Fermentation of Bacillus - PowerPoint Presentation

1. Fermentation of Bacillus subtilisWilson WuMicrobial Nutrition Angel Yeast 2017.5.23 Vietnam

2. Contents12Fermentation and downstream process of Bacillus subtilis Angel yeast nutrients and technical support 1

3. 1. Fermentation and downstream process of Bacillus subtilis 1.1 Features and application of Bacillus subtilis1.2 General fermentation process of Bacillus subtilis1.3 Influencing factors in fermentation of Bacillus subtilis1.4 Sporulation1.5 Downstream process of Bacillus subtilis production

4. 1.1 Features and application of Bacillus subtilis1.1.1 What’s Bacillus subtilis? Rod-shaped, facultative aerobic microorganism, Gram positive strainFound in soil and the gastrointestinal tract of ruminants and human, Bacillus is the first gram-positive bacterial be studiedWould form protective endospore to survive extreme environment of temperature and desiccationendospore

5. 1.1.2 Main features of Bacillus subtilisStrong activity of amylase and proteaseCan decompose pectin and polysaccharides from vegetableCan utilize protein, sugar and starchSome strains contain special enzymes which can decompose the nucleotidesSafety The US Food and Drug Administration (FDA) classifies Bacillus subtilis as a GRAS organism. That means it is generally recognized as safe and can be used problem-free in S1 laboratory

6. ApplicationSpecific productsProduce metabolite as microbe cell factoryNatto KinaseEnzymes\ amylase, protease,Nucleotides: AMP\IMP\GMP\CMPRiboflavin(Vitamin B2)\hyaluronic acidsAgricultureAntibioticBiopesticideAnimal feedEffective microorganismsWater treatmentProbiotic for animal1.1.3 Application of Bacillus subtilis?

7. 1.2 General fermentation process of Bacillus subtilis1.2.1 Main procedure of Bacillus subtilis development Strain development Optimize ferment-ation process Down-stream process studyProduction Classification and identificationSafety assessment Functional assesmentMedium optimizeChosen of Raw materialsProcess parameter: pH\DoFed-batch or batch fermen-tation CentrifugationFreeze dry or spray dryChosen of protective additivePreparation and packageStorage and stability IsolationScreen Properties

8. 1.2.2 Principal steps for pilot production of Bacillus subtilisLabotary scale studyTest and verify the optimal culture meidum ,incubation conditions Optimize the process parameter ventilation,control of DOPilot plant Production

9. 1.2.3 Optimization of process before industry production Medium optimization in flaskFermentation in 1 and 6LOptimize productionDown stream processVerify production costsFermentation in 600LProdution in 4000L … 12000L … 24000LRaw materialsFormulation optimizeInoculation effect on seeds Procedure of Scale-upLaboratory scale

10. Media prepare sterilizationCulture seed FermentationcentrifugationblendProtectivity agentsSpray or freezing dryingQuality testpackagestorageProcess of Bacillus subtilis production

11. 1.2.4 The difference between lab and industrial fermentationLab scale fermentationIndustrial fermentationTarget&StrategyPrepare for industrial fermentationResearch basic condition Select raw materialsFind out the key factorsObtain final productsStable production and quality controlScaleSmall scaleFrom 100ml to 50LLarge scaleFrom 100L to 100KLEquipmentShake flasksBench scale fermentorsDesk centrifugeOther laboratory devicesFermentation vesselsDisc centrifugeMany affiliated equipmentsProcess controlSimpleSuch as pH, Temperature, oxygen, culture media, etc.ComplicatedControl method is differentControl of stir, sterilization, ventilation, pressure, cooling, feeding, scale up, etc.

12. Plants with annual 200 tons bacillus subtilis preparation capacity5KL fermentation tank800kg H2O/h spray drying tower packageproduct50KLfermentation tanks 1.2.5 Devices for industry production use

13. Scale-up in plants 50L500L50T 5T

14. 5L fermenters and shaking culture 50mL and 1L centrifuge 500mL/h and 2000mL/h spray drying 1.2.5 Devices for labotary production use

15. Culture media Type Corn Soyabean meal Glucose Yeast extract MnSO4 AmylaseSeed culture（%） 2.0 3.0 0.5 1 0.02 0.02Fermentation（%）3.4 3.5 0.5 0.8 0.02 0.02 ProcessSeed culturePressure：0.1MpaTemperature：37±1℃Ventilation ratio： 1:1Thermostable amylase reaction temperature 90℃ 10min,and then sterilizationCondition of transferring to fermentor：after 12-14h culture，no contamination of other microorganisms, clear and tidy by microscope1.2.6 A typical process of Bacillus subtilis fermentation (10KL)

16. FermentationPressure：0.05MpaTemperature：0—4h 37℃4h—fermentation over: 38-39℃Temperature of thermo-stable amylase reaction 90℃ 10minVentilation ratio: 1:0.8Fermentation period 40-48hHow to judge the fermentation end：spores are almost observe by microscopy，Sporulation rate >95%Final product: After fermentation, centrifugation, protective agent should be added(6% white carbon black/SiO2·nH2O, 3% MgSO4、4% Saleratus), then go through spray drying process (Inlet temperature 165℃, outlet temperature 72-73℃，package

17. 1.3.1 Factors affecting the yield and quality of Bacillus subtilis1.3 Influencing factors in fermentation of Bacillus subtilis

18. ComponentOf Nitrogen SourceDNA,RNAionsAMP\TMP\GMP\CMP\UMPA\T\C\G\UvitaminsCarbohy-dratesproteinspeptidesAmino acidsNucleotidesTrace elementGrowth factorsbiotins1.3.4 Influence of nitrogen source on bacillus fermentation

19. 1.3.2 Features of ideal culture medium Facilitate the growth of microbes, easy to harvest sufficient viability counts in short timeEasy to separate culture medium and microorganism after fermentation High buffer capacity, reduce the feedback inhibition of metabolite Low cost, raw materials easy to get

20. 1.3.3 Formulation of culture medium Carbon source Common use: Corn Starch 、glucose、sucrose,Due to strong amylase ability of Bacillus subtilis, corn starch is the most popular Carbon source in industry production, also low cost Nitrogen source common use: yeast extract, inactive yeast ,soy meal, csl yeast extract, peptone, soy meal is popular in bacillus industry production Effects of ions Mn2+、Ca2+ enhance the sporulation rates Mg2+ improve the metabolism of carbon source and energy generatesEffect of growth factor Glu\Met improve the biomass Effect of Mn2+ content on fermentation of Bacillus subtilis Mn2+ content

21. 1.3.5 Influence of nitrogen source on bacillus fermentation Effect of adding Angel yeast extract FM902 on bacillus subtilis fermentationData from Bacillus subtilis producer from China Adding 0.5% FM902 dramatically improve the growth of bacillus, with 30% increase of spore counts

22. 1.3.6 Influence of different nitrogen source on bacillus fermentation Angel yeast peptone FP101 show the strong ability for promoting biomass of Bacillus subtilis

23. 1.3.7 Influence of amino acids content on bacillus fermentation Fig. 1.Time course of free-amino acids changed in the fermentation broth of B.subtilis SD-1

24. Adding 0.3% GLU\ASP\LYS\MET\ARG effectively enhance the biomass of Bacillus subtilis, especially Glu\LYS

25. 1.3.8 Ventilation and foam control Due to it’s aerobic specialty, Ventilation is critical essential for bacillus subtilis fermentation Keep Initial liquid medium volume not more than 50% of the fermentation tanksThe ventilation ratio must more than 1:0.8 vvm and the DO value must control above 20%Chosen appropriate defoaming agents is also important polyether modified silicone is suitable for bacillus fermentation

26. Once bacillus meets carbon, nitrogen or phosphorus deficiency, spores would form inside bacillus for adapt the bad environment Due to the resistance of high temperature, desicciation, low pH, radioactive features, Sporulation is essential for Bacillus subtilis production 1.4 Sporulation

27. 1.4.1 Influence of sporulation of Bacillus subtilis production Recipe of culture medium :appropriate formulation is the primary key factor for the form of spores,especially the C:N ratio, before industry production,the optimize of C:N ratio and formulation of culture medium must be optimize first.Screen of Nitrogen source: The chosen of nitrogen source is essential for sporulation,the component of nitrogen source is complicated, Different types of nitrogen source may cause great influence to sporulation. Yeast extract could increase biomass and shorten the fermentation period. petones may could improve the sporulation.Trace elements: Mn2+ could increase spores yield and enhance stability; Ca2+ could active some enzymes and is helpful for synthesis special substance CaDPA in spores; Mg2+ could promote the decomposition of carbon source, speed up energy metabolism and help to maintain aerobic oxidation of cells

28. 1.4.2 Influence of different types of peptone on the sporulation

29. Microscope image of fermentation broth with FP320 (Dilution rates 20)Microscope image of fermentation broth with FP326 (Dilution rates 20)endosporeexospore

30. FP320FP320FP326FP326Spore counts wiith peptone FP320 7X108/MLSpore counts with peptone FP3266X109/ML

31. 1.4.3 Test method of spores count  procedure of spores counts  25 g(mL)sample+225 mL diluent 80℃ water-bath 10 minGradient dilute Plate with proper gradientCulture in 37 ℃ 48hCountReagentDiluent: NaCl 18g，Tween-80 2mL，Distilled water 2000mL, completely dissolution, put it into 5*500ml triangular flask, sterilization.Nutrient agar culture medium and petri dish: peptone 10g，yeast extract 5g，NaCl 10g，Agar powder 15g, Distilled water 1000mL, pH 7.0-7.2. Calculation N = c/m*109c ——Average available count of plates, unit is cfu；m ——Weight of sample, unit is g；109——Dilution factor.Sporulation ratio=counts of spore/counts of vegetative cells Without water -bath obtain cou-nts of vegetative cells

32. 1.5.1 Centrifugation process Centrifugation process sporeOther effective constituent 1.5 Downstream process of Bacillus subtilis production

33. Different types of Centrifugation devices

34. Different types of Centrifugation devices-Table centrifuge Centrifuge for laboratory use

35. Different types of Centrifugation devices-tubular centrifuge Centrifuge for pilot plant use

36. Different types of Centrifugation devices-Disc centrifuge Centrifuge for industrial use High capacity；separate broth from 5000L-100000L/hContinuous centrifuge

37. Different types of Centrifugation devices-Disc centrifuge

38. Comparison of different Drying methodsstrength weaknessProtective agentsFreeze dry Lactobacillus High survival rate of thermos-sensitive probiotics during dry process Long shelf-life in cold chain logisticsTime consuming ,low capacity High cost six times more than Spray dry Skim milk\trehalose\sucrose \Glycerol\Spray dryBacillus Low cost High efficiencyHigh stability in room temperature storage conditionHigh process temperature Lower viability for thermo-sensitive microbesWhite carbon black Kieselguhr1.5.2 Drying process

39. Freezing dryer from Angel yeast pilot plant

40. 100L/h5L/h500mL/hDifferent scale devices of Spray-drying in bacillus producer from China 800L/h

41. SamplesViable counts Survival rate Fermentation broth 15×108cfu/mlFreezing dry454×108cfu/g84%Spray dry with White carbon black 940×108cfu/g92Spray dry without White carbon black 850×108cfu/g90Comparison of different Drying methods for Bacillus subtilis

42. Conclusion key-factor of bacillus fermentation Formulation of culture medium,C:N ratioChosen of nitrogen source Aeration and defoaming The ratio of Sporulation Appropriate down-stream processOptimize in lab before industrial production

43. 2. Angel Yeast nutrients and Technical support2.1 Introduction of Angel Yeast nutrients2.2 Introduction of R&D team and department of microbial nutrition research

44. Ultra-filtered Yeast Extract FM888 Powder Lab-Scale, BiopharmaceuticalYeast Peptone FP101 FP103 Powder Probiotics, Biopharmaceutical, Culture mediaNutrition-enriched Yeast Extract FM803 FM505 FM502 FM405 FM408 Powder Probiotics, Starter cultureStandard Yeast Extract FM808 FM902 LM800 LD00 Powder/Paste Industrial fermentationAutolyzed Yeast&Inactive Yeast FM801 YP600 Powder Antibiotics, Bioremediation, Bio-FertilizePeptones origin from animals&vegetables casein tryptone peptones soy peptone Powder Probiotics Antibiotics, APIs2.1 Angel yeast nutrients

45. 2.1 Angel yeast nutrientsBiomass products industry - Starter cultures and probiotics for human food products - Cultivation of mashroom and macrofungi Recommendation: FM808, FM803, FM505,FM502,FP101Culture media industry and lab reagent - Bench scale research - Dehydrated culture media, Diagnostics, Cell cuture Recommendation: FM888, FM808Bioactive molecules and chemicals industry - Amino acids and peptides - Organic acids, Vitamins, Nucleotides, Enzymes, Alcohol - Vitamins, Antibiotics, Hormones, Biological preservatives - Other types of large scale fermentation Recommendation: FM808, FM902, LM800, LD00, LP101, YP101Agricultural and environmental industry - Sewage treatment, Bio-pesticide, Bio-plastic - Probiotics for agriculture and environment Recommendation: FM902, FM801, FM701Main application

46. Rich in peptides, growth factors and amino acids.Shorten fermentation cycle Increase biomassCost effectiveAngel yeast extract FM902 is widely used for Bacillus subtilis fermentation as a good nitrogen source rich in peptides, amino acids and growth factors.Yeast Extract for Bacillus subtilis——FM902

47. Rich in short peptidesHelpful for vialbilty of sporesReplacement of peptones origin from animals and vegetables Safe Stable Angel yeast extract FP101 is widely used for Bacillus subtilis fermentation as a good nitrogen source rich in short peptides which is good for sporulation.Yeast Extract customized for Bacillus subtilis——FP101

48. Improve viability counts more than 30%Shorten fermentation cycle Enhance probiotics activity Angel yeast extract FM502 is customized developed for Lactic acid bacteria ,base on the genetic background and metabolism feature of Lactic acid bacteria, Rich in Vitamins and Nucleotides Yeast Extract customized for Lactic acid bacteria——FM502

49. Department of Microbial Nutrition ResearchResearch and development of new products Optimization of culture media formula Optimization of fermentation process Development of customized products 2.2 Technical supportExpert consultants of Angel

50. Test and scale up platformHPLCAtomic Absorption Spectrometer GCAmino acid analyzerGC-MS Powder InstrumentDrawing InstrumentUltrasonic Processors Pilot PlantPlatform for fermentation controlThe membrane separation system Spray drying system

51. Develop specialized nutrients for different microbes We customize nutrients for customersHyaluronic acidsErythritol LactobacillusCell culture Alanine

52. THANK YOU!