Issues related to Microorganisms for Bioprocessing

Issues related to Microorganisms for Bioprocessing

Contents:BioprocessingMicrobes in bioprocessingIssues of microorganism in bioprocessingIssues in isolationIssues in strain improvementIssues of production strainConclusion

Bioprocessing:

Application of natural or genetically manipulated whole cells/ tissues/ organs, or parts thereof, for the production of industrially or medically important product

Encompasses research, development, manufacturing, and commercialization of products prepared by using biological systems

Clementschitsch F. and K. Bayer (2006) Improvement of bioprocess monitoring: development of novel concepts. Microbial cell factories. 5:19

Bioprocessing:

Two types of processing

Upstream processing

Downstream processing

Clementschitsch F. and K. Bayer (2006) Improvement of bioprocess monitoring: development of novel concepts. Microbial cell factories. 5:19

Issues of microorganisms in bioprocessing

Initial Isolation1st Step: Isolation of Pure or Mixed Cultures from nature

2nd Step:Assesment to determine the capacity for the production

Krishnan S., S. Prapulla, D. Rajalakshmi, M. Misra and N. Karanth (1998) Screening and selection of media components for lactic acid production using PlackettBurman design. Bioprocess engineering. 19:61-65

Undesirable CharacteristicsExpensive ProcedureChances of contaminationLow ProductivityTime takingGenetic ChangeLoss of viabilityIssues:

Strain ImprovementSelection of natural variantsSelection of induced mutantsUse of recombinant technology

Lee S.Y., D.-Y. Lee and T.Y. Kim (2005) Systems biotechnology for strain improvement. Trends Biotechnol. 23:349-358

May require greater aerationProducts may pose new extraction challengesMay even require an entirely new fermentation mediumExpensive & Laborious Need more intensive foam controlIssues:

Production strain:

Physiological FactorsPhysical FactorsNutritional FactorsMicro-elementsMacro-elementsMorphologyMetabolic by-productsShear SensitivityGenetic StabilitypHViscosityHumidityTemperatureIssues in production strain:

1.Physical factors:

highly variable factor and important parameter of fermentation

directly affects growth rate of the microorganisms, and their final composition.

Temperature Based Classification of MicrobesTemperature Ranges For GrowthPsychrophiles-5oC to 30oCoptimum temperatures between 10oC and 20oC

Mesophiles20oC to 40oCThermophiles 50oC or more

Rivera E.C., A.C. Costa, D.I. Atala, F. Maugeri and M.R.W. Maciel (2006) Evaluation of optimization techniques for parameter estimation: Application to ethanol fermentation considering the effect of temperature. Process Biochemistry. 41:1682-1687

Effect of temperature:

Humidity is the amount of moisture content in the media.

Any change in optimal humidity will change the growth of microorganisms

Many enzymes work in aqueous environment due to change in water activity enzymes will not work effectively.

It will also cause problem of dryness.Humidity:Menges R.W., M.L. Furcolow, H.W. Larsh and A. Hinton (1952) Laboratory Studies on Histoplasmosis I. The Effect of Humidity and Temperature on the Growth of Histoplasma Capsulatum. J Infect Dis. 90:67-70

1. Physical factors:

pH:pH Based Classification of MicrobesNeutrophiles6.5-7.5Acidophiles0.0-6.5Alkalinophiles7.5-11.5

Issues due to pH:

FoulingDrift Hu Z.-C., Y.-G. Zheng, Z. Wang and Y.-C. Shen (2006) pH control strategy in astaxanthin fermentation bioprocess by< i> Xanthophyllomyces dendrorhous. Enzyme Microb Technol. 39:586-590

1. Physical factors:

pH:Issues due to pH:

FoulingDrift Hu Z.-C., Y.-G. Zheng, Z. Wang and Y.-C. Shen (2006) pH control strategy in astaxanthin fermentation bioprocess by< i> Xanthophyllomyces dendrorhous. Enzyme Microb Technol. 39:586-590

1. Physical factors:

Viscosity:

most important property of medium---rheological or viscosity characteristics

Viscosity is due to friction between neighboring particles in a fluid that are moving at different velocities

Converti A., M. Zilli, S. Arni, R. Di Felice and M. Del Borghi (1999) Estimation of viscosity of highly viscous fermentation media containing one or more solutes. Biochemical engineering journal. 4:81-85

1. Physical factors:

IMPACT:makes difficult to achieve proper and complete mixings.

Ultimately affect various mass transfer processes occurring in fermenter.

has a marked effect on many parameters and process conditions within the bioreactor, often resulting in changes in microbial behavior and product formation

Physiological FactorsMorphologyMetabolic by-productsShear SensitivityGenetic StabilityIssues in production strain:

Morphology:Morphology of microorganism is effected by:

Mixing

Aeration

Pumping

Metal ions

Carbon dioxide

2. Physiological Factors

Effect of Mixing:Mixing is done for:Homogenization of contents present in a bioreactor.Maintenance of uniform concentration of microbial cells.

Extensive mixing sometimes leads to whirlpool formation, causing mechanical damage to microbial cells.

This ultimately alter the morphology of microbial colonies.Morphology:

Effect of aeration:Aeration is done to ensure the availability of oxygen for microbial growth.

Impellers/ Sparger are used to achieve aeration.

O levels also determines morphology an organism will take.

Park J.P., Y.M. Kim, S.W. Kim, H.J. Hwang, Y.J. Cho, Y.S. Lee, C.H. Song and J.W. Yun (2002) Effect of aeration rate on the mycelial morphology and< i> exo-biopolymer production in< i> Cordyceps militaris. Process biochemistry. 37:1257-1262

Morphology:However, if cells get oxygenated rather than aerated then cell lysis takes place quickly, interfering with morphology.Oxygenated cell colonies are usually thicker.

Effect of aeration:

Effect of Pumping:Pumping is done for:Blending of mixturesAchieving constant cell growthExchange of heat from the bioreactor

Centrifugal/Rotary Pumps.

At higher pumping speed, morphological changes in microbial cell occur.

At lower pumping speed, cell get trapped between walls of pump and impellers causing morphological changes.

Kamilakis E.G. and D.G. Allen (1995) Cultivating filamentous microorganisms in a cyclone bioreactor: the influence of pumping on cell morphology. Process biochemistry. 30:353-360

Morphology:

Effect of Metal Ions:Metal ions (Mn) & chelators (EDTA) also interfere with morphological patterns by:Making pellets smaller and smooth rather than larger i.e. Aspergillus niger.Altering pellet diameters.

Mn ions has alter cell wall, changing colony growth from pelleted to filamentous.

This ultimately effect the biomass production after bioprocessing.

Morphology:Couri S., G.a.S. Pinto, L.F.D. Senna and H.L. Martelli (2003) Influence of metal ions on pellet morphology and polygalacturonase synthesis by Aspergillus niger 3T5B8. Brazilian Journal of Microbiology. 34:16-21

Effect of CO:Co is a by-product produce during aerobic respiration in fermenter.

Elevated levels of Co cause: intracellular pH changesIncreased branching Swollen roots, hyphaeLow growth ratesChitin production

This all will lead to morphological changes of microbial colonies.

Mori H., T. Kobayashi and S. Shimizu (1983) Effect of carbon dioxide on growth of microorganisms in fed-batch cultures. Journal of fermentation technology. 61:211-213

Morphology:

Genetic Stability:A measure of the resistance to change, with time, of sequence of genes within a DNA molecule or of the nucleotide sequence within a gene.

Issues:

Can lose genomic DNA over the time.Biochemical changes to final productsAbnormal glycosylationAbnormal post-translation modificationsLenski R. (1990) Quantifying fitness and gene stability in microorganisms. Biotechnology (Reading, Mass.). 15:173-192

2. Physiological Factors

Shear stress:

deformation of a material substance in which parallel internal surfaces slide past one another.

arises from theforce vector componentparallelto the cross section

takes place by two ways

by stirring devicesby fluid-mechanical stress from bursting gas bubbles

Silva-Santisteban B.O.Y. (2005) Agitation, aeration and shear stress as key factors in inulinase production by< i> Kluyveromyces marxianus. Enzyme Microb Technol. 36:717-724

2. Physiological Factors

impact on the

Disruption of cell membranes.the release of intracellular compounds.alternation of aggregate size.Morphological variationsLysisMidler M. and R. Finn (1966) A model system for evaluating shear in the design of stirred fermentors. Biotechnol Bioeng. 8:71-84

Shear stress:2. Physiological Factors

Solution:

Disposable bag reactors no stir cab be used

Junne S., T. Solymosi, N. Oosterhuis and P. Neubauer (2013) Cultivation of Cells and Microorganisms in WaveMixed Disposable Bag Bioreactors at Different Scales. Chemie Ingenieur Technik. 85:57-66

Shear stress:2. Physiological Factors

Metabolic by-products:

During the log phase microorganisms like yeast produce metabolites and metabolites by products as ethanol and carbon dioxide.

influenced by changing environment

Maintaining redox balance and sugar concentration is crucial for the metabolic activity.

To facilitate the industrial process, yeast can efficiently ferment sugar under aerobic conditions. Braun S. and S.E. Vecht-Lifshitz (1991) Mycelial morphology and metabolite production. Trends Biotechnol. 9:63-68

2. Physiological Factors

Metabolic by-products:

Yeast has the ability to grow at low pH but if:

thrive on high sugar content forms high amount of aromatic compounds

Spoils beer by giving it vinegary taste.

more acidic conditionlow level of oxygen

can be overcome by increasing amount of oxygen in medium

Braun S. and S.E. Vecht-Lifshitz (1991) Mycelial morphology and metabolite production. Trends Biotechnol. 9:63-68

3. Nutritional factors:Why need nutrients?

Nutritional requirements can be determined from chemical composition of microbial cells.Obtain energyMetabolic activityNew cellular component

replicate

growErtola R.J., A.M. Giulietti and F.J. Castillo (1994) Design, formulation, and optimization of media. Bioreactor system design.89

Issues :1. Improper nutrients will lead to the formation of abnormal molecules and hinders the growth of m/o. 2. The globular structure of enzymes will get alter and create problem in product formation.

3. The required amount of energy will not produce to carry out fermentation.4. Creates difficulty in determining the production strain.

3. Nutritional factors:Ertola R.J., A.M. Giulietti and F.J. Castillo (1994) Design, formulation, and optimization of media. Bioreactor system design.89

Conclusion:Thus, the optimization of conditions for isolation and for the biomass production of microorganisms is necessary.

Any change regarding to their physiology or morphology can inhibit microorganism to produce the required product causing a lot of loss regarding time, cost and labor.