Fermentation of Bacillus subtilis Wilson Wu Microbial Nutrition

Fermentation of Bacillus subtilis Wilson Wu Microbial Nutrition Angel Yeast 2017. 5. 23 Vietnam

Contents 1 Fermentation and downstream process of Bacillus subtilis 2 Angel yeast nutrients and technical support

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

1. 1 Features and application of Bacillus subtilis 1. 1. 1 What’s Bacillus subtilis? l Rod-shaped, facultative aerobic microorganism, Gram positive strain l Found in soil and the gastrointestinal tract of ruminants and human, Bacillus is the first gram-positive bacterial be studied l Would form protective endospore to survive extreme environment of temperature and desiccation endospore

1. 1. 2 Main features of Bacillus subtilis l Strong activity of amylase and protease l Can decompose pectin and polysaccharides from vegetable l Can utilize protein, sugar and starch l Some strains contain special enzymes which can decompose the nucleotides l Safety 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 S 1 laboratory

1. 1. 3 Application of Bacillus subtilis? Application Specific products Produce metabolite as microbe cell factory Natto Kinase Enzymes amylase, protease, Nucleotides: AMPIMPGMPCMP Riboflavin(Vitamin B 2)hyaluronic acids Agriculture Antibiotic Biopesticide Animal feed Effective microorganisms Water treatment Probiotic for animal

1. 2 General fermentation process of Bacillus subtilis 1. 2. 1 Main procedure of Bacillus subtilis development Classification and identification Safety assessment Optimize fermentation process Strain development ü Isolation ü Screen ü Properties ü ü Medium optimize Chosen of Raw materials Process parameter: p. HDo Fed-batch or batch fermentation Functional assesment Down-stream process study ü Centrifugation ü Freeze dry or spray dry ü Chosen of protective additive ü Preparation and package ü Storage and stability Production

1. 2. 2 Principal steps for pilot production of Bacillus subtilis Labotary scale study Pilot plant Test and verify the optimal culture meidum , incubation conditions Optimize the process parameter ventilation, control of DO Production

1. 2. 3 Optimization of process before industry production Procedure of Scale-up Medium optimization in flask Laboratory scale Fermentation in 1 and 6 L Raw materials Formulation optimize Inoculation effect on seeds Optimize production Fermentation in 600 L Down stream process Verify production costs Prodution in 4000 L … 12000 L … 24000 L

Culture seed sterilization Media prepare Fermentation centrifugation Process of Bacillus subtilis production blend Spray or freezing drying Quality test package storage Protectivity agents

1. 2. 4 The difference between lab and industrial fermentation Lab scale fermentation Industrial fermentation Target&Strategy Prepare for industrial fermentation Research basic condition Select raw materials Find out the key factors Obtain final products Stable production and quality control Scale Small scale From 100 ml to 50 L Large scale From 100 L to 100 KL Equipment Shake flasks Bench scale fermentors Desk centrifuge Other laboratory devices Fermentation vessels Disc centrifuge Many affiliated equipments Process control Simple Such as p. H, Temperature, oxygen, culture media, etc. Complicated Control method is different Control of stir, sterilization, ventilation, pressure, cooling, feeding, scale up, etc.

1. 2. 5 Devices for industry production use Plants with annual 200 tons bacillus subtilis preparation capacity 50 KLfermen tation tanks 5 KL fermentation tank 800 kg H 2 O/h spray drying tower package product

50 T Scale-up in plants 5 T 500 L 50 L

1. 2. 5 Devices for labotary production use 5 L fermenters and shaking culture 50 m. L and 1 L centrifuge 500 m. L/h and 2000 m. L/h spray drying

1. 2. 6 A typical process of Bacillus subtilis fermentation (10 KL) Culture media Type Corn Soyabean meal Glucose Yeast extract Mn. SO 4 Amylase Seed culture（%） 2. 0 3. 0 0. 5 1 0. 02 Fermentation（%）3. 4 3. 5 0. 8 0. 02 Process Seed culture Pressure： 0. 1 Mpa Temperature： 37± 1℃ Ventilation ratio： 1: 1 Thermostable amylase reaction temperature 90℃ 10 min, and then sterilization Condition of transferring to fermentor： after 12 -14 h culture， no contamination of other microorganisms, clear and tidy by microscope

Fermentation Pressure： 0. 05 Mpa Temperature： 0— 4 h 37℃ 4 h—fermentation over: 38 -39℃ Temperature of thermo-stable amylase reaction 90℃ 10 min Ventilation ratio: 1: 0. 8 Fermentation period 40 -48 h How 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/Si. O 2·n. H 2 O, 3% Mg. SO 4、4% Saleratus), then go through spray drying process (Inlet temperature 165℃, outlet temperature 72 -73℃，package

1. 3 Influencing factors in fermentation of Bacillus subtilis 1. 3. 1 Factors affecting the yield and quality of Bacillus subtilis • Purity • Genetic stability • Drying process • Centrifugation • Protective agents • • Strain properties Fermentati on process Downstrea m process Preparation Formulation Carbon source Nitrogen source Process • Package • Storage condition

1. 3. 4 Influence of nitrogen source on bacillus fermentation proteins peptides Amino acids DNA, RNA AMPTMPGMP CMPUMP ATCGU Nucleotides Component Of Nitrogen Source Trace element Growth factors ions vitamins biotins Carbohydrates

1. 3. 2 Features of ideal culture medium l Facilitate the growth of microbes, easy to harvest sufficient viability counts in short time l Easy to separate culture medium and microorganism after fermentation l High buffer capacity, reduce the feedback inhibition of metabolite l Low cost, raw materials easy to get

1. 3. 3 Formulation of culture medium l Carbon source Common use: Corn Starch 、glucose、sucrose, Due to strong amylase ability of Bacillus subtilis, corn starch is the Effect of Mn 2+ content on fermentation of Bacillus subtilis most popular Carbon source in industry production, also low cost l Nitrogen source common use: yeast extract, inactive yeast , soy meal, csl yeast extract, peptone, soy meal is popular in bacillus industry production l Effects of ions Mn 2+、Ca 2+ enhance the sporulation rates Mg 2+ improve the metabolism of carbon source and energy generates l Effect of growth factor GluMet improve the biomass Mn 2+ content

1. 3. 5 Influence of nitrogen source on bacillus fermentation Effect of adding Angel yeast extract FM 902 on bacillus subtilis fermentation Data from Bacillus subtilis producer from China Adding 0. 5% FM 902 dramatically improve the growth of bacillus, with 30% increase of spore counts

1. 3. 6 Influence of different nitrogen source on bacillus fermentation Influence of different nitrogen source of Bacillus subtilis effect of p. H on baciilus subtilis fermentation 9 6 8 7 4 6 3 p. H OD 600 5 2 1 0 0 1 FP 103 2 FP 101 3 t/h SOY MEAL 4 5 6 FP 101 4 3 soy meal 2 tryptone 1 0 TRYPTONE FP 103 5 0 2 4 t/h 6 8 Angel yeast peptone FP 101 show the strong ability for promoting biomass of Bacillus subtilis 10

1. 3. 7 Influence of amino acids content on bacillus fermentation Time course of free-amino acids changed in the fermentation broth of B. subtilis SD-1 Fig. 1.

effect of amino acids on bacillus fermentation 5 4 DCW(gL) 3. 5 3 2. 5 2 1. 5 1 0. 5 0 Control 0. 3%Glu 0. 3%Asp 0. 3%Met 0. 3%Lys 0. 3%Arg Adding 0. 3% GLUASPLYSMETARG effectively enhance the biomass of Bacillus subtilis, especially GluLYS

1. 3. 8 Ventilation and foam control l Due to it’s aerobic specialty, Ventilation is critical essential for bacillus subtilis fermentation l Keep Initial liquid medium volume not more than 50% of the fermentation tanks l The ventilation ratio must more than 1: 0. 8 vvm and the DO value must control above 20% l Chosen appropriate defoaming agents is also important polyether modified silicone is suitable for bacillus fermentation

1. 4 Sporulation 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 p. H, radioactive features, Sporulation is essential for Bacillus subtilis production

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. l 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. l Trace elements: Mn 2+ could increase spores yield and enhance stability; Ca 2+ could active some enzymes and is helpful for synthesis special substance Ca. DPA in spores; Mg 2+ could promote the decomposition of carbon source, speed up energy metabolism and help to maintain aerobic oxidation of cells l

1. 4. 2 Influence of different types of peptone on the sporulation

endospore exospore Microscope image of fermentation broth with FP 320 (Dilution rates 20) Microscope image of fermentation broth with FP 326 (Dilution rates 20)

Spore counts wiith peptone FP 320 FP 326 7 X 108/ML Spore counts with peptone FP 326 6 X 109/ML

1. 4. 3 Test method of spores count procedure of spores counts Reagent Diluent: Na. Cl 18 g，Tween-80 2 m. L，Distilled water 2000 m. L, completely dissolution, put it into 5*500 ml triangular flask, sterilization. Nutrient agar culture medium and petri dish: peptone 10 g，yeast extract 5 g，Na. Cl 10 g，Agar powder 15 g, Distilled water 1000 m. L, p. H 7. 0 -7. 2. Calculation N = c/m*109 c ——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 25 g(m. L)sample+225 m. L diluent 80℃ water-bath 10 min Gradient dilute Plate with proper gradient Culture in 37 ℃ 48 h Count Without water bath obtain counts of vegetative cells

1. 5 Downstream process of Bacillus subtilis production 1. 5. 1 Centrifugation process spore Other effective constituent

Different types of Centrifugation devices

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

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

Different types of Centrifugation devices-Disc centrifuge Centrifuge for industrial use l High capacity；separate broth from 5000 L-100000 L/h l Continuous centrifuge

Different types of Centrifugation devices-Disc centrifuge

1. 5. 2 Drying process Comparison of different Drying methods strength weakness Freeze dry High survival rate of thermos- Time consuming , low capacity Lactobacillus sensitive probiotics during dry High cost six times more than Skim process Spray dry milktrehalosesucrose Long shelf-life in cold chain Protective agents Glycerol logistics Spray dry Low cost High process temperature White carbon black Bacillus High efficiency Lower viability for thermo- Kieselguhr High stability in room sensitive microbes temperature storage condition

Freezing dryer from Angel yeast pilot plant

Different scale devices of Spray-drying in bacillus producer from China 800 L/h 100 L/h 500 m. L/h

Comparison of different Drying methods for Bacillus subtilis Samples Viable counts Survival rate Fermentation broth 15× 108 cfu/ml Freezing dry 454× 108 cfu/g 84% Spray dry with White 940× 108 cfu/g 92 850× 108 cfu/g 90 carbon black Spray dry without White carbon black

Conclusion key-factor of bacillus fermentation l Formulation of culture medium, C: N ratio l Chosen of nitrogen source l Aeration and defoaming l The ratio of Sporulation l Appropriate down-stream process Optimize in lab before industrial production

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

2. 1 Angel yeast nutrients p Ultra-filtered Yeast Extract FM 888 Powder Lab-Scale, Biopharmaceutical p Yeast Peptone FP 101 FP 103 Powder Probiotics, Biopharmaceutical, Culture media p Nutrition-enriched Yeast Extract FM 803 FM 505 FM 502 FM 405 FM 408 Powder Probiotics, Starter culture p Standard Yeast Extract FM 808 FM 902 LM 800 LD 00 Powder/Paste Industrial fermentation p Autolyzed Yeast&Inactive Yeast FM 801 YP 600 Powder Antibiotics, Bioremediation, Bio-Fertilize p Peptones origin from animals&vegetables casein tryptone peptones soy peptone Powder Probiotics Antibiotics, APIs

2. 1 Angel yeast nutrients Main application Ø Biomass products industry - Starter cultures and probiotics for human food products - Cultivation of mashroom and macrofungi Recommendation: FM 808, FM 803, FM 505, FM 502, FP 101 Ø Culture media industry and lab reagent - Bench scale research - Dehydrated culture media, Diagnostics, Cell cuture Recommendation: FM 888, FM 808 Ø Bioactive 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: FM 808, FM 902, LM 800, LD 00, LP 101, YP 101 Ø Agricultural and environmental industry - Sewage treatment, Bio-pesticide, Bio-plastic - Probiotics for agriculture and environment Recommendation: FM 902, FM 801, FM 701

Yeast Extract for Bacillus subtilis——FM 902 Rich in peptides, growth factors and amino acids. Shorten fermentation cycle Increase biomass Cost effective Angel yeast extract FM 902 is widely used for Bacillus subtilis fermentation as a good nitrogen source rich in peptides, amino acids and growth factors.

Yeast Extract customized for Bacillus subtilis——FP 101 Rich in short peptides Helpful for vialbilty of spores Replacement of peptones origin from animals and vegetables Safe Stable Angel yeast extract FP 101 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 Lactic acid bacteria——FM 502 Improve viability counts more than 30% Shorten fermentation cycle Enhance probiotics activity Angel yeast extract FM 502 is customized developed for Lactic acid bacteria , base on the genetic background and metabolism feature of Lactic acid bacteria, Rich in Vitamins and Nucleotides

2. 2 Technical support Department of Microbial Nutrition Research • Research and development of new products • Optimization of culture media formula • Optimization of fermentation process • Development of customized products Expert consultants of Angel

Test and scale up platform HPLC GC-MS Pilot Plant Atomic Absorption Spectrometer Powder Instrument Platform for fermentation control GC Drawing Instrument Amino acid analyzer Ultrasonic Processors The membrane separation system Spray drying system

We customize nutrients for customers Develop specialized nutrients for different microbes Hyaluronic acids Erythritol Lactobacillus Alanine Cell culture

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