Biomass Production of the Bacteria Oenococcus oeni Ashton, R., Juenke, C., Kennedy, B., and Leitschuh, J., School of CBEE

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].

1 μm

Cell density (X, g/L) changes over time at a rate proportional to the current cell density.

dXX

dtMathematically this is represented as:

Modeling Biomass Production

where μ represents the specific growth rate. During exponential growth the specific growth rate reaches its maximum and remains constant at μ = μmax.

Therefore, during exponential growth:max

0( ) tX t X eThe 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.

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 growthGoal: maximize the cell density at the stationary phase.

Time, t

ln(X

)

Stationary phase

Log(exponential)phase

Death phase

Lag phase

A typical growth curve for a bacterial culture in a bioreactor.

Analysis of growth curves from O. oeni yielded: 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.

Biomass Production Results

1

max 0.07hr

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.

The growth curves for O. oeni. Inoculum for the fed-batch run had been previously refrigerated which caused the extended lag phase.

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.

Problem Statement

Background

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.

Business Plan

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.

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

Centrifuge Pre freeze-drytreatment Freeze-dryer

Bioreactor

Cell brothAdaptedcell broth

Freeze-driedcells

Inoculum

Concentratedcell broth

Process flow diagram for the biomass production of Oenococcus oeni.

• Dr. Christine Kelly• Dr. Phil Harding• Coralie Backlund• Kelsey Yee• Andy Brickman

Acknowledgements

Biomass Production Process

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.

[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-wine-making-genome.html

[2] African Trading Company. http://www.africantradingco.com/winebarrels.html

[3] Feast NH. http://blogs.nashuatelegraph.com/livefreeordine/2009/04/

References

Industry Production Scale-Up

D = 0.9 m

Hcone = 0.5 m

Htotal = 1.8 m

Lab scale bioreactor. Scale-up bioreactor design.

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).

MotorAgitator

Air sparger

Baffle

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.

Vacuum pump attached to the Lyo-centre lyophilizer. 

Lyo-centre vacuum chamber and vacuum flask.

Vacuum pump

Vacuum chamber

Freeze-drying cells

Vacuum chamber

Fed batch started here. It did not significantly increase growth.

Malate Lactate + CO2

O. oeni

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].

Extended lag phase due to refrigeration of cells prior to inoculation.