standardizing microbiome research – culture from discovery ... · acid/base addition via pump ph...
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Standardizing Microbiome Research – Culture from Discovery to ProductionEppendorf AG Bioprocess Center, Jülich, Germany Eppendorf AG, Hamburg, GermanyEppendorf Inc., Enfield, CT, USAContact: [email protected]
Your local distributor: www.eppendorf.com/contact · Eppendorf AG · Barkhausenweg 1 · 22339 Hamburg · Germany · www.eppendorf.com
Various microbiomes
Productivitywww.eppendorf.com
ELABTM is a registered trademark of Eppendorf AG. eLabJournal® is a registered trademark of Bio-ITech B.V., Netherlands. Eppendorf®, the Eppendorf Brand Design, and BioBLU® are registered trademarks of Eppendorf AG, Germany. DASbox® and DASGIP® registered trademark of DASGIP Information and Process Technology GmbH, Germany. BioFlo® is a registered trademark of Eppendorf Inc., USA. All rights reserved, including graphics and images. Copyright © 2019 by Eppendorf AG.
Microbiome workflow
Eppendorf supports the microbiome workflow from sample management, to sample preparation and analysis, to bacterial culture
Liquid addition Temperature control Oxygen control pH control
Microbiome culturing needs are very diverse
Bioprocess systems allow monitoring and control of the dissolved oxygen (DO) concentration in the culture medium
> DO monitoring using a DO sensor > Monitoring of redox potential using redox sensors under an-aerobic conditions > Gassing with air, O2, N2, CO2
> Anaerobic conditions by gassing with anaerobic gas mixtures > Automated gas mix via thermal mass flow controllers (TMFC)
Scalable bioprocess systems for microbiome culturing needs from research to production
> Presence of oxygen: Aerobic
> No presence of oxygen: Anaerobic
> Temperature
> pH
> Nutrients and metabolites
> ...
> Soil
> Gut
> Vaginal
> Skin
> ...
Store
An
alyze
Growth conditions can vastly differ depending on sample origin They require ideal match to source the environment
Automated DO control via a cascade. Oxygen transfer is increased by sequentially increasing the agitation speed, the flow rate, and the oxygen concentration in the gas mix.
The growth of the cells will cause the pH to drift. Bioprocess systems allow online monitoring and control.
> pH monitoring using pH sensor > pH control by automatic addition of pH agent to the culture using the system’s integrated pumps > One-sided (usually only base) or two-sided (acid and base) pH control is possible.
Schematic representation of pH control loop.
Most processes require a mechanism to control temperature at a set point that encourages optimum cell growth.
> Microbial communities need physiological temperature > Heat-generating processes require cooling within the culture > Different devices available to control the temperature in a bioprocess: Heat blanket, water jacket or thermowell
Example for temperature control in a fermentation process in a DASGIP® Parallel Bioreactor System. The temperature setpoint (T1.SP), the process value (T1.PV) and the controller output percentage (T1. Out) are shown.
Liquids can be added to a bioprocess using the system’s integrated pumps.
> pH agent > Feed solutions > ...
Schematic representation of experimental concept to mimick the gastrointestinal tract in a bioreactor. Liquids (saliver, gastric juice, pH agent, bile, trypsin) werde added to a DASGIP vessel using the DASGIP MP8 multi pump module. Adapted from Luiking et al., Clin. Nutr. 35(1): 48-58. 2016.
DASbox Mini Bioreactor System
> Parallel operation of up to 24 vessels > Working volume: 65 mL – 250 mL > Small working volumes save on the amount of cell material, media and supplements required
DASGIP Parallel Bioreactor System
> Parallel operation of up to 16 vessel > Working volume: 0.2 L – 3.8 L > Modular design of control units allows for flexible system configurations that meet the demands of specific applications
Small-scale parallel bioreactor systems
> Ensure maximum reproducibility between runs
> Save time
Parallel experimentation to compare different culture conditions. Cultivation of Lactobacillus sp. in an 8-fold DASGIP Parallel Bioreactor System. Bacterial growth at various temperatures was compared. NaOH consumption was used as a proportional indicator for growth-dependent acidification (Eppendorf Application Note 299).
Usually, process development is carried out at small volumes and the process is subsequently scaled up to larger production volumes.
Bench-scale systems > BioFlo® 120 and BioFlo 320 bioprocess control station > Working volume (fermentation): 0.4 L – 10.5 L > Universal connections for digital and analog sensors
Large-scale systems > BioFlo 510, BioFlo 610, BioFlo Pro > Working volume (fermentation): 10.5 L – 1,200 L > Sterilize-in-place systems > Modular desing for system flexibility
Stirred-tank bioreactors > Comparable vessel geometries across scales support scale-up > Vessel characterization to identify OTR capabilities, tip speed range, and impeller power numbers > The vessel capabilities need to be taken into account to make sure that process conditions at small scale can be reproduced at larger scales.
Scale-up of E. coli fermentation from 1 L to 10 L to 100 L. Biomass growth curves were comparable at all scales. Fermentations were carried out using a constant power input per volume (P/V) value.
Soil microbiome
Leaf microbiomeOral microbiome
Gut microbiome
Skin microbiome
Lung microbiome
Vaginal microbiome
“We start to understand that microbes are important to our ecosystem and our health”
“Bacteria, if we change the ones inside you have the potential to cure diseases”
(Dr. Jack Gilbert, Professor in the Department of Pediatrics and the Scripps Institution of Oceanography, UCSD - Center of Mirobiome Innovation)
Feedback
Loop
Pump
Bioreactor
pH measurement
Transfer sensor readingto bioprocess control
software
Bioprocesscontrol software
Acid/base additionvia pump
pH sensor
acidic~neutral
37 °C
> Saliva> Gastric juice> pH agent
> Pancreatin/bile> Trypsin> pH agent
Liquids added DASGIP MP8
multi pump module
Processparameters
~neutral
Time
Stomach Intestine
-15
-5
5
15
25
35
45
25
27
29
31
33
35
37
39
0:00:00 2:00:00 4:00:00 6:00:00 8:00:00 10:00:00 12:00:00 14:00:00 16:00:00 18:00:00 20:00:00
T1.O
ut [%
]
T1.P
V [°
C], T
1.SP
[°C]
T1.PV T1.SP T1.Out
Inocula�on Time
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7 8 9
OD
600
Time (h)
Bioblock 1 L
BioFlo 320 10 L
BioFlo 610 100 L
Sample
Data analysis
NextGenerationSequencing
Preparelibrary
Ampli-fication
DNA/RNApurification
Samplemanagement
Samplestorage
Data analysis
Mass spectrometry
Samplepreparation
Proteinseparation
Protein/metabolitepurification
BioprocessingCell
growthMicro-
culturesCell
identification
Culture