid          bacterium isolated from estonian type cheese...
The parameters determined                Outgrowth percentage (%)                   how many of the cells inoculated will ...
Rakkude koguse kasv              3.50E+10                                                                                 ...
The concentration of colonies in volumetric unit of  sample depending on agar and glucose concentrations                18...
Outgrowth percentage of lactic acid bacteria at various                glucose and agar concentrations     Glucose, g/ L  ...
Dependance of lag-phase length on glucose concentration                                                   Lag- faas i pik ...
Determination of growth rate of Lactobacillus paracasei S1R1 in solid-  state fermentations at various glucose concentrati...
Outgrowth percentage, duration of lag phase            dependant on glucose and agar concentration          It was shown t...
Preparation of inocula with different concentration of                              bacteriaXIVth International Society fo...
Thermal Activity Monitor TAM 2277                                    Combination measuring unit
Growth curves at high colony number and low colony              number are different        180        160                ...
Growth at large number of colonies can be                  approximated to the growth in liquid culture            180    ...
Growth in large number of colonies can be              approximated to the growth in liquid culture        180        160 ...
Biomass growth rate is proportional to the heat                       production rate (in exponential phase)              ...
Calculation of specific growth rate µ•   In exponential growth phase        dX/dt = µX                          (1)•   If ...
Specific growth rates and heat production of Lb. paracasei                  and L. lactis in agaroseExperiment           1...
The difference in growth curve is            dependent on                    growth limitation by diffusion               ...
At high colony number (40 - 50) Growth curves in liquid medium were similar to those with higher number of colonies (~40) ...
Cultivation of Lb.casei at high inoculation rate (103 – 107)                                                              ...
At low colony number (< 10) Maximum specific growth rate µmax is lower (0.15-0.30 h-1) Provided that inhibition of growth ...
Colony growth limitation by diffusionXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sop...
Heat production during lag-phase and exponential phase is   independant of the number of colonies in the ampoule;         ...
Conclusions (outgrowth percentage)         The outgrowth percentage dependance on glucose         concntration has “three ...
Conclusions (microcalorimetric data)         At high number of colonies (40-50) in a closed volume bacterial         growt...
Special thanks...                           Vallo Kõrgmaa                           Romi Mankin                           ...
Thank you!
Upcoming SlideShare
Loading in …5
×

XIVth International Society for Biological Calorimetry Conference, 2006

700 views

Published on

Presentation on the XIVth International Society for Biological Calorimetry Conference on June 2 - 6, 2006 in Sopot, Poland

Published in: Technology, Travel
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
700
On SlideShare
0
From Embeds
0
Number of Embeds
5
Actions
Shares
0
Downloads
0
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

XIVth International Society for Biological Calorimetry Conference, 2006

  1. 1. Microcalorimetric Monitoringof Microbial Growth in Solid- State FermentationsMenert, A., Kazarjan, A., Stulova, I., Lee, C.C., Vilu, R. Tallinn University of Technology
  2. 2. Why calorimetry? Calorimetry is an extremely appropriate method for studying microbiological processes. Thermal power-time curves are influenced by the metabolic activity and can be related to the different physiological states of bacteria (Kemp and ( Lamprecht, 2000). From microcalorimetric data the thermodynamic (∆H) as well as kinetic (µ=dX/(X·dt)) parameters of a process can be calculated.XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  3. 3. Ice calorimeter of Lavoisier-Laplace The quantities of heat that are produced or absorbed are proportional to the extent of the processes involved.XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  4. 4. Isothermal microcalorimeter 2277 TAMXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  5. 5. General advantages of calorimetry low specificity good reproducibility non-destructive analysis continuous registration of processes possibility to analyze turbid or coloured samples high throughput of samplesXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  6. 6. Multichannel calorimeters TAM III SystemXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  7. 7. Reproducibility of data on TAM III Lab Assistant Results Report Ampoule (5-25-06)-lactic acid bacteria.rslt Summary Name: Ampoule (5-25-06)-lactic acid bacteria.rslt Start time: May 25, 2006 21:10:59 End time: Jun 01, 2006 12:44:15 Operator: AM Results file path: C:Documents and SettingsAnneMy DocumentsTAM III experimentsAmpoule (5-25-06)-lactic acid bacteria.rslt General Experiment Info 150 Bath temperature: 25 °C Sample - Ch 3:1 Sample - Ch 3:2 Name: Lactic acid bacteria in MRS 100 Hea t flo w (µ W) µmax1= 0.2658 h-1 µmax2= 0.2580 h-1 50 Qtot1= 29,408 J Qtot2= 29,514 J 0 May 27 May 29 Jun 01XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  8. 8. Introduction Solid-phase fermentations are of great interest in: Cheese ripening Spoiling of meat products etc. Solid matrix forces the cells to grow in colonies, not free flowing Solid-phase fermentations have been less studiedXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  9. 9. Structure of agar β-(1-3)-D and α-(1-4)-L bonded galactose Scheme of agar gelatination Repeating unit in agar structureXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  10. 10. Bacterial growth curve 1 – Lag- phase 2 – Exponential phase 3 – Declining growth phase 4 – Stationary phase 5 – Lysis phase Usually more attention is paid to phases 1-3, as biomass growth takes place there which is essential in biotechnological industry. The most important is phase 2 (exponential growth) where productivity is the greatest.XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  11. 11. Bacterial growth curve Lag- phase Exponential phase Stationary phase Van Impe et al., 2005XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  12. 12. Bacterial growth in colonies Every colony starts to grow from one single cell: the radius of colony Rcol increases in time by addition of new cells Lag-phase – acclimatization phase Rcol Exponential phase – the radius of colony increases Stationary phase – the increase of the radius of colony has stoppedXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  13. 13. Parameters used to describe the growth of colonies (Malakar et al, 2002) Rboundery – boundery of living space Rcol Rcol Rcol – radius of growing colony Rboundery dX dX dRcol = µX =c dt dt dt Malakar, P. K., Martens, D.E., Van Breukelen, W., Boom, R. M., Zwietering, M. H., Van ,t Riet, K. Appl. Environ. Microbiol., 2002, 3432-3441XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  14. 14. Every colony starts to grow from one single cell Morphology of colonies is dependant on many factors; the simpliest geometrical shapes are sphere and ellpisoidXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  15. 15. Determination of bacterial growth in solid state dependant on the concentration of glucose and agar 104 cfu/flaskXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  16. 16. Bacteria studied Lactobacillus paracasei S1R1 – lactic acid bacterium isolated from estonian type cheese, belonging to NSLAB (non-starter lactic acid bacteria) Lactococcus lactis – typical lactic acid bacteriumXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  17. 17. The parameters determined Outgrowth percentage (%) how many of the cells inoculated will form the colonies duration of lag-phase (t – hours) Growth rate (µ – h-1) The dependance of these parameters on glucose (2-50 g/L) and agar concentration (1, 3, 5%) was measured The growth of individual colonies was monitored in the experiments The deep inoculation was made with low inoculation rate The aim was to achieve long distances between the colonies, to guarantee the independant growth of individual colonies Constant incubation temperature 31oC was keptXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  18. 18. Rakkude koguse kasv 3.50E+10 17 h 3.00E+10 2.50E+10 Change of bacterial number in timekogus 1 cm3 2.00E+10 1.50E+10 1.00E+10 5.00E+09 0.00E+00 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 17 h aeg OD muutus ajas 1.7 1.6 1.5 1.4 Change of bacterial 1.3 OD in time 1.2 OD (540 nm) 1.1 1 0.9 0.8 0.7 XIVth International Society for Biological Calorimetry 0.6 0.5 Conference June 2 - 6, 2006 Sopot, Poland 0.4 0.3 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Tunnid
  19. 19. The concentration of colonies in volumetric unit of sample depending on agar and glucose concentrations 1800 1600 1400 1200 1 % agar col/mL 1000 3% agar 800 5 % agar 600 400 200 0 0 10 20 30 40 50 60 glucose g/LXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  20. 20. Outgrowth percentage of lactic acid bacteria at various glucose and agar concentrations Glucose, g/ L 2 5 10 15 20 30 40 50 Agar, % 1 2,60 3,40 3,12 2,72 6,29 0,05 0,05 0,03 3 1,03 2,63 2,45 1,77 4,15 0,04 0,05 0,03 5 0,71 0,53 0,41 0,47 0,55 0,04 0,04 0,02 Glucose, g/ L 2 5 10 15 20 22 24 26 28 30 40 50 Agar, % 3 0,10 0,17 0,58 1,46 1,44 1,44 1,66 1,60 1,50 0,04 0,05 0,03 1,80 1,60 Outgrowth percentage, % 1,40 1,20 1,00 0,80 0,60 0,40 0,20XIVth International Society for Biological Calorimetry 0,00 0 5 10 15 20 25 30 35 40 45 50 55Conference June 2 - 6, 2006 Sopot, Poland Glucose, g/L
  21. 21. Dependance of lag-phase length on glucose concentration Lag- faas i pik k us e rine vate l glük oos i k onts . 3% agar (in 3% agar) 100 90 80 70 Lag- faasi pikkus, t 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 glük oos , g/lXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  22. 22. Determination of growth rate of Lactobacillus paracasei S1R1 in solid- state fermentations at various glucose concentrations (3% agar) 0,04 0,03 -1 Growth rate, h 0,02 0,01 0,00 0 5 10 15 20 25 30 35 40 45 50 55 Concentration of glucose, g/LXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  23. 23. Outgrowth percentage, duration of lag phase dependant on glucose and agar concentration It was shown that the outgrowth percentage of bacteria studied increases with the increase of glucose concentration 2-15g/L, the outgrowth percentage is maximal and practically the same at limiting substrate concentrations 15-30g/L, but it is dependant on the concentration of agar. The maximum outgrowth percentage was measured in 1% agar at 20g/L glucose -– 6%. At Gucose concentrations > 30g/l the outgrowth percentage decreased dramatically. At the same glucose concentrations the outgrowth percentage decreased with increasing the agar concentration. Almost in the same manner behaved the duration of lag-phase. The measured minimum lag-phase duration was 20 hours.XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  24. 24. Preparation of inocula with different concentration of bacteriaXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  25. 25. Thermal Activity Monitor TAM 2277 Combination measuring unit
  26. 26. Growth curves at high colony number and low colony number are different 180 160 Lb paracasei 7 kol Lb. paracasei 7 kol L. lactis 6 kol 140 L. lactis 40 kol L. lactis 45 kol 120 Lb. paracasei 40 kol 100dQ/dt 80 60 40 20 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 XIVth International Society for Biological Calorimetry Time, h Conference June 2 - 6, 2006 Sopot, Poland
  27. 27. Growth at large number of colonies can be approximated to the growth in liquid culture 180 160 L. lactis 40 col L. lactis 45 col Lb. paracasei 40 col 140 Lb. paracasei 45 col 120 100 dQ/dt 80 60 40 20 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Time, hXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  28. 28. Growth in large number of colonies can be approximated to the growth in liquid culture 180 160 L. lactis 40 col L. lactis 45 col Lb. paracasei 40 col 140 Lb. paracasei 45 col 120 150 100dQ/dt 80 100 Hea t flo w (µW) 60 50 40 0 20 May 27 May 29 Jun 01 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Time, h XIVth International Society for Biological Calorimetry Conference June 2 - 6, 2006 Sopot, Poland
  29. 29. Biomass growth rate is proportional to the heat production rate (in exponential phase) Specific growth rate of X Ansorbance microorganisms µ Cellcount Biomass ln X dX/dt = µX µ=(lnXt-lnX0)/t µ=1/X*dX/dt Xt=X0*eµt lnXt =lnX0 + µt Time qdQ/ Qs1 my1 Q µµW/mL µJ/mL 1/hdt 2.5e+06 0.50150 6 5 5 ln dQ/dt = 0.648 + 0.272 t µmax = 0.272 h-1 3120 2e+06 0.40 4 3 190 1.5e+06 0.30 2 ln dQ/dt 1 ln Q 0 5 10 15 20 -1 060 1e+06 0.20 0 5 10 15 20 -1 ln Q = - 3.382 + 0.254 t -3 -2 µmax = 0.254 h-130 500000 0.10 -3 -5 hours -4 0 0 0 0 4 8 12 16 20 -5 -7 time Time / h Region for calculation of maximum specific growth rate ln (dQ/dt) = ln (dQ/dt)t=0 + µt
  30. 30. Calculation of specific growth rate µ• In exponential growth phase dX/dt = µX (1)• If the stoichiometry of biomass growth does not change during the growth (dX/dt) is proportional to dQ/dt and (X-X0) is proportional to Q.• The rate of biomass increase is proportional to the rate of increase in the heat production (where YQ is the proportionality factor): dX/dt = YQ * dQ/dt (2)• From definition of specific growth rate (Eq. 1) and replacing it into Eq. 2 we get the relationship between µ and dQ/dt: µX = YQ * dQ/dt (3)• The increase of biomass in the exponential growth phase is an exponential function: X = X0 * eµt (4)• Replacing X from Eq. (4) into Eq. (3) µ * X0 * eµt = YQ * dQ/dt (5) dQ/dt = 1/YQ * µ * X0 * eµt (6) • After integrating :ln (dQ/dt) = ln (dQ/dt)t=0 + µt (7) where ln (dQ/dt)t=0 = ln (1/YQ * µ * X0 * eµ).
  31. 31. Specific growth rates and heat production of Lb. paracasei and L. lactis in agaroseExperiment 101005 -1 Channel Inoculum µmax, h Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies 1 sm1 Lb.paracasei S1R1 10 0,3128 36,15 213,59 1,5063 10,581 2,2967 19,809 30,390 31,981 40 1 sm2 Lb.paracasei S1R1 10 0,2922 30,21 271,48 1,2587 11,215 2,0147 23,490 34,705 36,000 40 2 sm3 Lb.paracasei S1R1 10 0,3128 40,33 145,67 1,6806 12,767 2,7251 22,171 34,938 39,035 40 2 sm4 Lb.paracasei S1R1 10 0,3004 40,39 136,50 1,6830 10,434 2,662 19,946 30,380 34,717 40Experiment 171005 Channel Inoculum µmax, h-1 Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies 0 sm1 Lb.paracasei S1R1 10 0,1501 52,52 96,13 2,1882 10,800 39,330 2 0 sm2 Lb.paracasei S1R1 10 0,2398 55,90 59,48 2,3292 9,526 34,415 5 1 sm3 Lb.paracasei S1R1 10 0,2016 47,26 95,34 1,9691 11,372 40,528 3 1 sm4 Lb.paracasei S1R1 10 0,3052 50,94 67,78 2,1226 9,044 32,238 7Experiment 091105 -1 Channel Inoculum µmax, h Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies 1 sm1 L. lactis S1R1 10 0,1943 52,58 96,07 2,1910 12,402 41,741 6 sm2 L. lactis S1R1 102 0,4243 38,27 165,76 1,5944 12,902 2,6637 23,709 36,611 40,178 45 1 sm3 Lb.paracasei S1R1 10 0,1795 67,23 65,89 2,8014 17,631 42,585 7 2 sm4 Lb.paracasei S1R1 10 0,2130 37,95 148,11 1,5813 11,259 2,5773 20,323 31,582 35,173 40Experiment 151105 Channel Inoculum µmax, h-1 Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies 1 sm1 Lb.paracasei S1R1 10 0,2373 49,33 89,86 2,0556 11,421 40,522 8 2 sm2 Lb.paracasei S1R1 10 - 190,07 8,08 7,9194 - 0 1 sm3 L. lactis S1R1 10 0,3015 37,70 170,86 1,5708 11,672 2,5363 22,085 33,757 43,237 50 2 sm4 L. lactis S1R1 10 0,3961 39,50 150,76 1,6458 8,261 2,5534 19,591 27,851 31,623 40
  32. 32. The difference in growth curve is dependent on growth limitation by diffusion screening effect – in large colonies bacteria grow only on the outer layer growth limitation by the production of lactic acidXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  33. 33. At high colony number (40 - 50) Growth curves in liquid medium were similar to those with higher number of colonies (~40) in solid state. Initially, after the lag-phase the bacteria grow with maximum growth rate µmax= 0.30- 0.40 h-1. After that limitation of substrate (glucose) starts retarding the growth. The growth is finally hindered by the production of lactic acid. The limiting factor at large number of colonies or in liquid culture is production of lactic acidXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  34. 34. Cultivation of Lb.casei at high inoculation rate (103 – 107) 8 10 300 8 10 200 B 7 9 A 100 7 9 200 100 8 8 -1 6 dQ/dt, µW mL -1 -1 log cfu mL 6 0 0 log cfu mL -1 dQ/dt, µW mL 7 7pH pH -100 5 -100 5 6 6 -200 5 5 4 -200 4 -1 -1 dQ/dt, µW mL dQ/dt, µW mL -300 -1 -1 log cfu mL log cfu mL 4 pH 4 pH 3 -300 3 -400 0 10 20 30 40 50 0 10 20 30 40 50 Tim e, h Time, h 8 10 300 9 C 7 200 Bacterial count (♦), thermal power (--) and pH ( ) for 8 100 inoculum size dQ/dt, µW mL-1 6 log cfu mL-1 7 A - 103 cfu mL-1 (MRS + agarose)pH 0 B - 105 cfu mL-1 (MRS + agarose) 5 6 C - 107 cfu mL-1 (MRS + agarose) -100 5 4 -1 dQ/dt, µW -1 mL -200 log cfu mLXIVth International Society for Biological Calorimetry 4 pH 3Conference June 2 10 6, 2006 Sopot, Poland 0 - 20 30 40 50 Time, h
  35. 35. At low colony number (< 10) Maximum specific growth rate µmax is lower (0.15-0.30 h-1) Provided that inhibition of growth is limited by diffusion the growth is retarded mainly due to substrate (glucose) defficiency If the number of colonies is small (~7), the measured growth rate µ< µmax as the growth rate is determined by the diffusion rate as well as (possibly) by the screening effect of outer layer cells. The growth is limited by diffusion of glucose until the end, when it stops as a result of ending the glucose supply and/or inhibition by formation of lactic acidXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  36. 36. Colony growth limitation by diffusionXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  37. 37. Heat production during lag-phase and exponential phase is independant of the number of colonies in the ampoule; Q1 = const Q1=9-12 J / per ampouleP,µW Pin[1](t) Pin[2](t) Pin[3](t) Pin[4](t) P,µW Pin[1](t) Pin[2](t) Pin[3](t) Pin[4](t) 10.581 J 9.0436 J 150 45 colonies 7 colonies 400 100 200 30.390 J Q3 50 Q1 Q2 Q1 Q2 31.981 J 31.875 J 0 0 0 20 40 60 Time,hour 0 2 4 Time,day Qtot= Q1 + Q2 + Q3 Total heat production in ampoule is constant, Qtot= const Qtot=30-40 J / per ampole XIVth International Society for Biological Calorimetry Conference June 2 - 6, 2006 Sopot, Poland
  38. 38. Conclusions (outgrowth percentage) The outgrowth percentage dependance on glucose concntration has “three phases” – 2-15 g/L: with increasing the glucose concentration the outgrowth percentage increases; 15-28 g/L: the region with with maximum outgrowth percentage 30-50 g/L: high glucose concentrations inhibit outgrowth With increasing the concentration of agar the outgrowth percentage decreases.XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  39. 39. Conclusions (microcalorimetric data) At high number of colonies (40-50) in a closed volume bacterial growth in solid state is similar to the growth in liquid medium. The growth is mainly limited by the production of lactic acid. At low number of colonies (<10) the growth rate is dependant on the number of colonies in the closed volume. The growth is mainly limited by hindered diffusion of glucose. Heat production during lag-phase and exponential phase is independant of the number of colonies in the ampoule. Total heat production in a closed volume (ampoule) is constant and independant of the number of colonies.XIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  40. 40. Special thanks... Vallo Kõrgmaa Romi Mankin Glafira Shkaperina Natalja Kabanova Signe AdambergXIVth International Society for Biological CalorimetryConference June 2 - 6, 2006 Sopot, Poland
  41. 41. Thank you!

×