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1、4.1 Introduction A fermentation product is produced by the culture of a certain organism,or organisms,in a nutrient medium.If the fermentation is invaded by a foreign micro-organism then the following consequences may occur:1.The medium would have to support the growth of both the production organis
2、m and the contaminant,resulting in a loss of productivity.2.If the fermentation is a continuous one then the contaminant may outgrow the production organism and displace it from the fermentation.3.The foreign organism may contaminate the final product,e.g.single-cell protein where the cells,separate
3、d from the broth,constitute the product.4.The contaminant may produce compounds which make subsequent extraction of the final product difficult.5.The contaminant may degrade the desired product;this is common in bacterial contamination of antibiotic fermentations where the contaminant would have to
4、be resistant to the normal inhibitory effects of the antibiotic and degradation of the antibiotic is a common resistance mechanism,e.g.the degradation of-lactam antibiotics(-内内酰酰胺胺类类抗生素抗生素)by-lactamase producing bacteria.6.Contamination of a bacterial fermentation with phage could result in the lysi
5、s of the culture.Avoidance of contamination may be achieved by:1.Using a pure inoculum to start the fermentation.2.Sterilizing the medium to be employed.3.Sterilizing the fermenter vessel.4.Sterilizing all materials to be added to the fermentation during the process5.Maintaining aseptic condition du
6、ring the fermentation.The extent to which these procedures are adopted is determined by the likely probability of contamination and the nature of its consequences.Some fermentations are described as protected-that is,the medium may be utilized by only a very limited range of micro-organisms,or the g
7、rowth of the process organism conditions,such as a reduction in pH.The brewing of beer falls into this category;hop resins tend to inhibit the growth of many micro-organisms and the growth of brewing yeasts tends to decrease the pH of the medium.Thus,brewing worts are boiled,but not necessarily ster
8、ilized,and the fermenters are thoroughly cleaned with disinfectant solution but are not necessarily sterile.Also,the precautions used in the development of inoculum for brewing are far less stringen than,for example,in an antibiotic fermentation.However,the vast majority of fermentations are not pro
9、tected and,if contaminated,would suffer some of the consequences previously listed.The approaches adopted to avoid contamination will now be discussed in more detail.Basic terms sterilization灭菌灭菌disinfection消毒消毒Antisepsis 防腐防腐Bacteriostasis：bk,tiristeisis抑菌抑菌 Asepsis 无菌无菌 Factors influencing antimic
10、robial activity the concentration and kind of an agent usedthe length of exposure to the agentthe temperature at which the agent is used the number of microorganisms present the kinds of microorganisms present the existence of organic materials4.2 4.2 物理消毒灭菌法物理消毒灭菌法Controlling Microorganisms By Phys
11、ical AgentsControlling Microorganisms By Physical Agents一、热力灭菌法一、热力灭菌法 (Heat)(Heat)二、辐射灭菌法二、辐射灭菌法 (Radiation)(Radiation)三、滤过除菌法三、滤过除菌法 (Filtration)(Filtration)四、超声灭菌法四、超声灭菌法 (SupertronicSupertronic)五、低温干燥抑菌法五、低温干燥抑菌法 (Desiccation)(Desiccation)六、低温（六、低温（Low Temperature Low Temperature）4.2.1.2 Moist h
12、eat 4.2.1.2 Moist heat 湿热灭菌方法：湿热灭菌方法：denature proteins and melt lipids;more effective Autoclaving:高压蒸汽灭菌高压蒸汽灭菌 Boiling water煮沸灭菌煮沸灭菌Pasteurization巴氏消毒法巴氏消毒法Fractional sterilization间歇蒸气灭菌法间歇蒸气灭菌法Continuous sterilization连续蒸汽灭菌法连续蒸汽灭菌法 1)Steam heating to 100 for 30 min常压常压/流动蒸流动蒸气消毒法气消毒法 Vegetative cel
13、ls are destroyed but endospores survive 2)Incubate at 30 -37 overnight Most bacterial endospores germinate 3)Second heat treatment,100 ,30 min Germinated endospores are killed.4)Second incubation at 30-37 overnight Remaining endospores germinate 5)Third heat treatment,100 ,60 min Last remaining germ
14、inated endospores are killed Fractional sterilization间歇蒸气灭菌法间歇蒸气灭菌法1:Ionizing Radiation 电离辐射电离辐射 X-rays and gamma rays more energy and penetrating power than UVused to sterilize pharmaceuticals and disposable medical supplies such as syringes,surgical gloves,catheters导尿管,and sutures缝合线used to retard
15、 spoilage in sea foods,meats,poultry,and fruits 射线射线、高能量的电子束（阴极射线）、高能量的电子束（阴极射线）不升高温度且穿透力强不升高温度且穿透力强适于忌热物品的灭菌或消毒适于忌热物品的灭菌或消毒Microwave penetrating non-metal materials(glass,plastics,china)Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents 4.2.3 滤过除菌滤过除菌 Filtr
16、ation l主要用于一些主要用于一些不耐热不耐热的血清、毒素、抗的血清、毒素、抗生素、药液以及空气等除菌。生素、药液以及空气等除菌。l不能除去病毒、支原体和型细菌不能除去病毒、支原体和型细菌Heavy metals(Hg2+、Ag+)denaturing proteins and inactivating enzymes2%mercurochrome（红汞）or 0.1%merthiolate（硫柳汞）skin,mucosa and woundbacteriostatic,ineffective against endospores1%silver nitrate（硝酸银）eye drops
17、 for newborns to prevent gonococcal ophthalmia（淋菌性眼炎）Acids and alkaliesAlter membrane permeability and denature proteins and other moleculesSalts of organic acids:food preservatives Undecylenic acid十一烯酸:dermatophyte皮肤真菌 infections In virtually all fermentation processes,it is mandatory for a cost-ef
18、fective operation to have contamination-free seed cultures at all stages,from the preliminary culture to the production fermenter.A bioreactor can be sterilized either by destroying the organisms with some lethal agent such as heat,radiation,or a chemical,or by removing the viable organisms by a phy
19、sical procedure such as filtration.1、由于杂菌的污染，使生物反应中的基质或产物因杂、由于杂菌的污染，使生物反应中的基质或产物因杂菌的消耗而损失，造成生产能力的下降；菌的消耗而损失，造成生产能力的下降；2、由于杂菌所产生的一些代谢产物，或在染菌后改变、由于杂菌所产生的一些代谢产物，或在染菌后改变了培养液的某些理化性质，使产物了培养液的某些理化性质，使产物 的提取和分离变得的提取和分离变得困难，造成收率降低或使产品的质量下降；困难，造成收率降低或使产品的质量下降；3、杂菌会大量繁殖，会改变反应介质的、杂菌会大量繁殖，会改变反应介质的PH值，从而使值，从而使生物反
20、应发生异常变化；生物反应发生异常变化；4、杂菌可能会分解产物，从而使生产过程失败；、杂菌可能会分解产物，从而使生产过程失败；5、发生噬菌体污染，微生物细胞被裂解，而使生产失、发生噬菌体污染，微生物细胞被裂解，而使生产失败，等等败，等等 在发酵生产中，为什么要进行灭菌操作？在发酵生产中，为什么要进行灭菌操作？问题1：在一定温度下，微生物的受热死亡在一定温度下，微生物的受热死亡遵照分子反应速度理论。在灭菌过程遵照分子反应速度理论。在灭菌过程中，活菌数逐渐减少，其减少量随残中，活菌数逐渐减少，其减少量随残留活菌数的减少而递减，即微生物的留活菌数的减少而递减，即微生物的死亡速率与任一瞬时残存的活菌数成
21、死亡速率与任一瞬时残存的活菌数成正比，正比，4.4.1.14.4.1.1 微生物的死亡速率和理论灭菌时间微生物的死亡速率和理论灭菌时间 The process of destroying a population generally follows first-order kinetics.Using the initial number of cell N0/ml,the number of destroyed organisms N at time t(min),and the surviving cells N,the death rate can be calculated as fo
22、llows:(k is the specific death constant/min)When integrated between N0 at time t=0 and N at time t=t,the following equation is obtained:kt-=InN/N0 Or InN/N0=-kt The ratio of N/N0 is the inactivation factor,the ratio of N/N0 is the survival factor and the InN/N0=V is the design criterion,a parameter
23、which encompasses the contamination level of the medium to be sterilized,N0,and the desired sterility level,N.The ideal curve expressing the exponential decline in survivors with time is plotted in follow Figure:This line has a negative slope and in following Figure part2 corredponds to the death cu
24、rve for vegetative cells of Escherichia coli at 54-60.This type of killing kinetics is referred to as a logarithmic death curve.In actuality,this curve is not always linear,as shown for the death curve of Bacillus stearothermophilus spore.Batch sterilizationMost nutrient media are presently steriliz
25、ed in batch volumes in the bioreactor at 121.Approximate sterilization times can be calculated from the nature of the medium and the size of the fermenter.Not only the nutrient media,but also the fittings,valves and electrodes of the fermenter itself must be sterilized.Therefore,actual sterilization
26、 times are significantly longer than calculated ones and must be empirically determined for the specific nutrient solutions in the fermenter.One method of sterilization is to inject steam into the fermenter mantle or interior coils(indirect sterilization).Another method is to inject steam into the n
27、utrient solution itself(direct procedure),in which case pure steam(free of chemical additives)is a prerequisite pri:rekwizit.Many industrial steam supplies contain potentially toxic chemicals derived from anti-corrosive(腐腐 蚀蚀 的的)additives used in the steam-manufacturing process.With direct steam inj
28、ection,condensate accumulates within the fermenter and the volume of liquid thus increases during the sterilization process.The drawbacks of the heat-sterilization process are shown for a typical sterilization of a 3000 L batch fermenter in follow Figure.It takes 2-3 hours to reach the sterilization
29、 temperature of 121,depending on the steam conduction and fermenter size.Once the proper temperature has been reached,another 20-60 minutes are required for the actual killing process,followed by cooling for one hour.The energy required for heating must subsequently be removed in order to cool the f
30、ermenter and if the hot water obtained during the cooling be put to some use,batch sterilization become very costly.Another disadvantage of heat sterilization(and from the standpoint of microbiology the most significant shortcoming)can be seen in above Figure.The heating,sterilization and cooling ph
31、ases not only kill microorganisms but also severely alter nutrient solutions.Discoloration and change in the pH value result from caramelization(生生 产产 焦焦 糖糖)and Maillard reactions.Vitamins are destroyed and the quality of the culture medium deteriorates.The extent to which the subsequent fermentatio
32、n is affected depends on the organism and the processContinuous sterilizationThe two main disadvantages of batch sterilization just mentioned,culture medium damage and high energy consumption,can be largely avoided by use of a continuous sterilization procedure.Although continuous sterilization is t
33、he logical preliminary step for continuous fermentation in industrial scale,it is also of value in batch fermentations,making greater yields possible for the time and space allotted.The reason for this is because of the exponential relationship between death rate and temperature,making the time requ
34、ired for the complete elimination of life shorter if higher temperatures are used.While batch sterilization is carried out in 30-60 minutes at 121,continuous sterilization is normally accomplished in 30-120 seconds at 140.The heating of culture media for continuous sterilization can be done either b
35、y injection of steam or by means of heat exchangers.Sterilization with steam injection is done by injecting steam into the nutrient solution.The temperature is raised quickly to 140 and is maintained for 30-120 seconds.Due to the formation of condensate,the nutrient solution becomes diluted;to corre
36、ct this,the hot solution is pumped through an expansion valve(膨膨胀胀 阀阀)into a vaporizer and the condensate is removed via vacuum pumps so that the sterilized nutrient solution has the same concentration after the cooling process as before.The disadvantage of this process is the sensitivity it exhibit
37、s to changes in the viscosity of the medium and to pressure variations.In the continuous process using heat exchangers(as Figure),the nutrient solution in the first heat exchanger is preheated to 90-120 within 20-30 seconds by the exiting previously sterilized nutrient solution.Then in the second he
38、at exchanger,it is heated indirectly with steam to 140 .This temperature is maintained for 30-120 second in a holding pipe before it is placed in the first exchanger for preliminary cooling and then in a third exchanger for cooling to the temperature of the fermenter.The cooling phase is only 20-30
39、seconds.The following Figure shows the temperature profile of the nutrient solution during sterilization.In the process using heat exchangers,90%of the energy input is recovered.The disadvantage of this method is that with some nutrient solutions,insoluble salts(calcium phosphate or calcium oxalate
40、草草 酸酸 钙钙 )are formed and crusts appear in the first heat exchanger,due to the extreme temperature differences between the sterilized nutrient solution and the cold incoming solution.If precipitation occurs,the heat transfer coefficient decreases and the system must then be stopped,treated with clean
41、ing agent(acid or base),and resterilized.By sterilizing the critical components of the nutrient solution separately,the value of K can be kept constant and the useful period can be extended for weeks.Starch-containing solutions which become viscous when heated are difficult to use in continuous ster
42、ilization processes.Before the actual sterilization,a liquefaction and partial hydrolysis through acids or amylases must be carried out.Moreover,if there are suspended particles in the nutrients solutions,the short sterilization times in the continuous process may not be sufficient for the heat to p
43、ermeate them thoroughly.The heating time for 1 mm particles is 1 second;for 1 cm size should be restricted to 1-2 mm in continuous sterilization processes.Most industrial fermentations are operated under vigorous aeration and the air supplied to the fermenter must be sterilized.The number of particl
44、es and microorganisms in air varies greatly depending on location of the plant,air movement,and previous treatment of the air.4.5 空气净化（除菌）空气净化（除菌）Sterilization of fermentation air 4.5 空气净化（除菌）空气净化（除菌）Sterilization of fermentation airOn the average,outdoor air has 10-100,000 particles per m3 and 5-2,
45、000 microorganisms/m3.Of these,50%are fungus spores and 40%are Gram-negative bacteria.Fermenters generally work with aeration rates of 0.5-1.0 vvm(air volume/liquid volume minute).A fermenter having a working volum of 50 m3 with an aeration rate of 1 vvm needs 3000 m3 sterile air per hour.The critical importance of air sterilization in industrial microbiology can be seen from these values.4.5 空气净化（除菌）空气净化（除菌）Sterilization of fermentation air