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Biomass Production of the Bacteria Oenococcus oeni
Ashton, R., Juenke, C., Kennedy, B., and Leitschuh, J., School of CBEE
Problem Statement
Biomass Production Process
Modeling Biomass Production
Commercial biomass production of the bacteria Oenococcus Oeni (a bacteria that
performs malolactic acid fermentation in wine) is currently inefficient and
expensive. The growth, processing and packaging of O. oeni will be optimized in
order to create a cost effective strategy for biomass production.
O. oeni biomass is grown to its maximum cell density in a bioreactor. The cell broth is then
concentrated by centrifugation. Prior to freeze-drying the concentrated cells are exposed to
pre freeze-drying treatments such as cryoprotectants. The cells are then freeze-dried for
packaging and storage.
Cell density (X, g/L) changes over time at a rate proportional to the current cell density.
Background
X (t )  X e
Adapted
cell broth
Cell broth
Centrifuge
Pre freeze-dry
treatment
The specific growth rate is effected by a number of factors including temperature, pH,
dissolved oxygen and growth media composition. Optimization of μmax is integral to
optimizing biomass production.
Freeze-dryer
Bioreactor
Freeze-dried
cells
Process flow diagram for the biomass production of Oenococcus oeni.
1 μm
Electron microscopy image of the bacteria
Oenococcus oeni (~1 μm), which is used to
reduce tartness in wine by converting malic acid
from grapes to a softer tasting lactic acid [1].
Industry Production Scale-Up
The Oregon market calls for 1000 lb of bacteria per year. Using growth parameters
determined in a 3L bioreactor (left), nine 1000L bioreactors would be required to meet this
demand. The scaled-up design (right, not to scale) takes into account freeboard space, a
conical bottom of height, Hcone, and a height, Htotal, to diameter, D, ratio of 2 (a general
heuristic for bacterial bioreactors).
Motor
Air sparger
Agitator
Baffle
O. oeni
Scale-up
O. oeni performs ML fermentation in wine production, reducing tartness by
converting malate (malic acid) in grapes to lactate (lactic acid) and giving the wine a
“buttery” flavor and aroma [2], [3].
Htotal = 1.8 m
Time, t
A typical growth curve for a bacterial culture in a
bioreactor.
Fed batch started here.
It did not significantly
increase growth.
Scale-up bioreactor design.
Downstream Processing and Packaging
The effect of lyophilization (freeze-drying) in varying sucrose solutions on cell viability is
currently being investigated. Lyophilization is being considered for production scale storage
and packaging.
1.5
Extended lag phase due to
refrigeration of cells prior
to inoculation.
1
0.5
Fed-Batch Operation (Run 4)
Batch Operation (run 3)
Vacuum chamber
0
References
Freeze-drying cells
[1] Yarris, L. (n.d.). Science Beat. Secrets of the wine cellar: the
genome of a wine-making microbe . Retrieved March 2, 2010, from
Berkely Lab: http://www.lbl.gov/Science-Articles/Archive/JGI-winemaking-genome.html
[3] Feast NH.
http://blogs.nashuatelegraph.com/livefreeordine/2009/04/
Lag phase
2
Vacuum chamber
[2] African Trading Company.
http://www.africantradingco.com/winebarrels.html
Log
(exponential)
phase
2.5
Cell Density (g/L)
Market and Strategy: Oregon’s Willamette Valley will be the target market. Our
strategy will be to provide the following:
• Superior quality and service
• Culture variety including indigenous bacteria for the wine industry
• Execute ML ferments with upwards of 95% confidence
• Localized service
• Reduced costs
Lab scale bioreactor.
Death phase
Oenococcus oeni was grown in a 3L lab-scale bioreactor containing the rich medium Luria
Broth at pH 4.0 and 25°C (optimum values determined from literature) under both batch and
fed-batch conditions. Fed-batch operations did not appear to effect the maximum cell
density.
D = 0.9 m
Mission statement: To cultivate microorganisms for the wine and beer industry in the
Pacific Northwest and provide a superior product that will maximize fermentation
efficiency by reducing energy and biomass costs.
Stationary phase
Biomass Production Results
Hcone = 0.5 m
Business Plan
• Dr. Christine Kelly
• Dr. Phil Harding
• Coralie Backlund
• Kelsey Yee
• Andy Brickman
Cell growth can be categorized
into 4 stages:
• Lag phase: the organism
becomes used to the
environment.
• Exponential phase: cells grow
exponentially.
• Stationary phase: biomass
production ceases
• Death phase: cell death
exceeds cell growth
Goal: maximize the cell density
at the stationary phase.
ln(X)
Concentrated
cell broth
Malate Lactate + CO2
Acknowledgements
Mathematically this is represented as:
where μ represents the specific growth rate. During exponential growth the specific growth
rate reaches its maximum and remains constant at μ = μmax.
max t
Therefore, during exponential growth:
0
Inoculum
Oenococcus oeni is an important
bacterial species used for malo-lactic
(ML) fermentation in the wine
industry. ML fermentation is a
secondary fermentation in wine in
which malic acid (a naturally occurring
acid in grapes) is converted to lactic
acid, and occurs after the primary
alcoholic fermentation performed by
yeast. It improves wine quality by
reducing the total acidity, thereby
softening the wine. In addition, it
infuses a favorable buttery aroma from
an intermediate of ML fermentation
called diacetyl.
dX
 X
dt
0
50
100
Time (hr)
150
The growth curves for O. oeni. Inoculum for the fed-batch run had
been previously refrigerated which caused the extended lag phase.
Analysis of growth curves from O. oeni yielded:  max  0.07hr
which correlates to the highest specific growth rate for this organism found in literature.
Maximum cell densities occurred around 1.7-1.9 g/L. This appeared to be independent of
substrate levels as indicated by the fed-batch operation. These values are also verified by
literature as being high yields for O oeni.
1
Vacuum pump
Vacuum pump attached to
the Lyo-centre lyophilizer.
Lyo-centre vacuum
chamber and vacuum flask.
200