發 酵 學 概 論

Biochemical engineering is no longer an academicchallenge

•US: most new professorships are biomolecularly oriented•Europe: moving in same direction

–Long searches to find new professors of biochemicalengineering

–Most comes from industry (Patents, few publications)–Solution: move towards biomolecular focus

•Metabolic engineering•Complex control systems to re-engineer eucaryotic cells•Tissue engineering

Biotechnology –major sectors

•Pharma•Agriculture•Environment•Chemistry•Energy Green Chemistry

New pharma compoundsand materials, devices,

Control systems, genetherapy

New materials

New compounds

New processes

Biomass

Biofuels

Bioelectricity

Biodegradation

End-of-pipe solution

Integrated processes

Agro-efficiency

Food quality

Agrochemicals

經濟部篩選國內具潛力之生化產品•原料藥與製劑•血液製品•檢驗試劑與檢驗儀器•生物晶片•人用疫苗•生物農藥•特用化學品•科學化中藥•食用色素

•菇類保健食品•益生菌保健食品•食品用酵素•免疫食品•傳統發酵食品•保健食品•酒類製品•香料及化妝品•機能性素材及食品

什麼是發酵？發酵(fermentation)一詞來自於拉丁語“發”(fervere) 意為煮沸描述酵母菌將糖轉變成二氧化碳造成如沸騰之現象。

巴斯德認為發酵是酵母在無氧狀態下的呼吸過程。生物化學上之定義為微生物在無氧時的代謝過程。

現在：人們把利用微生物在有氧與無氧條件下的生命活動來製備微生物菌體或其代謝產物的過程通稱發酵

•Industrial Microbiology is the disciplinethat uses microorganisms, usually grown ona large scale, to produce valuablecommercial products or carry outimportant chemical transformations.

•Fermentation Technology is the technologyto grow cells in a large scale with highefficiency, it also includes product recoveryprocesses.

Industrial Microbiology andFermentation Technology

Microbial Products: Microbial Cells, Enzymes, PharmaceuticalProducts, Specialty Chemicals and Food Additives

發酵的演化與歷史

基因改質菌種篩選較佳生產菌種

基因工程萌芽

利用突變及篩選改進菌種

使用純種生產菌種

使用代代相傳之麴

使用菌種

回饋控制、人工智慧控制控制

電腦控制可滅菌pH及溶氧探針

溫度控制、pH控制

憑經驗製程控制

流動化床與氣舉式醱酵槽

細胞固定化連續式離心機

機械攪拌、無菌操作

鋼製儲桶、通氣木桶、銅器設備

抗生素、必須胺機酸、胰島素

蛋白質分解酵素、單細胞蛋白質、抗生素

盤尼西林、維他命B12、纖維分解酵素

麵包酵母、丙酮、丁醇、檸檬酸

酒精、醋(龍山文化、埃及)

產品

第五階段1970-

第四階段1960-70

第三階段1945-1960

第二階段1900-1945

第一階段1900年以前

階段

發酵的分類

• 微生物菌體發酵：益生菌、藥用真菌• 微生物產物發酵（一級代謝物）：氨基酸、核酸、醣類• 微生物代謝產物發酵（二級代謝物）：抗生素、維生素• 微生物轉化發酵：抗生素轉化、中草藥發酵• 細胞培養：動、植物細胞培養

發酵特點

•1 發酵過程為全生命反應，數十個生化反應可串聯發生•2 常溫、常壓、耗能少•3 原料需求簡單，成本低•4 微生物種類繁多，具特異功能•5 生產條件標準化，可cGMP化•6 培養時間短，量產化簡單•7 可調控代謝途徑，產品多樣化

傳統發酵技術應用發酵應用範圍包括：食品工業—

•製造麵包，澱粉分解為較小的片段，同時放出二氧化碳

•釀酒過程，使糖類分解成酒精同時放出二氧化碳•茶製工藝，使茶葉中的沒食子茶素分解再合成為茶黃

素，使茶葉成為紅茶•醬油、豆腐乳、乳酪、酸奶、紅麴、味增等都需要發

酵過程。

製藥工業—•藉由大型生物反應器-醱酵槽，來進行抗生素、氨基酸

等生產。

近代發酵技術應用〔一〕

•製藥界：基因工程蛋白質藥物（胰島素）、新一代的抗生素及非抗生素（降血脂）產品。

•保健食品：益生菌產品、乳酸菌產品、發酵乳品、食用藥用菇菌類產品、新式發酵飲料、機能性飲料

•環保領域：環境微生物製劑、農漁牧業用的處理微生物、廢棄物、污染源處理、廢水處理、水質淨化、廚餘有機堆肥

近代發酵技術應用〔二〕

•農業產業：動物疫苗、動物用生長荷爾蒙、生物農藥及土地改質的生物製劑

•化妝品領域：保濕產品如：透明質酸細胞活性因子如：ＳＫⅡ的pitera

醱酵技術之目標

•沒有雜菌•最佳生長狀態 –培養菌快速生長

–代謝產物快速累積

•有效率之回收純化•最有效率之生產程序（單位時間、單位成本生產最大產量）

影響發酵之參數

菌種特性 培養基組成 發酵槽體設計菌種年齡 發酵條件 環境刺激繼代次數 代謝調控接菌體積

生物參數 物理參數化學參數

•Strain selection•Laboratory process development•Pilot Scale up•Industrial Scale up•Downstream process development•Product packaging techniques•Other commercial consideration•Examples

Flowsheet for developing an industrialmicrobial fermentation process

FrozenSeed

SeedFermemter

ProductionFermenter

SeedFlask

Pre-SeedFermenter

Typical Fermentation TrainTotal time 3-20 days

Fermentation Process

Strain Selection

–Purchase from Culture Collections–Screening of nature circumstances–Genetic engineering–Mutations–Cell biology techniques

International Culture Collections

Domestically, strains can be purchased from: CGMCC orChina General Microbiological Culture Collection Center

Screening of nature circumstances

GeneticEngineering

Incorporation intoartificial plasmids ofgenes from a widevariety of sources hasmade possible thetransfer of geneticmaterial acrossvirtually any speciesbarrier

Various high valueadded products havebeen produced fromGenetic engineeringmethods

MutationVia chemicalor physical,and biologicalmeans

Cell Biology Techniques

•Protoplast fusion (promote high frequenciesof genetic recombinants)–removing the cell wall with lytic enzymes in the

presence of osmotic stabilizers.–In the presence of fusogenic agent such as

polyethylene glycol (PEG), protoplasts areinduced to fuse and form transient hybrids ordiploids.

–Regeneration of viable cells from the fusedprotoplasts.

Laboratory Process DevelopmentShake Flask Experiments

Optimization of conditionsfor cell growth and productformation using shake flaskexperiments:

1. pH2. Temperature3. Dissolved oxygen (DO)4. Substrate choice5. Maximal and optimal

substrate concentration6. Others

LaboratoryProcess

DevelopmentFermentor

Experiments

•Agitation•Cooling and heating•Air inlet and outlet•pH control•Nutrient addition• Inoculation•Viewing port

Laboratory Process DevelopmentLab scale fermentor experiments

•Batch process•Fed-batch process•Continuous process•Semi-continuous

process

Process control and monitoring•Process parameters to be monitors

Sugar consumption

pH

Temperature

Fermentation time (h)

Agitation

Cell Dry Weight

Product

Computer softwares have been developed to monitor and change the process on line

Pilot Scale Up•Scale up: The transfer of a process from small-

scale laboratory equipment to large-scalecommercial equipment

•Pilot experiment–To test the feasibility of the lab scale

fermentation process in a semi-industrialscale

–Pilot fermentors normally have a size rangingfrom 100 L to 10,000 L, depending on theproducts to be mass produced later.

Problems emerging during the scale up•As the size of the equipment is increased, the surface-

volume ratio changes•Large fermentor has much more volume for a given

surface area, it is obviously more difficult to mix the bigtank than the small flask

•In scale up studies on aerobic fermentations, oxygenrate in the fermentor is best kept constant as the size ofthe fermentor is increased.–How to keep DO constant?

•Increase stirring rate•Increase air pressure•Use pure oxygen•Increase air inlet

桌上型5L發酵槽

100L發酵槽

1噸發酵槽

1噸發酵槽

抗生素生產工廠

Product Recovery

•What purity is necessary?–acetic acid or alcohol little purification–industrial enzymes: moderate purity–food additives have high purity–pharmaceuticals super purity

•What is the concentration of the product?

Product Recovery

•Is the product in the cell or secreted?–enzymes and other proteins in cell require cell

harvest and extraction–secreted products allow for continuous

fermentation–secreted products reduce costs of purification.

Why?

•Can we improve secretion?

Downstream ProcessDevelopment

Chromatography

Filter (press filter)

DownstreamProcessDevelopment

Continuouscentrifuges

Unit Operations in Downstream Separation•Microfilters•Ultrafiltration•Gel chromatography•Reverse osmosis•Dialysis•Electrodialysis• Ion exchange•Distillation/freeze dry•Solvent extraction•Foam and bubble fractionation•Ultracentrifugation•Centrifugation•Liquid cyclones•Gravity sedimentation

Size

Diffusivity

Ion exchangeVapour temperature, pressure

SolubilitySurface activity

Density

Processing fermentation brothFermentation

Dilute slurry

CentrifugationFiltrationSedimentation

Clarified liquor

Filtration

Bright liquor

Concentrated slurry

Dewatering

Drying

Dry Biomass

Centrifugation: Continuouscentrifugation

Disc-Centrifuges

Solids-retainingcentrifuge

Nozzle with pressurizeddischarge of concentrate

Nozzle with peripheralnozzles

Periodicallysolids-ejectingcentrifuge

Periodicallysolids-ejectingcentrifuge withaxial channels

Product PackagingTechniques

•Steril Packaging Techniques for MedicalApplications and Food Preservation

•Pyrogen Free and Steril Packaging for InjectionPurpose

•Freeze Dry Packaging for Foods or Medicines•Dewatered Packaging (such as Dry Yeast)•Normal Food Packaging (such as Na-glutamate)•Salty Packaging•Preservation Chemicals•Ordinary Packaging

Other Commercial ConsiderationStrengths and weaknesses of biotechnological processes

•Strengths–Reliance on renewable

feedstocks–Versatility with different

feedstocks–Food, feed and drug

applications–Fine chemicals to bulk

chemical applications–Low temperature–Operation in aqueous media–Several reactions achieved in

a single fermentation step–High level of automation–Stereospecificity–Complex molecules

converted and/or produced–“Benign”effluents produced

•Weaknesses–Feedstocks are oxidized and

unsuitable for many applications–Feedstock costs fluctuate–Sterilization is a major cost–Product often in dilute aqueous

solutions–Product recovery costly–Equipment costs high–Reactions slow leading to poor

volumetric productivity–Complicated reaction conditions–Mainly batch operation–High cell (catalyst) regeneration

costs–High BOD wastes

Other Commercial ConsiderationCost Evaluation

•Outline of total capital investment•Fixed capital

–Direct costs (Land, site development, buildings,processing, services)

–Indirect costs (Engineering, construction,Contingency, Fees)

–Start-up costs

•Working capital–Inventory, accounts receivable, account payable