INDUSTRIAL

MICROBIOLOGY​ ​MCB 406

Course Lecturer  
Dr. Adeleke Osho  

Industrial Microbiology

Scope of Industrial Microbiology. Microorganisms of industrial importance. Fermentation processes including

culture methods – solid and submerged. Strain selection and development, media formulation and economics.
Optimization of the fermentation process including fermentation design, fermenters and operations. Primary and
secondary metabolites. Microbiological quality control of foods, drugs and cosmetics. Use of enzymes in the
industries

INDUSTRIAL MICROBIOLOGY  

1.0 ​Scope of Industrial Microbiology  

Industrial Microorganisms and Their Products  

The major organisms used in industrial microbiology are fungi (yeasts and molds) and certain 
prokaryotes, in particular species of the genus Streptomyces. Industrial microorganisms can 
be thought of as metabolic specialists, capable of synthesizing one or more products in high 
yield. Industrial microbiologists often use classical genetic methods to select high-yielding 
microbial variants, with the goal being to increase the yield of the product to the point that an 
economically feasible process is possible. Thus the behaviour of the actual production strain 
may be far removed from that of the original wild-type strain.  

The  use  of  microbes  to  obtain  a  product  or  service  of  economic  value  constitutes  industrial 
microbiology​.  ​Any  process  mediated  by  or  involving  microorganisms  in  which  a  product  ​of 
economic  value  is  obtained  is  called  fermentation​.  The  terms  industrial  microbiology  ​and 
fermentation  are  virtually  synonymous in their scope, objectives and activities. The ​microbial 
product may be  
∙ ​microbial cells (living or dead), microbial biomass, and components of microbial cells,​ ​∙
microbial metabolites,  
∙ ​intracellular or extracellular enzymes or  
∙ ​chemicals produced by the microbes utilizing the medium constituents or the provided 
substrate  
∙ ​and/or modified compound that has been microbiologically transformed​ ​∙
Recombinant products through the DNA recombinant technology.  
The services generated by microorganisms range from the  
∙ ​degradation of organic wastes, 
∙ ​detoxification of industrial wastes and toxic compounds,  
∙ ​the degradation of petroleum to manage oil spills, etc.  
 ∙ ​Industrial microbiology also encompasses activities like production of biocontrol​ ​agents, 
inoculants used as biofertilizers, biofuel: etc.  
The  activities  in  industrial  microbiology  begin  with  the  isolation  of  microorganisms  from 
nature,  their  screening  for  product  formation, improvement of product yields, maintenance of 
cultures,  mass  culture  using  bioreactors,  and  usually  end  with  the  recovery  of  products  and 
their purification.  

 
Properties of a Useful Industrial Microorganism  
A microorganism used in an industrial process must have other features besides just being 
able to produce the substance of interest in high yield.   

1. ​First and foremost, the organism must be capable of growth and product formation in 
large-scale culture.   
2. ​It  should  produce  spores  (if  fungi or yeast) or some other reproductive cell form so that 
it  can  be  easily  inoculated  into  the  large  vessels  used  to  grow the producing organism 
on an industrial scale.   
3. ​It must also grow rapidly and produce the desired product in a relatively short period of 
time.  
4. ​It  must  also  be  able  to  grow  in  a  liquid  culture  medium  obtainable in bulk quantities at 
a  low  price.  Many  industrial  microbiological  processes  use  waste  carbon  from  other 
industries as major or supplemental ingredients for large-scale culture media. These  
include  corn  steep  liquor  (a  product  of  the  corn  wet-milling  industry  that  is  rich  in 
nitrogen  and  growth  factors)  and  whey (a waste liquid of the dairy industry containing 
lactose and minerals).  
5. ​An  industrial  microorganism  should  not  be  pathogenic,  especially  to  humans  or 
 economically  important  animals  or  plants.  Because  of  the  high  cell  densities  in 
industrial  microbial  processes  and  the  virtual  impossibility  of  avoiding  contamination 
of  the  environment  outside  the  growth  vessel,  a  pathogen  would  present  potentially 
disastrous problems.  
6. ​Finally,  an  industrial  microorganism  should  be  amenable  to  genetic  manipulation 
because  increased  yields are often obtained by means of mutation and classical genetic 
selection  techniques.  A  genetically  stable  and easily engineered microorganism is thus 
a clear advantage for an industrial process.  

Problems often associated with Industrial Microbial Processes  

1. ​Finding the ​least expensive medium ​in which to grow the microbe so as to ​maximize 
yield and profits.  
o ​Often this is a ​waste product ​from another industrial process, such as corn steep 
liquor, sugar processing wastes or whey.  
2. ​Maintaining strain purity and developing better strains for ​improving the yield​. o ​ ​A 
single mutation may decrease the yield by a significant percentage or result in undesirable 
substances being produced. The industrial research laboratories constantly seek better 
strains for the production of their product.  
3. ​Preventing contamination by other microbes and by viruses (phage) that live on the 
microbe involved.  
o ​The media must be sterilized prior to being inoculated with the desired organism 
and  purity  must  be  maintained  throughout  the  production  process.  A  small 
quantity  of  a  contaminant  may produce an enzyme that can destroy the product 
in  thousands  of  gallons  of  medium.  For  many  microbes,  viruses  present  a 
constant  danger  as  a  single  virus  can  infect  and  destroy  the  desired microbe in 
an  entire  tank.  The  sterilization  of  large  containers  and  huge  quantities  of 
media represent both an engineering and microbial challenge.  
4. ​Developing rapid and efficient methods for purification of the desired produce in a 
stable form that is safe to use.  
o ​The  products  of  many  fermentations  are  often  unstable  in  the  IMPURE  FORM 
or  subject  to  unwanted  modifications  if  they are not purified quickly. The final 
growth  mixture  may  contain  dangerous  substances  from  which  the  desired 
product  must  be  separated.  As  every  step  in  the  purification results in a loss of 
the  product,  the  search  for  more  efficient  purification  procedures  is  never 
ending.  
5. ​Always striving to improve yield by modifying the strain, nutrients or environmental 
conditions.  
o ​As  product  yields  are  ​sensitive  ​to  subtle  modifications  in  the  nutrient  and  the 
environmental  conditions​,  these  are  constantly  monitored.  For  example,  the 
pH,  oxygen  content,  nitrogen/phosphorous  ratio  etc.  may  be  adjusted  during 
the production process.  
6. ​Safe and inexpensive disposal of the massive quantities of ​waste products ​remaining 
after the product is formed. The waste products of these large fermentations present  
major  waste  disposal  problems  as  they  are  rich  in  organic  matter  that  are  highly 
polluting  if  released  untreated  into  the  environment.  However,  the  cost  of  treatment 
cuts into the profit margin and increases the cost of the product.  
Microbes History  
- Microbes have been employed for ​product generation​, e.g., wines, bread, etc., since 
thousands​ ​of years, but these activities were purely art.  
- The science of industrial microbiology is only about 150 years old.  
- The first observations of microorganisms by Anthony Leeuwenhoek were published in 
1677.  
-  The  experiments  of  Spallanzani  in  1799  and  of  Schwan  in  1837  not  only  disproved  ​the 
idea  of  spontaneous  generation  of  microorganisms,  but  also  provided  a  means  of 
sterilization of liquids (by heat) and air (by heat), respectively.  
- Schwan's findings also suggested that alcoholic fermentation was due to a fungus or 
mold, i.e., yeast, and inoculation resulted in quicker fermentation.  
-  But  microbiology  is  widely  considered  to  have  begun  in  1857  when  Luis  Pasteur 
reported  his  studies  on  lactic  acid  fermentation,  including  the  microscopic features of 
the  microorganisms  and  a  suitable  medium  for  the  process;  the  scientific  basis  of 
industrial microbiology began with this paper.  
- In 1860, Pasteur reported the first synthetic medium for microorganisms, and used it​ ​to 
study alcohol fermentation.  
-  In  1861,  Luis  Pasteur  showed  that  growth  and  physiology  of  yeast  (and  hence  the 
accumulation  of  fermentation  product,  alcohol)  differs  depending  on  the  presence  or 
absence  of  CO​2​.  This  phenomenon  is  known  as  Pasteur  effect  and  is  applicable  to 
other​ ​microorganisms as well.  
- In 1878, Lister described the dilution technique for obtaining the first pure microbial 
culture of lactic acid bacterium.  
-  A  simpler  and  more  effective  technique  for  obtaining  pure  cultures  from  isolated 
separate  colonies  developed  on  solidified  medium  was  described  by  Robert  Koch  in 
1881; this technique is widely followed even today  
- In 1876, Cohn showed that bacterial spores have a high level of heat resistance and 
developed the technique of 'intermittent sterilization' for their inactivation.​ ​- In 1897, 
Buchner demonstrated alcohol fermentation by cell-free yeast juice; he​ ​suggested that a 
proteinaceous enzyme was responsible for fermentation.​ ​- Wildiers demonstrated in 1901 
that yeast required growth factors (vitamins) for growth,​ ​especially at low inoculum 
levels; vitamins are used in fermentation even today.​ ​- In 1929, Alexander Fleming 
accidentally discovered penicillin produced by ​Penicillium​ ​growing as contaminant in a 
Petri plate of ​Staphylococcus​. Fleming developed the​ ​technique for assay of antibacterial 
activity of penicillin using bacteria and showed its​ ​low toxicity to man and animals. This 
was followed by an intensive search for​ ​antibiotics during the Second World War leading 
to the discovery of streptomycin,​ ​chloramphenicol, tetracyclines, etc. Later developments 
have resulted in the use​ ​of metabolically blocked mutants of microorganisms, which 
accumulate large amounts​ ​of metabolic intermediates.  

2.0​ STRAIN SELECTION AND DEVELOPMENT 

Isolation and screening of microorganisms  
Isolation of Microorganisms  
The  first  step  in  developing  a  producer  strain  is  the  isolation  of  concerned  microorganisms 
from  their  natural  habitats.  Alternatively,  microorganisms  can  be  obtained  as  pure  cultures 
from  organisation,  which  maintain  culture  collections,  e.g.,  ​American  Type  Culture 
Collection  ​(​ATCC​)  Rockville,  Maryland,  U.S.A.;  ​Commonwealth  Mycological  Institute 
(CMI),  Kew,  ​Surrey,  England;  ​Fermentation  Research  Institute  ​(FERM),  Tokyo,  Japan; 
U.S.S.R.​ ​Research Institute for Antibiotics ​(RIA), Moscow, U.S.S.R., etc.  
The  ​microorganisms  of  industrial  importance  a​ re,  generally,  ​bacteria,  actinomycetes,  fungi 
and  ​algae​.  These  organisms  occur  virtually  everywhere,  e.g.,  in  air,  water,  soil,  surfaces  of 
plants  and  animals,  and  plant  and  animals  tissues.  But  most  common  sources  of  industrial 
microorganisms  are  soils,  lake  and  river  mud.  Often  the  ecological  habitat  from  which  a 
desired  ​microorganism  is  more  likely  to  be  isolated  will  depend  on  the  characteristics  of  the 
product  ​desired  from  it,  and  of  process  development.  For  example,  if  the  objective  is  to 
isolate  a  source  ​of  enzymes,  which  can  withstand  high  temperatures,  the  obvious  place  to 
look  will  be  hot  water  ​springs.  A  variety  of  complex  isolation  procedures  have  been 
developed,  but  no single method ​can reveal all the microorganisms present in a sample. Many 
different  microorganisms  can  ​be  isolated  by  using  specialized  enrichment  techniques,  e.g., 
soil  treatment  (UV  irradiation,  ​air  drying  or  heating  at  120°C,  filtration  or  continuous 
percolation,  washings  from  ​root  systems,  treatment  with  detergents  or  alcohols, 
pre-inoculation  with  toxic  agents),  ​selective  inhibitors  (antimetabolites,  antibiotics,  etc.), 
nutritional  (specific  C  and  N  sources),  ​variations  in  pH,  temperature,  aeration,  etc.  The 
enrichment  techniques  are  designed  for  ​selective  multiplication  of  only  some  of  the 
microorganisms  present  in  a  sample.  These  ​approaches  however  take  a  long  time  (20-40 
days),  and  require considerable labour and money. ​The main isolation methods used routinely 
for  isolation  from  soil  samples  are:  sponging  ​(soil  directly),  dilution,  gradient  plate,  aerosol 
dilution,  flotation,  and  differential  ​centrifugation.  Often  these  methods  are  used  in 
conjunction with an enrichment technique.  

Screening of Microorganisms for New Products  

The next step after isolation of microorganisms is their screening.  
A  set  of  highly  selective  procedures,  which  allows  the  detection  and  isolation  of 
microorganisms  producing  the  desired  metabolite,  constitutes  ​primary  screening​.  Ideally, 
primary  screening  should  be  ​rapid,  inexpensive,  predictive,  specific  b​ ut  effective  for  a  broad 
range  of  compounds  and  applicable  on  a  large  scale.  Primary  screening  is  time  consuming 
and  labour  intensive  since  a  large  number  of  isolates  have  to  be  screened  to  identify  a  few 
potential  ones.  However  this  is  possibly  the  most  critical  step  since  it  eliminates  the  large 
bulk  ​of  unwanted  useless  isolates,  which  are  either  non  producers  or  producers  of  known 
compounds.  Computer  based  databases  play  an  important  role  by  instantaneously  providing 
detailed  information  about  the  already  known  microbial  antibiotic  compounds.  Rapid  and 
effective  screening  techniques  have  been  devised  for  a  variety  of  microbial  products,  which 
utilize  either  a  property  of  the  product  or  that  of  its  biosynthetic  pathway  for  detection  of 
desirable  isolates.  Some  of  the  screening  techniques  are  relatively  simple,  e.g.,  for 
extracellular  ​enzymes  and  enzyme  inhibitors.  However  for  most  microbial  products  of  high 
value, the ​screening is usually complex and tedious, and often may involve two or more steps, 
e.g.,  for  ​antimicrobials.  In  some  cases,  it  may  be  desirable  to  concentrate  on  a  group  of 
organisms  ​expected  to  yield  new  products.  For  example,  the  search  for  new  antibiotics  now 
focusses​ ​on rare Actinomycetes, i.e., Actinomycetes other than those belonging to the genus 
Streptomyces. Suitably designed specialized screening techniques may be used to detect 
compounds having various pharmacological activities other than antibiotics.  

Inoculum Development  
-  The  preparation  of  a  population  of  microorganisms  from  a  dormant  stock  culture  to  an 
active  ​state  of  growth  that  is  suitable  for  inoculation  in  the  final  production  stage  is  called 
inoculum  ​development.  As  a  first  step  in  inoculum  development,  inoculum  is  taken  from  a 
working  ​stock culture to initiate growth in a suitable liquid medium. Bacterial vegetative cells 
and  ​spores  are  suspended,  usually,  in  sterile  tap  water,  which  is  then  added  to  the  broth.  In 
case  of  ​non-sporulating  fungi  and  actinomycetes  the  hyphae  are  fragmented  and  then 
transferred  to  ​the  broth.  Inoculum  development  is  generally  done  using  flask  cultures;  flasks 
of  50  ml  to  12  ​litres  may  be  used  and  their  number  can  be  increased  as  per  need.  Where 
needed,  small  ​fermenters  may  be  used.  Inoculum  development  is  usually  done  in  a  stepwise 
sequence  ​to increase the volume to the desired level. At each step, inoculum is used at 0.5-5% 
of  the  ​medium  volume;  this  allows  a  20-200-fold  increase  in  inoculum  volume  at  each  step. 
Typically,​ ​the inoculum used for production stage is about 5% of the medium volume.  

3.0 ​Media Formulation and Economics  

Culture Media  
Inoculum  preparation  media  are  quite  different  from  production  media.  These  media  are 
designed  for  rapid  microbial  growth,  and  little  or  no  product  accumulation  will  normally 
occur.  ​Many  production  processes  depend  on  inducible  enzymes.  In  all  such  cases,  the 
appropriate  ​inducers  must  be  included  either  in  all  the  stages  or  at  least  in  the final stages of 
inoculum  ​development.  This  will  ensure  the  presence  of  the  concerned  inducible enzymes at 
high levels​ ​for the production to start immediately after inoculation.  

Contamination  
-  The  inoculum  used  for  production  tanks  must  be  contamination  free.  But  the  risk  ​of 
contamination  is  always  present  during  inoculum  development.  Therefore,  every  effort  must 
be made to detect as well as prevent contamination.  
Sterilization  
-  Sterilization is the process of inactivating or removing all living organisms from a substance 
or  surface.  In  concept,  it  is  regarded  as  absolute  in  all  living  cells  must  be  inactivated  / 
removed,  ​usually  in a single step at the given time. But in practice, the success of sterilization 
procedures  ​is  only  a  probability.  Therefore,  the  probability  of  a  cell  escaping 
inactivation/filtration  ​does  exist  although  it  is  usually  very  small.  When  a  closed  system  is 
sterilized  once,  it  remains  ​so  indefinitely  since  it  has  no  openings  for  the  entry  of 
microorganisms.  But  most  fermentation  ​vessels  are  open  systems;  such  systems  are  initially 
sterilized  and  must  be  kept  sterilized  by  ​ensuring  the  removal  of  living  cells  at  their  entry 
points, e.g., the cotton plug of a culture flask.​ ​Common Contaminants  
- The most common contaminants of different industrial processes are considerably different. 
Some examples are given below  
1.  In  canning  industry,  ​Clostridium  butylicum  i​ s  the  chief  concern.  This  obligate  anaerobe 
can  ​grow  in  sealed  cans,  and  produce  heat  resistant  spores  and a deadly toxin. However, it is 
not  a  ​problem  for  catsup  (too  acidic),  jam  and  jellies (too high sugar concentration) and milk 
(stored​ ​at low temperature).  
2. Organisms like lactobacillus are a problem in production of wine.  
3. In antibiotic industry, potential contaminants are many, e.g., molds, yeast, and many 
bacteria, including Bacillus. 
4.The most dreaded contaminants of fermentation industry are phages. The only effective 
protection against phages is to develop resistant strains.  
Sterilization Procedures  
- Sterilization involves either inactivation or removal of living organisms. This may be 
achieved by  
(i) heating,  
(ii)irradiation.  
(iii) Chemicals or  
(iv) filtration; these are briefly discussed below.  
Heating.  
- It is the most commonly used and the least expensive sterilizing agent.​ ​- Dry heat is used 
in ovens and is suitable for sterilization of solids, which can withstand​ ​the high 
temperatures needed for sterilization, e.g., laboratory glassware, talc, etc.​ ​Steam, i.e., 
moist or wet heat, is used for sterilization of media and fermenter vessels.​ ​An autoclave 
uses steam for sterilization (at 121°C and 15p.s.i.). the period of time at​ ​this temperature 
pressure depending on medium volume, e.g., 12-15 min for 200 ml.​ ​17-22 min for 500 
ml, 20-25 min for 1 L and 30-35 min for 2 L.  
-  But  sterilization  of  oils will require a few hours, and concentrated media (10-20% solid) 
must  be  agitated  for  effective  sterilization.  Autoclaves  can  also  be  used  to  sterilize 
laboratory  vessels,  small  volumes  of  media  and  even  small  fermenters.  Large 
fermenters  are  sterilized  by  either  a  direct  injection of steam or by indirect heating by 
passing  steam  through  heat  exchange  coils  or  a  jacket.  The  steam  should  always  be 
saturated.  Media  sterilization  may  be  achieved  in  a  continuous  flow  sterilization 
system  ​either  by  direct  steam  injection  or  by  indirect steam heating, and then filled in 
a  sterile  ​fermenter.  Alternatively,  the  medium  may  be  filled  in  the  fermenter  and 
steam  sterilized  with  the  latter.  Heat killing in most part is due to protein inactivation. 
In​ ​general, moist heat is far superior to dry heat.  
- Bacterial spores are the most heat resistant, e.g., spores of thermophilic bacteria can 
survive steam at 30p.s.i. at 134°C for 1-10 min and dry heat at 180°C for up to 15 min. 
Radiation.  
High  energy  X-rays  are  used  for  sterilization  of  a  variety of lab ware and of food. In general, 
vegetative  cells  are  much  more  susceptible  than  bacterial  spores  (Clostridium  spores  can 
resist​ ​nearly 0.5 M rad). But ​Deinococcus radiodurans v​ egetative cells can survive 6 M rad.  

Viruses  are  usually  similar  to  bacterial  spores  but  some  viruses,  e.g.,  encephalitis  virus 
require  ​up  to  4.5  M  rad.  In  practice,  2.5  M  rad  is  used  for  sterilizing  pharmaceutical  and 
medical  ​products.  X-rays  cause  inactivation  by  inducing  single  and  double  strand  DNA 
breaks,  and  by  ​producing  free  radicals  and  peroxides,  to  which -SH enzymes are particularly 
susceptible.  
Chemicals  
.  The  chemicals  used  for  sterilization  cause  inactivation  by  oxidation  or  alkylation; these ​are 
formaldehyde,  H​2​O2​ ​,  ethylene  oxide,  propylene  oxide  etc.  H​2​O2​   ​(10-25%  w/v)  is  ​being 
increasingly  used  in  the  sterilization  of  milk  and  of  containers  for  food  products.  It  is  a 
powerful  oxidizing  agent,  kills  both  vegetative  cells  and  spores  and  is  very  safe.  Ethylene 
oxide  ​is  used  for  sterilizing  equipment,  which  are  likely  to  be  damaged  by  heat,  and  is  very 
effective,​ ​but highly toxic and violently explosive if mixed with air.  
Filtration.  
Aerobic  fermentation  requires  a  very  high  rate  of  air  supply  often  amounting  to  1  vol  of  air 
(equal  to  medium  volume)  every  minute.  Air contains both fungal spores and bacteria, which 
are  ordinarily  removed  by  filtration  using  either  a  depth  filter  or  a  screen  filter. Depth filters 
are made from fibrous or powdered materials pressed or bonded together in a relatively thick 
layer;  the  materials  used  are  fiberglass,  cotton,  mineral  wool,  cellulose  fibers, etc. in form of 
mats,  wads  or  cylinders.  Modern  depth  filters  are  cylinders  of  bonded  borosilicate 
microfibers.  ​Depth  filters  allow  higher  filtration  rates  and  efficiencies  than  screen filters, but 
are  not  suitable  ​for  filtration  of  moist  air.  Screen  filters  are  membranes  of  cellulose esters or 
other  polymers  ​with  pores  of  0.45  µm  or  smaller  (bacterial  contaminants  are  0.5  µm  or 
larger).  Usually,  a  ​microfibers  profiler  is  used  with  such  filters  to  remove  gross 
contamination.  All  filters  ​themselves  must  be  sterilized  before  they  can  be  used  to  sterilize 
the  air.  Filters  are  also  used  ​to  sterilize  the  effluent  gasesfrom  fermenters,  especially  in  case 
of pathogenic microorganisms.  

4.0 ​Strain Selection and Development  

Strain Improvement  
-  After  an  organism  producing  a  valuable  product  is  identified,  it  becomes  necessary  to 
increase  ​the product yield from fermentation to minimise production costs. Product yields can 
be​ ​increased by  
(i) developing a suitable medium for fermentation,  
(ii) refining the fermentation process and  
(iii) improving the productivity of the strain  

Generally,  major  improvements  arise  from  the  last  approach;  therefore,  all  fermentation 
enterprises  place  a  considerable  emphasis  on  this  activity.  The  techniques  and  approaches 
used  ​to  genetically modify strains, to increase the production of the desired product are called 
strain  ​improvement  or  strain  development.  Strain  improvement  is  based  on  the  following 
three  ​approaches:  (i)  mutant  selection,  (ii)  recombination,  and  (iii)  recombinant  DNA 
technology.  

(i) Mutant Selection  

Large  scale  mutant  selection  programmes  begin  when  favourable  reports  of clinical trials are 
obtained.  In  the  early  stages,  selection  of  ​spontaneous  mutants  ​may  be  helpful,  but  induced 
mutations  are  the  most  common  sources  of  improvements.  Many  mutations  bring  about 
marked  ​changes  in  a  biochemical  character  of  practical  interest;  these  are  called  major 
mutations.  Some ​major mutations can be useful in strain improvement. For example, a mutant 
strain  (S-604)  of  ​Streptomyces  aureofaciens  ​produces  6-demethyl  tetracycline  in  place  of 
tetracycline;  this  ​demethylated  form  of  tetracycline  is  the  major  commercial  form  of 
tetracycline.  In  contrast,  ​most  improvements  in  biochemical  production  have  been  due to the 
stepwise  accumulation  of  ​so  called  minor  genes​.  These  genes  lead  to  small  increases  (or 
decreases)  in  the antibiotic or ​other biochemical production, and selection may be expected to 
result  in  a10-15%  increase  in  ​yield.  The  selected  strains  are  usually  subjected  to  successive 
cycles  of  mutagenesis  and  ​selection;  after  several  cycles,  a  large  increase  is  yield  is  likely to 
be  obtained.  Mutants  of  ​Penicillium  chrysogenum  w ​ ere  selected  for  increased  penicillin 
production;  each  cycle  of  ​selection  was  preceded  by  Mutagen  (chemical)  treatment  and 
resulted  in  only  small  changes  in  ​penicillin  yield.  The  two types are associated with different 
strains  of  ​Agrobacterium.  ​tumefaciens.​  There is also a related disease ("hairy root disease") in 
which  infected  tissues  ​proliferate  in  root  tissue  and  produce  an  opine.  This  disease  is 
associated  with  the  bacterium  ​A.  r​ hizogenes.​   Formation  of  opines  explains  the  ecological 
significance  of  tumor  formation.  Each  ​strain  of  Agrobacterium  synthesizes  enzymes 
(permease,  dehydrogenase)  that  allow  it  ​to  metabolize  the  specific  type  of  opine  formed  by 
the  tumor  it  induces.  Thus  by  stimulating  ​the  plant  to  form  opines,  the  bacteria  insure 
themselves  a  supply  of  nutrients  specifically  ​designed  for  them.  After  several  (about  dozen) 
cycles  of  selection,  a  strain  (E  15-1)  was  ​obtained  that  yielded  55%  more  penicillin  than  the 
original strain (Fleming strain). 
Selective Isolation of Mutants  
A  majority  of  desirable  mutants,  especially  the  'minor  gene'  mutants,  showing  increased 
production  are  isolated  by  screening  a  large  number  of  clones  surviving  the  mutagen 
treatment;  ​this  is  called  secondary  screening.  But  this  approach  requires  a  large  amount  of 
work. ​Therefore, efforts have increasingly focussed on developing techniques for the isolation 
of​ particular classes of mutants, which are likely to be overproducers.  
1.  Isolation  of  auxotrophic  mutants  is  the  basis  for  commercial  amino  acid  production  in 
Japan  ​from  the  bacterium  ​Corynebacterium  glutamicus​.  For  example,  phe-mutants  of  ​C 
glutamicus​ ​accumulate tyrosine.  
2.  Many  analogue-resistant  mutants  have  feed-back  insensitive  enzymes  of  the  biosynthetic 
pathway  the  analogue  of  whose  product  was  used  for  selection  of  such  cells.  Such  mutants 
tend​ to overproduce the end product of the concerned pathway.  

3.  Sometimes  revertants  from  nonproducing  mutants  of  a  strain  are  high  producers,  e.g., ​one 
such  reversion  mutant  of  ​Streptomyces  viridifaciens  ​showed  over  6-fold  increase  in 
chlortetracycline production over the original strain from which the nonproducing mutant was 
obtained.  
4. Reversion mutants of appropriate auxotrophs may often be high producers.​ ​5. In some 
cases, selection for resistance to the antibiotic produced by the organism itself may​ ​lead to 
increased yields.  
6. Sometimes, mutants with altered cell membrane permeability show high production of 
some​ ​metabolites.  
7.  Mutants  have  been  selected  to  produce  altered  metabolites,  especially  in  case  ​of 
aminogycoside  antibiotics.  For  example,  ​Pseudomonas  aureofaciens  p​ roduces  the  antibiotic 
pyrrolnitrin;  a  mutant  of  this  fungus  yields  4'-  fluoropyrrolnitrin.  Mutant  selection  has  been 
the  ​most  successful  approach  for  strain  improvement,  but  major  advances  are  being  made  in 
the​ ​exploitation of other strategies, i.e., recombination and recombinant DNA technology. 
  
5.0 ​Optimization of Fermentation Process  

Bioreactors -  
A bioreactor is a device in which a substrate of low value is utilized by living cells or enzymes 
to  generate  a  product  of  higher  value​.  Bioreactors  are  extensively  used  for  food  processing, 
fermentation,  waste  treatment,  etc.  On  the  basis  of  the  agent  used,  bioreactors  are  grouped 
into​ ​two broad classes:  
(i) those based on living cells and,  
(ii) ​those employingenzymes.  
But in terms of process requirements, they are of the following types:  
(i) aerobic,  
(ii) ​anaerobic,  
(iii) ​solid state, and  
(iv) ​immobilized cell bioreactors.  
All bioreactors deal with heterogeneous systems having two or more phases, e.g., liquid, gas, 
solid. Therefore, optimal conditions for fermentation necessitate efficient transfer of mass, 
heat​ ​and momentum from one phase to the other. A bioreactor should provide for the 
following:​ ​(i) agitation (for mixing of cells and medium),  
(ii) ​aeration (aerobic fermenters; for O​2 ​supply),  
(iii) ​regulation of factors like temperature, pH, pressure, aeration, nutrient feeding,​ ​liquid 
level, etc.,  
(iv) ​sterilization and maintenance of sterility, and 
(v) ​withdrawal  of  cells/medium  (for  continuous  fermenters).  Modem  fermenters  are 
usually  integrated  with  computers  for  efficient  process  monitoring,  data 
acquisition,​ ​etc.  
The  size  of  fermenters  ranges  from  1-2  Litres  laboratory  fermenters  to  500,000  litres  or, 
 occasionally,  even  more;  fermenters  of  up to 1.2 million litres have been used. Generally, 
20-25%  of  fermenter  volume  is  left  unfilled  with  medium  as  "head  space"  to  allow  ​for 
splashing,  foaming  and  aeration.  The  fermenter  design  varies  greatly  depending  on  the 
type of fermentation for which it is used.  

Immobilized Cell Bioreactors  

Bioreactors of this type are based on immobilized cells. Cell immobilization is advantageous 
when  
(i) the enzymes of interest are intracellular,  
(ii) extracted enzymes are unstable,  
(iii)  the  cells  do  not  have  interfering  enzymes  or  such  enzymes  are  easily 
inactivated/removed,  and  ​(iv)  the  products  are  low  molecular  weight  compounds 
released into the medium.  
Under these conditions, immobilized cells offer the following advantages over enzyme 
immobilization:  
(i) enzyme purification is not needed,  
(ii) ​high activity of even unstable enzymes,  
(iii) ​high operational stability,  
(iv) ​lower cost and  
(v) ​Possibility of application in multistep enzyme reactions.  
(vi) ​In  addition,  immobilization  permits  continuous  operation  of  bioreactor,  which 
reduces  the  reactor  volume  and,  consequently,  pollution  problems.  Obviously, 
immobilized  cells  are  used  forsuch  biotransformation  of  compounds,  which 
require​ ​action of a single enzyme.  

Cell immobilization may be achieved in one of the following ways:  

1.  Cells  may  be  directly  bound  to  water  insoluble  carriers,  e.g.,  cellulose,  dextran,  ion 
exchange  resins,  porous  glass,  brick,  sand,  etc.,  by  adsorption,  ionic  bonds  or  covalent 
bonds.  
2. They can be cross linked to bi- or multi-functional reagents, e.g., glutaraldehyde, etc.​ ​3. 
Polymer matrices may be used for entrapping cells; such matrices are polyacrylamide 
cell, ҝ-carrageenan (a polysaccharide isolated from a seaweed), calcium alginate (alginate 
is extracted from seaweed), poly glycol oligomers, etc. Out of these approaches, calcium 
alginate immobilization is the most commonly used since it can be used for even very 
sensitive cells. Cell immobilization has been used for commercial production of amino 
acids, organic acids, etc.  

Bioreactor Media  
The  medium  composition  is  as  critical  to  product  yields  as  high  producing  strains  of 
microorganisms. The medium not only provides the nutrients needed for microbial growth but 
also  for  the  metabolite  production.  The  organisms  vary  greatly  in  their  nutrient requirements 
from  autotrophs,  which  produce  all  the  biochemical  required  from simple inorganic nutrients 
deriving  their  energy  from  oxidation  of  some  inorganic  component  of  the  medium  to  the 
difficult  organisms  like  lactic  acid  bacteria,  which  require  many organic compounds for their 
growth.  
The various media for bioreactors may be grouped into two broad categories: 
(i) ​Synthetic ​and (ii) ​Complex​.   
A  synthetic  or  chemically  defined  medium  is desirable for various studies, but product yields 
from  such  media  are  generally  low.  Foaming is not a problem with such media. The complex 
media  contain undefined constituents like soybean meal, molasses, corn steep liquor, etc., and 
give  much  higher  yields  of  metabolites.  Carbon  source  can  be  simple,  e.g.,  sugar,  alcohol, 
etc.,  ​or  complex  carbohydrates,  proteins,  molasses,  potatoes,  sweet  potatoes,  etc.  In  many 
processes,  ​precursors  need  to  be  provided,  e.g.,  phenylacetic  acid  for  penicillin  G,  inorganic 
cobalt  for  vit.  ​B1​ 2​.  Buffers  are  also  added  to  prevent  drastic  changes  in  pH,  and  anti-foam 
would  often  be  ​needed  when complex media are used. For much fermentation, e.g., antibiotic 
production,  ​medium  suited  for  rapid  cell  growth  is  unsuitable  for  product  formation.  In such 
cases,​ ​specialized media for production have to be devised.  

Downstream Processing  
-  ​The  various  processes  used  for  the  actual  recovery  of  useful  products  from  a  fermentation 
or  a​ ny  other  industrial  process  is  called  downstream  processing.  T ​ he  cost  of  ​downstream 
processing  ​(DSP) is often more than 50% of the manufacturing cost, and there is product loss 
at each step of DSP.  
Therefore, the DSP should be efficient, involve asfew steps as possible (to avoid product
loss),​ ​and be cost-effective. The various steps in DSPareasfollows:

(i) ​separation of particles,

(ii) ​disintegration of cells,
(iii) ​extraction,
(iv) ​concentration,
(v) ​purification and
(vi) ​drying.

(i) Separation of Particles  

- The first step in DSP is the separation of solids, usually cells, from the liquid medium. This 
is generally achieved as follows.  
(a) Filtration  
.  It  is  used  for  the  separation  of  filamentous  fungi  and  filamentous  bacteria,  e.g., 
streptomycetes,  and  often  for  yeast  flocks.  The  various techniques of filtration employed ​are, 
surface  filtration,  depth filtration, centrifugal filtration, cross flow filtration, and rotary ​drum 
vacuum filtration.​   
Centrifugation.  
It may be used to separate bacteria and usually protein precipitates. But difficulties arise due 
to small differences in the densities of the particles and the medium. In addition, equipment 
cost, power consumption, temperature, etc. are the other disadvantages.​ ​Flocculation and 
Floatation  
Flocculation,  i.e.,  sticking  together  of  cells,  can  be  induced  by  inorganic  salts,  mineral 
hydrocolloids  are  organic  polyelectrolytes.  Since  sedimentation  rate  of  a  particle  increases 
with  size,  flocculated  cells  can  be  recovered by centrifugation. In cases, where flocculation is 
not  effective,  very  fine  gas  bubbles  can  be  created  by  sparging,  release  of  overpressure  or 
electrolysis.  The  gas  bubbles  adsorb  to  and  surround  the  cells,  raising  them  to  the  surface  of 
medium  in  form  of  foam  (floatation);  long  chain  fatty  acids  or  amines  promote  stable  foam 
formation. The cells collected in the foam are readily recovered. Flocculation and floatation 
are used for the most efficient recovery of microbial biomass in some single cell protein 
production systems.  

6.0 ​Fermentation Processes  

Fermentation  
Inoculum  is  developed  in  several  stages.  The  organisms  are  often  multiplied  in  one  or  more 
seed  tank  stages. The medium used for these stages is rather rich to support good growth. The 
seed  tank  culture  is  finally  inoculated  into  the  production  stage  fermenter  (25,000-100,000 
1).The production medium is almost always complex and is devised to maximise yields of ​the 
enzyme.  Appropriate  measures  must  be  employed  to  ensure  contamination  control.  ​Biomass 
of  the  organism  begins  to  increase  early  during  fermentation,  while  enzyme  activity  ​shows 
significant  increases  about  midway  through  the  fermentation;  the  end  of  fermentation  is 
signalled by cessation of enzyme synthesis.  

 
Isolation and Purification  
Isolation  and  purification  is  done  immediately  after  termination  of  fermentation  in  a  manner 
that  retains  the  enzyme  activity.  If  the  cells  are  to  be  used for immobilization, the biomass is 
isolated  and  treated to make it ready for use. The extracellular enzymes are recovered directly 
from  broth,  while  enzymes  localized  within  cells  are  isolated  by  rupturing  the cells. Enzyme 
purification  is  based  on  various techniques whose efficacy and cost differ widely; the process 
used will mainly depend on the purity needed and the cost, which is acceptable.  

Types of Fermentation Process  

The  fermentation unit in industrial microbiology is similar to a chemical plant in the chemical 
industry.  A  fermentation  process  is  a  biological  process  and,  therefore,  has  requirements  of 
sterility  and  use  of  cellular  enzymatic  reactions  instead  of  chemical  reactions  aided  by 
inanimate  catalysts,  sometimes  operating  at  elevated  temperature  and  pressure.  Industrial 
fermentation  processes  may  be  divided  into  two  main  types,  with  various  combinations  and 
modifications.  
Some of the most important types of fermentation are as follows:
1. Solid State Fermentation
2. Submerged Fermentation
3. Anaerobic Fermentation
4. Aerobic Fermentation
5. Immobilized Cell Bioreactors
6. Immobilized Enzyme Bioreactors.
1. Sub-merged Fermentation (SLF)  
(Submerged Liquid Fermentation)  

(a) Batch Fermentation Process  

Fermentation is derived from the latin word "​fervere"​ which means to boil, a process typically
manifested by the action of yeasts on malted grain during the production of alcoholic beverages.
Fermentation could be regarded as a metabolic process involved in the conversion of sugar to acids,
gases and/or alcohol​. The growth of microorganisms generally results in metabolite production but
specific metabolite requires optimal cultural conditions at a particular growth rate for save delivery.
A closed loop system where yeasts or bacteria grow in a culture media and an additional material is
not added such is called BATCH CULTURE. Growth in a batch culture undergo some series of steps.
There is the initial phase that seems to record no growth, the lag phase, followed by the gradual
increase that builds up into constant, maximum rate often referred to as the logarithmic phase or
exponential phase.
A  tank  of  fermenter  is  filled  with  the  prepared  mash  of  raw  materials  to  be  fermented.  The 
temperature  and  pH  for  microbial  fermentation  is  properly  adjusted,  and  occasionally 
nutritive  ​supplements  are  added  to  the  prepared  mash.  The  mash  is  steam sterilized in a ​pure 
culture  process.  The  inoculum  of  a  pure  culture  is  added  to  the  fermenter,  from  a  separate 
pure  culture  vessel.  Fermentation  proceeds,  and  after  the  proper  time  the  contents  of  the 
fermenter,  are  taken  out  for  further  processing.  The  fermenter  is  cleaned  and  the  process  ​is 
repeated. Thus each fermentation is a discontinuous process divided into batches  

 
(b) Continuous Fermentation Process  
Growth  of  microorganisms  during  batch  fermentation  conforms  to  the  characteristic  growth 
curve,  with  a  lag  phase  followed  by  a  logarithmic  phase.  This,  in  turn,  is  terminated  by 
progressive  decrement  in  the  rate  of  growth  until  the  stationary  phase  is  reached.  This  is 
because  of  limitation of one or more of the essential nutrients and /or production of inhibitory 
substances.  In  continuous  fermentation,  the  substrate  is  added  to  the  fermenter  continuously 
at  ​a  fixed  rate.  This  maintains  the  organisms  in  the  logarithmic  growth  phase.  The 
fermentation  ​products  are  taken  out  continuously.  The  continuous  fermentation  is  also 
referred to as​ ​chemostat.  

(c) Fed-Batch Culture:  

When a batch culture is subsequently led with fresh nutrient medium without removing the 
growing microbial culture, it is called fed-batch culture. Fed-batch culture allows one to 
supplement the medium with such nutrients that are depleted or that may be needed for the 
terminal stages of the culture, e.g., production of secondary metabolites. 
Therefore, the volume of a fed- batch culture increases with time. Fed-batch cultures achieve 
higher cell densities than batch cultures. It is used when high substrate concentration causes 
growth inhibition. It allows the substrate to be used at lower nontoxic levels, followed by 
subsequent feeding. It allows the maximum production of cellular metabolites by the culture.  

2. Solid State Fermentation (SSF)  

Solid-state fermentation (SSF) processes can be defined as “the growth of microorganisms  
(mainly fungi) on moist solid materials in the absence of free-flowing water”.  

Semi  Solid  OR  Solid  State  Methods  -  In  this,  the  culture  medium  is  impregnated in a carrier 
such  as  bagasse,  wheat  bran,  potato  pulp,  etc.  and  the  organism  is  allowed  to  grow  on  this. 
This  ​method  allows  greater  surface  area  for  growth.  The  production  of  the  desirable 
substance  and  ​the  recovery  is  generally  easier  and  satisfactory.  In  the  development  of  a 
fermentation  process,  ​the  composition  of  the  culture  medium  plays  a  major  role  and  will 
determine  to  a  very  great  ​extent  the  level  of  end  product.  For  example,  a  culture  medium 
containing  sucrose  enables  ​better  production  of  citric  acid  by  ​A.  niger  ​than  any  other 
carbohydrate.  The  pH,  temperature  ​of  incubation,  aeration  etc.,  are  all  important  factors  in 
fermentations  and  these  have  to  be  ​optimized  for  each  type  of  fermentation.  Emphasis  is 
generally  placed  on  the  use  of  cheap  raw  ​materials  so  that  the  cost  of  production  is  low.  In 
such  fermentations,  microbial  growth  and  ​product  formation  occur  at  the  surface  of  solid 
substrates.  Examples  of  such  fermentations  are  ​mushroom  cultivation, mold ripened cheeses, 
starter  cultures,  etc.  More  recently,  this  approach  ​has  been  used  for  the  production  of 
extracellular  enzymes,  certain  valuable  chemicals,  fungal  ​toxins,  and  fungal  spores  (used for 
biotransformation).Traditional  substrates are several ​agricultural products, rice, wheat, maize, 
soybean,  etc.  The  substrate  provides  a  rich  and  ​complex  source  of  nutrients,  which  may  or 
may  not  need  to  be  supplemented.  Such  substrates  ​selectively  support  mycelial  organisms, 
which  can  grow  at  high  nutrient  concentrations  and  ​produce  a  variety  of  extracellular 
enzymes,  e.g.,  a  large  number  of  filamentous  fungi,  and  a  ​few  bacteria  (Actinomycetes  and 
one strain of Bacillus).  
According  to  the  physical  state,  solid  state  fermentations  are divided into two groups: (i) low 
moisture  solids fermented without or with occasional/continuous agitation, and (ii) suspended 
solids  fermented  in  packed  columns,  through  which  liquid  is  circulated.  The  fungi  used  for 
solid  state  fermentations  are  usually obligate aerobes. Solid state fermentations on large scale 
use  stationary  or  rotary  trays.  Temperature  and  humidity  controlled  air  is  circulated  through 
the  stacked  solids.  Less  frequently,  rotary  drum  type  fermenters  have  been  used.  Solid  state 
fermentations  offer  certain unique advantages, but suffer from some important disadvantages. 
However,  commercial  application  of  this  process  for  biochemical  production  is  chiefly 
confined to Japan.  
Solid State Fermentation, SSF  
The following are some the advantages of SSF over submerge liquid fermentation (SLF): 1. 
Comparative studies between submerged liquid fermentation (SLF) and SSF claim higher 
yields than those obtained in the corresponding submerged cultures 2. The low availability 
of water reduces the possibilities of contamination by bacteria and yeast. This allows 
working in aseptic conditions in some cases.  
3. Similar environment conditions to those of the natural habitats for fungi, which 
constitute the main group of microorganisms used in SSF.  
 4. Higher levels of aeration, especially adequate in those processes demanding an 
intensive oxidative metabolism. 
5. The inoculation with spores (in those processes that involve fungi) facilitates its 
uniform dispersion through the medium.  
6. Culture media are often quite simple. The substrate usually provides all the nutrients 
necessary for growth.  
7. Simple design reactors, with few spatial requirements can be used due to the 
concentrated nature of the substrates.  
8. Low energetic requirements (in some cases autoclaving or vapour treatment, 
mechanical agitation and aeration are not necessary).  
9. Small volumes of polluting effluents. Fewer requirements of dissolvents are necessary 
for product extraction due to their high concentration.  
10. The low moisture availability may favour the production of specific compounds that 
may not be produced or may be poorly produced in SLF.  
11. In some cases, the products obtained have slightly different properties (e.g. 
more Thermo-tolerance) when produced in SSF in comparison to SLF.  
12.​Due to the concentrated nature of the substrate, smaller reactors in SSF with respect to 
SLF can be used to hold the same amounts of substrate  

In the same way, SSF has some disadvantages when compared with the 
submerged-liquid cultures:  

1. Only microorganisms that can grow at low moisture levels can be used. 2. Usually the 
substrates require pre-treatment (size reduction by grinding, rasping or chopping, 
homogenisation, physical, chemical or enzymatic hydrolysis, cooking or vapour 
treatment).  
3. Biomass determination is very difficult.  
4. The solid nature of the substrate causes problems in the monitoring of the process 
parameters (pH, moisture content, and substrate, oxygen and biomass concentration). 5. 
Agitation may be very difficult. For this reason static conditions are preferred. 6. 
Frequently needs high inoculum volumes.  
7. Many important basic scientific and engineering aspects are poorly characterized. 
Information about the design and operation of reactors on a large scale is scarce. 8. 
Possibility of contamination by undesirable fungi.  
9. The removal of metabolic heat generated during growth may be very difficult. 10. 
Extracts containing products obtained by leaching of fermented solids are often viscous 
of nature.  
11. Mass transfer limited to diffusion.  
12. In some SSF, aeration can be difficult due to the high solids 
concentration. 13. Spores have longer lag times due to the need for 
germination.  
14.​Cultivation times are longer than in SLF  

3. Anaerobic Fermentation  
Basically  a  fermenter  designed  to  operate  under  microacrophilic  or anaerobic conditions will 
be the same as that designed to operate under aerobic conditions, except that arrangements for 
intense  agitation  and  aeration  are  unnecessary  Many  anaerobic  fermentations  do,  however, 
require  mild  aeration  for  the  initial  growth  phase,  and  sufficient  agitation  for  mixing  and 
maintenance  of  temperature.  In anaerobic fermentation, a provision for aeration is usually not 
needed. But in some cases, aeration may be needed initially for inoculum build up. In most 
cases,  a  mixing  device  is  also  unnecessary;  while  in  some  cases  initial  mixing  of  the 
inoculum  ​is  necessary.  Once  the  fermentation  begins,  the  gas  produced  in  the  process 
generates  sufficient  ​mixing.  The  air  present  in  the  headspace  of  the  fermentor  should  be 
replaced  by  CO​2​,  H​2​,  N​2  ​or  a  suitable  mixture  of  these;  this  is  particularly  important  for 
obligate  anaerobes  ​like  Clostridium.  The  fermentation  usually  liberates  CO​2  ​and  H​2​,  which 
are  collected  and  used,  ​e.g.,  CO​2  ​for  making  dry  ice  and  methanol,  and  for  bubbling  into 
freshly  inoculated  fermenters.  ​In  case  of  acetogens  and  other  gas  utilizing  bacteria,  O​2  ​free 
sterile  CO​2  ​or  other  gases  ​are  bubbled  through  the  medium.  Acetogens have been cultured in 
400 Litre-fermenters by​ ​bubbling sterile CO​2 ​and 3kg cells could be harvested in each run.  
Recovery  of  products  from  anaerobic  fermenters  does  not  require  anaerobic  conditions.  But 
many  enzymes  of  such  organisms  are  highly  O​2​,  sensitive. Therefore, when recovery of such 
enzymes is the objective, cells must be harvested under strictly anaerobic conditions.  

4. Aerobic Fermentation  
A  number  of  industrial  processes,  although  called  'fermentations',  are  carried  on  by 
microorganisms  under  aerobic  conditions.  In  older  aerobic  processesit  was  necessary  to 
furnish  ​a  large  surface  area  by  exposing  fermentation  media  to  air.  In  modern  fermentation 
processes  ​aerobic  conditions  are  maintained  in  a  closed  fermenter  with  submerged  cultures. 
The  contents  ​of  the  fermenter  are  agitated  with  an  impeller  and  aerated  by  forcing  sterilized 
air  The  main  ​feature  of  aerobic  fermentation  is  the  provision  for  adequate  aeration;  in  some 
cases,  the  ​amount  of  air  needed  per  hour  is  about  60-times  the  medium  volume.  Therefore, 
bioreactors  ​used  for  aerobic  fermentation  have  a  provision  for  adequate  supply  of  sterile  air, 
which  is  ​generally  sparged  into  the  medium.  In  addition,  these  fermenters  may  have  a 
mechanism  for  ​stirring  and  mixing  of  the  medium  and  cells.  Aerobic  fermenters  may  be 
either of the (i) stirred ​tank type in which mechanical motor driven stirrers are provided or (ii) 
of  air lift type in which ​no mechanical stirrers are used and the agitation is achieved by the air 
bubbles  generated  by  ​the  air  supply.  Generally,  these  bioreactors  are  of closed or batch type, 
but  continuous  flow  ​reactors  are  also  used, such reactors provide a continuous source of cells 
and are also suitable​ ​for product generation when the product is released into the medium  

5. Surface Culture Method  

In  this  method  the  organism  is  allowed  to  grow  on  the  surface  of  a  liquid  medium  without 
agitation.  After  an  appropriate  incubation  period  the  culture  filtrate is separated from the cell 
mass  and  is  processed  to  recover  the  desirable  product.  Sometimes  the  biomass  may  be 
reused.  ​Examples  of  such  fermentations  are  the  alcohol  production,  the  beer  production  and 
citric  ​acid  production.  This  method  is  generally  time  consuming  and  needs  large,  area  or 
space.  
6. Submerged Culture Method  
In  this  process,  the  organism  is  grown  in  a  liquid  medium  which  is  vigorously  aerated  and 
agitated  in  large  tanks  called  fermentors.  The  fermentor  could  be  either  an  open  tank  or  a 
closed  ​tank  and  may  be  a  batch  type  or  a  continuous  type  and  are  generally  made  of 
non-corrosive  ​type  of  metal  or  glasslined  or  of  wood.  In  batch  fermentation,  the  organism is 
grown  in  a  known  ​amount  of  culture  medium  for  a  defined  period  of  time  and  then  the  cell 
mass  is  separated  from  ​the  liquid  before  further  processing  while  in  the  continuous  culture, 
the  culture  medium  is  ​withdrawn  depending  on  the  rate  of  product  formation  and  the  inflow 
of  fresh  medium.  Most  ​fermentation  industries  today  use  the  submerged  process  for  the 
production of microbial​ ​products.