CH6705 Biochemical Engineering Unit-V 2016-2017

1. Centrifugation

The process of seperation of two imiscible liquid on the basis of their density.

 The instrument called centrifuge machine

 Mainly based on the centrifugal force and Angular velocity

2K  rpm
A.V  radius / sec s
60
Type:
 Zonal centrifugation
 Density gradient centrifugation
 Differential centrifugation

2. Filtration:
It mechanical mode of seperation of two substances on the basis of size. Mainly based on that smaller size
molecules can separated from the large size by this method.
Type:

 Simple filtration
 Ultra filtration – by pressure

3. Sedimentation:
The method used to separate the water from contamination.
It is done when water consists of large sized organic material like leaves & gravel.

They mainly settle down depending on their size and weight.

4. Chromotography.
Tswett define chromatography as separation of coloured substances.
Common feature:
 Stationary phase
 Mobile phase

5. Ion exchange chromotography.

 Seperation based on their surface charges.
 Two types of exchanger:
 Cation exchanger – carboxymethyl / sulfonate
 Anion exchanger – DEAE
 For this pH of the medium is very crucial.

6. Reverse osmosis?
 The method of solvent flow from high concentration to solution of low concentration
 Need ATP to work.
 Mainly due to osmotic pressure & semipermeasable membrane.

7. Electrophoresis?
Movement of charged particle is an electric field resulting in migration toward the opposite charged
electrode called electrophoresis.

Types of electrophoresis:

 Zone electrophoresis
 ISO electric focusing

Immobilization of molecule at isoelectric pH

 Immunoelectrophoresis.
CH6705 Biochemical Engineering Unit-V 2016-2017

8. Steps involved in waste water treatment?

1. Primary treatment: Usually carried out by sedimentation, involved in the removal of organic acids.
2. Secondary treatment: Removal of fine suspended and dissolved organic material.
3. Tertiary treatment: Treatment carriedout by disinfectant (Chlorination)
9. Anaerobic digestion:

1. Mostly useful for stabilization of concentrated sludges for industrial waste.

2. Mainly carried out in the air tight reactor.

10. Methanogenesis.
 Third and final stages of biodegradation.
 Involve production of methane & CO2
 Mainly in this broken process methane gas is departure from the digestor represents the stabilization of
sewage/sludge.

11. Major process of tertiary treatments.

 Solids removal
 Biological nitrogen removal
 Biological phosphorous removal
 Disinfection

12. Anaerobic ponds

 It is useful for the treatment of high strength organic acid contains waste.
 Ponds completely devoids of the dissolved O2.
 30 feet deep (approx. 9m) so that heat conversion is possible.

13. BOD (Biological oxygen demand)

 Mainly used to detect the organic pollution
 Required five days of Incubation
 Yeast like Trichosporon cutancum take 15 minute to check the organic pollution.

14. Setting:
 The process by which particulates settle to the bottom of a liquid and form a sediment.
 Particle experience a force due to gravity or due to centrifugal motion.
 They form the slurry at the bottom.
 Application: Water waste treatment mining particle mechanics.

15. Froth floatation method:

 Process for selecting hydrophobic material from hydrophilic.
 Historically first used in mining.
 William Hayes in 1869 patented a process for seperating sulphide & gangue material .
 Also called bulk-oil floatation.

16. Extraction
 General method of seperation.
 Discovered by Franz von soxhlet
 Mainly by general dynamic reduction method.

17. ION-chemically oxygen demand

 Used to measure O2 requirement of a sample that is susctible to oxidtaion by strong chemical oxidant.
 Mainly Kmno4 used due to its superior oxidized ability.

18. Product recovery process

Mainly due to:
CH6705 Biochemical Engineering Unit-V 2016-2017

 Reduce the loss of valuable products

 Reduce production down time.
 Reduce consumption of rinsing water
 Reduce consumption of cleaning media.
 Reduce the waste water 1 rad.

Product Recovery operations step with one example:

Fermentation broths are complex aqueous mixtures of cells, soluble extracellular products, intracellular products,
and unconverted substrate or unconvertible components therein. Characteristically, a bioprocess includes,
centrally, both the bioreactor and a subsequent product recovery section. The particular separation techniques
useful for any given bioprocess depend not only on the location of the product (intracellular vs. extracellular) and
size, charge, and solubility of the product but also on the size of the process itself and the product value. For
example, various forms of chromatography are usefully applied to purification of high value pharmaceuticals or
biological such as hormones, antibodies, and enzymes, but are both expensive and difficult to scale up beyond
laboratory size.

The present chapter on separations pertinent to bioprocess operations is structured in the following
sequence. We first examine individual recovery methods useful in whole or in part for recovery of microbial (or
plant or animal) products. Next, we illustrate how these processes are arranged in series to produce the quality of
separation needed for a given product (e.g., from filtered yeast cells, to crude enzyme extracts, to pyrogen-free
insulin from recombinant E. coli). We also take note of the appreciable cost of many separation operations and
reflect upon interactions between choice of bioreaction process and consequent required product recovery; for
example it is often desirable from the viewpoint of product recovery to choose a strain which produces an
extracellular, rather than intracellular, product. Also, impurities or residues in various possible substrates may
affect overall process costs more in the separatin section than in the fermentation itself. The processes and
principles for product recovery apply effluents from enzyme and cell culture systems, as well as the prototype
process most often considered, a microbial fermentation process.

The ultimate challenge is to select the best combination of substrate, enzyme or organism, bioreactor, and
separation for a given product. A common basis, the economics of bioprocess design, is the mechanism by which
this choice is achieved and is considered in the next chapter.

Each separation needed depends on initial broth characteristics (viscosity, product concentration,
impurities, undesired particulates, etc.) and final product concentratin and form needed (crystallized product,
concentrated liquid, crude solution, dried powder). For example, a crude product may be obtained as a dried
residue of the fermentation broth. The operations sequence through which a bioreactor broth must pass for a
highly purified product is typically as follows:

1. Removal of particulates (insolubles). Common operation here are filtration, centrifugation, and/or
setting/sedimentation/decanting.
2. Primary isolation. Solvent extraction, sorption, precipitation, and ultrafiltration are best known. The latter
offers filtration which discriminates at the level of molecular size. During primary isolation, desired
product concentration increases considerably, and substances of widely differing polarities are separated
from the product.
3. Purification. These operations often select for impurity removal as well as further product concentration.
Approaches include fractional precipitation, many kinds of chromatography, and adsorption.
4. Final product isolation. The last step(s) must provide the desired product in a form suitable for final
formulation and blending, or for direct shipping. Processes here include centrifugation and subsequent
drying of a crystallized product, drum or spray drying, freeze drying (lyophilzation), or organic solvent
removal.

A characteristic processing profile for pharmaceuticals via bioprocessing is given in Table, which indicates
both concentration and relative purity (%)

Table Typical processing profile

CH6705 Biochemical Engineering Unit-V 2016-2017

Pr oduct
Step Concentration,g / L Quality, %
Harvest broth 0.1-5 0.1-1.0
Filtration 0.1-5 0.1-2.0
Primary isolation 5.0-10 1.0-10
Purification 50-200 50-80
Crystallization 90-100

through the product recovery process consisting solids removal, primary isolation, purification, and final isolation
(crystallization) steps.

Figure: Process flowsheet for antibiotic recovery.

An example of an overall separation system is shown in Figure. Here fifteen different separation
techniques, plus a fermentation brother pretreatment thank, are used to produce both a crude and a highly purified
antibiotic product, the former as a spray of continuously dried crude solid, and the latter as a crystalline, essentially
pure material.
Unit operations in down streaming process of Filtration and centrifugation.
Filtration:
Small fermentation batches can be handled in a plate-and-frame filter, which gradually accumulates
biomass, then is opened and cleared of filter cake. Larger processes rely on continuous filters. One of these is the
CH6705 Biochemical Engineering Unit-V 2016-2017
rotary vacuum filter which in some cases requires precoat, The type of continuous rotary vacuum filter
schematically illustrated in figure uses strings to lift of the rotating filter cake (a layer of concentrated solids) which
has accumulated on the drum. While string discharge is satisfactory for removing penicillium mycelia,
streptomyces mycelia are more difficult to process and required precoat of the filter cloth with filter aid, e.g.,
diatomaceous earth, and cake removal with a knife-blade to scrape the cake from the rotating drum.

Filtering characteristic of the solid-liquid slurry are often described in terms of the elementary theory of
filtration. Assuming laminar flow of filtrate liquid through the cake, we may write

Figure: Schematic diagram of a string filter in operation.

1 dVf p

A dt c [(W / A)  r]

where A = area of filtering surface

Vf  volume of filtrate collected
t = time
p =pressure drop across filter
 c = filtrate viscosity
 = average specific cake resistance
W = mass of accumulated dry cake solids = [w /(1  mw)]Vf , where  is filtrate density, w is the mass
fraction of solids in the slurry, and m is the ratio of wet-cake to dry-cake mass

r = resistance coefficient of filter medium

Pressure drop across the filter is constant for the rotary vacuum filters commonly used in the fermentation
industry. If we assume that the cake is incompressible, α is constant and Equation can be integrated to obtain

t c w Vf  c r
 
Vf / A 2 p(1  mw) A p

indicating that t / Vf under the conditions assumed.

Figure a shows a plot of t / Vf against Vf for a Streptomyces griseus fermentation broth filtered at various
pH value using a cotton cloth, diatomaceous – earth filter aid, and a p of 28.4lb/in . Two important features
2
CH6705 Biochemical Engineering Unit-V 2016-2017
are revealed by this data: (i) the cake is not incompressible since the data for each pH do not fall on a straight line.
Generally cells and other organic material from

(a) (b)
Figure:
(A) pH has a profound influence on the filtration rate of S. griseus broth. (b) Heating pretreatment of S.
Griseus broth changes the specific resistance of resulting cake.

Fermentations form compressible cakes. (ii) The data in figure show a strong dependence of filtering properties
on broth pretreatment and filtration condition. Clearly the pH during filtration has a major influence on filtration
rates. Figure reveals that broth preheating can substantially lower the specific cake resistance, presumably by
coagulating mycelia protein.

Cell recycle is receiving increasing emphasis in order to increase reactor volumetric productivity (recall
Chap.9, sec). In large-scale aerobic domestic waste treatment (Chap.14), the flocculent multi-population biomass
is conveniently settled and recycled as settled cell sludge. For small processes involving nonflocculent organisms,
a cross-flow filtration is possible. Here continuous fluid motion parallel to the filter surface continuously removes
the accumulating cell mass, thereby allowing a nearly steady filtrate flow [vs. the time dependent behavior of a
batch system, Equation]. The transmembrane flux of filtrate depends both on the applied transmembrane pressure
p , and the steady resistance of the cellular layer, α(W/A). Accordingly, Equation is appropriate for cross-flow
filtration where now the steady-state filter cake weight W and specific resistance α are functions of the operating
fluid cross-flow (or tangential-flow) rate.

The cake composition may differ in conventional vs. cross-flow filtration, especially if the feed contains a
variety of particles besides cells. Cell concentration factors of more than one order of magnitude are achievable;
values reported for cross-flow with a 100,000 molecular weight cutoff membrane are 15-50 for harvesting of E.
coli, mycoplasma (for veterinary vaccines), and influenza virus (whole virus vaccine). When a batch volume of
cells is to be cross-flow filtered, the cell concentration in the continuously recycled cell stream builds up in time;
an analysis of this circumstance requires inclusion of an increasing tration which time.

AS with high-speed centrifugation, some prefiltering may be require prior to cross-flow filtration. For
example, in antibiotic fermentations using soy grits and calcium carbonate, incomplete utilization of these nutrient
components leaves particulate which are best removed by a coarse screen or filter prior to cross flow filtration.

Where an extracellular product is extractable directly from a bioreactor brother by an immiscible solvent
phase, the processes of cell elimination and product separation from the broth may be accomplished in a single
operation. This combined function processing is considered in a later section.
Centrifugation:
Centrifugation may be used to remove cells from fermentation broths; yeasts, for example, are sometimes
CH6705 Biochemical Engineering Unit-V 2016-2017
harvested in this fashion. A schematic diagram of one type of continuous centrifuge is given in figure. For dilute
suspensions, each cell may be treated as a single particle in an infinite fluid, In this case the analysis of Example
applies. Such an approach is not valid for concentrated slurries, in which a given particle’s motion is influenced
by neighboring particles.

Correlations of particle velocity u h in such hindered-settling situations with the single-particle velocity u0
and the volume fraction of particles  p have been developed. Possessing the general form

uh 1

u0 1  1/p 3

the empirical relationships derived between β and  p are

1  3.05 2.84
p 0.15 <  p  0.5, irregular particles

  1  2.29 3.43
p 0.2 <  p  0.5, spherical particles
1  2 dilute suspensions ( p < 0.15)


Centrifuges may be classified by their internal structures, which have been developed to handle somewhat
differing suspensions leading to different methods of solids discharge (Table). An important feature of the
decanter or scroll-type centrifuge is that it can easily handle large solid particles, in contrast to there forms of
continuous centrifuges. Accordingly, this type of centrifuge may be used in series with another, fine particle
centrifuge, to allow full capacity utilization of the latter without clogging or overloading.

Example applications of these centrifuges appear in Table; note the scroll (decanter) centrifuge for
recovery of large mold pellets, and the larger throughput capacity of the nozzle-type centrifuges.
Noncellular solids often occur in biological process fluids. For example, when enzymes are harvested from
plant biomass, the latter is typically first

Figure:
In this continuous centrifuge, solid particulates are removed in flow between closely stacked cones.
Clarified effluent is withdrawn from the top of the unit.

Crushed and extracted at cool temperatures into a high ionic strength solution, following which the major solid
content must be removed. Similarly, partial utilization of solid substrates (e.g., cellulosics) may result in a
fermentation broth containing macroscopic particles. Where an appreciable density difference exists, the scroll
CH6705 Biochemical Engineering Unit-V 2016-2017
centrifuge is used first to remove large or easily settled solids.
Down Streaming process of membrane separation methods
MEMBRANE SEPARATIONS :

The principal advantage of membrane separations is that operation is achieved without change of phase
or interphase transfer; thus any desired product is continually maintained in an aqueous environment. Size
difference provide one basis on which membrane separations occur figure. Given the following characteristic
dimensions,

Diameter, nm
Yeast and fungi 10 3  10 4
Bacteria 300  10 3
Colloidal solids 100-1000
Macromolecules (proteins, polysaccharides) ( 2-10
10 4  10 6 MW)
Antibiotics (300-1000 MW) 0.6-1.2
Mono-, disachaccarides (200-400 MW) 0.8-1.0
Organic acids (100-500 MW) 0.4-0.8
Inorganic ions (10-100 MW) 0.2-0.4
Water (18 MW) 0.2

Two appropriate membrane separations are available, reverse osmosis and ultra-filtration.

Reverse Osmosis
Osmosis occurs when a solution and a volume of pure solvent are separated by a solute-impermeable membrane;
this circumstance leads to diffusion of pure solvent through the membrane, into the solution phase, in order to
equalize solvent chemical potentials in each phase. If an increasing pressure p is applied to the solution phase,
osmosis halts when the applied pressure p equals the osmotic pressure,  , of the solution, where

  cRT[1  B2 [c]  B3 [c]2  ...]

Where B2 , B3 are virial coefficients for the solute in solution.

Thus, at low concentrations and zero solvent flux, p =   cRT. When p exceeds  , a solvent flux
occurs from the dilute solution into the pure solvent, giving rise to reverse osmosis, a process which produces a
more concentrated solution.

In reality, membranes are not perfectly size selective, and it is convenient to consider both a passive solute
permeability p as well as a flow-related reflection coefficient  for each solute. The latter represents the fraction
of solute molecules which are not passed through the membrane; thus  = 1.0 is perfect reflection and =0 is
complete solute passage. These reflection coefficients are membrane dependent; the apparent pore size of the
membrane provides and indication f the size-selective nature of this process.

Under an applied pressure, the transmembrane solvent flux rate and solute transfer rate are represented
by Equations.
N1 (solvent)  Lp (p  )
N2 (solute)  C2 (1  )N1  Pc 2
Where Lp and P are membrane permeabilities for solvent and solute, respectively, c 2 is the solute concentration
difference across the membrane, and c 2 is the average solute concentration is solution. The corresponding solute
concent5ration in the liquid exiting the membrane is thus

N2  pc 2 
 c 2 (1  )  
N1  N1 
CH6705 Biochemical Engineering Unit-V 2016-2017

Which, for  near 1.0 and small P and/ or large N 1 , will be much smaller than the upstream solute concentration.

Reflection coefficient
Substance Mol wt Molecular Dialysis Cellophane Wet gel
radius, A tubing
D2 20 1.9 0.002 ……. 0.001
Urea 60 2.7 0.024 0.006 0.004
Glucose 180 4.4 0.20 0.044 0.016
Sucrose 342 5.3 0.37 0.074 0.028
Raffinose 595 6.1 0.44 0.089 0.035
Inulin 991 12 0.76 0.43 0.23
Bovine serum albumin 66,000 37 1.02 1.03 0.73
Calculated pore radius, …….. …….. 23 41 82
A
Membrane constant, Lp …….. …….. 1.7 6.5 25
10 5 g.(cm 2 .s.atm)1

Data from R.P. Durbin, j. Gen. Physiol., 44: 315 (1960)

Visking cellulose.
Dupont 450-PT-62 cellophane.
Sylvania 300 viscose wet gel.

Figure:
Concentration polarization caused by buildup of solute(s) near the upstream membrane surface.

Liquids are nearly incompressible, thus provision of appreciable pressures is not costly per se. However,
as the flux increases, a layer of solute-rich fluid builds up at the interface, giving rise to a greater solute permeation
rate, as well as a diminished solvent flux by virtue of the locally in creased osmotic pressure. This concentration
polarization provides, effectively, an upper limit to membrane flux rate: upstream stirring near the membrane
surface can reduce this ultimate resistance, as indicated in Figure (880 vs. 1830 rpm stirring with 6.5 percent
protein), in a manner analogous to the fluid motion in cross-flow filtration which diminishes the filtration cake
resistance near the upstream membrane face.
CH6705 Biochemical Engineering Unit-V 2016-2017
t   i   ci RT[1  B2i [c]i B3i [c]i ...]
2

i i

The solvent flux is again proportional to  p  t  , so now each solute flux depends on the total osmotic pressure
as well as its own permeability and rejection coefficients: hence concentration polarization of any solute decrease
the flux of all solutes.

In the absence of any appreciable solvent flux, if the downstream fluid is circulated and continuously
replaced, dialysis occurs in which small solutes are continuously diffused into the circulating fluid and large solutes
retained. This technique underlies operation of artificial kidney machines, which can remove salts and small waste
organics, while retaining large molecules such as proteins and sugars. If removal of one low molecular weight
species is not desired, it must be provided in the circulating dialysate fluid; thus the used the use of 10 3 M
phosphate in the dialysis purification (for removal of other small solutes) in the enzyme purification example.

Figure:
The influence of concentration polarization on deviations from linearity and approach to a constant flux
as transmembrane pressure is increased.

Ultrafiltration

The use of larger pore membranes than found in reverse osmosis allows flow passage of 1-10 A molecules and
retention of proteins or other macromolecules. For molecules from A to 500-1000 A diameter, ultrafiltration is
useful both for product concentration (by solvent removal) and purification (by removal of low molecular weight
impurities).

When macromolecules account for the concentration polarization layer, the osrnotic pressure is typically
negligible, as Equation. Suggests for MW large. However, the accumulating macromolecular solute polarization
layer can create an appreciable mass-flow resistance.

Several analytical approaches are available, depending on the physical situation.

1. For very dilute feeds, membrane intrinsic resistance sets the solvent flux according to a simplified form of the
reverse osmosis equation which neglect osmotic pressure, thus

N1 (solvent)  Lpp
N2 (solute) c2 (1  )LpP  P c2
CH6705 Biochemical Engineering Unit-V 2016-2017

2. The accumulating macromolecular polarization layer may, in its more concentrated volume, form a gel phase
which adds and appreciable, perhaps even dominant, resistance of the ultrafiltration process.

Defining a molecular sieving parameter   c p / c w and the observed rejection of a solute R as (

(c f  c p ) / c f where c w ,cf , and c p are gel, feed, and permeate concentrations, respectively, then

 1     1  R  
N1  M k ln   
    R  

Where k = liquid phase mass transfer coefficient and M = molar density of solvent. The amylase data of Figure
support the linearity of ln 1-R  / R vs. N1 . Addition of a second polymer, β-lactoglobulin, again provides a
linear plot of N1 vs. 1-R  / R , but with large value of (1  ) /  . Thus, for a given solvent flux, the rejection
facto R of amylase is larger for the mixture. Note that the slopes vary somewhat as well, reflecting a composition-
dependent gel layer resistance.

Further evidence of gel resistance is given in figure; here operation at the β-lactoglobulin pK value of pH
= 5.2 gives maximum gel concentration. Intuitively, a denser gel would be expected from these neutral (isoelectric)
molecules than from solutes of net charge other than zero; this notion is born out by the enormously increased
rejection of α-amylase with increasing β-lactalbumin concentration at pH=pK for β-lactalbumin.

Figure:
Variation of flux with amylase rejection (upper scale), pH and presence of β-lactoglobulin.
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure:

And solute rejection may be correlated by Equation. However, the slope M k ln [(1-) / ] are concentration
dependent, and the ultrafiltration efficiencies are not a function of molecular size alone: for the example above, the
dilute, higher molecular size alone: for the example above, the dilute, higher molecular weight α-amylase (MW =
48,000) was passed through the membrane in preference to the more concentrated, gel-forming β –lactoglobulin
(MW = 36,000).

Down streaming process with any two example

Commercial Enzymes :

Enzyme products are available as crude, dried preparations, dilute or concentrated liquids, or purified
(even crystallized) solids. Figure provides a general press recovery scheme for enzyme derived from animal, plant,
surface, or submerged fermentations. The former sources requires immediate pretreatment to release enzyme
into an extracting buffer, followed by the appropriate solids removal steps when liquid or purified products are
required. A more detailed process recovery example for a plant enzyme shows the necessity of good mixing (flow
in coil) at a low temperature (stabilize intracellular products) to maximize initial extraction. The two serial
centrifuges perform a solids fractionation, removing large particles first by scroll centrifugation so that the more
expensive, higher rpm bowl centrifuge is not clogged with large particles. Subsequent acidification shifts pH
sufficiently to precipitate much originally soluble protein, provided a sufficient residence time is allowed in the
cooled holding coil to form a centrifugal precipitate the desired protein, recovered as wet solid from the second
disc centrifuge. Thus, this example contains two instances where similar or identical processes are placed serially
to carry out a fractionation, first of solids by centrifugation and second by acidification/ precipitation.

As subsequent smaller scale operations occur in protein purification (as processed volume diminishes),
recovery steps may logically shift from continuous to batch as shown in Figure. For protease production. Note
additional steps to enhance enzyme yield: (1) repeated washing of biomass, (2) two-stage ultrafiltration to carry
out sequential 5-fold volume reductions, (3) a switch from continuous to batch ultrafiltration to effect a further
40-fold volume reduction.
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure: Preparation of commercial enzymes.

Figure:
Continuous isolation of enzyme proly-tRNA synthetase from mung bean.
CH6705 Biochemical Engineering Unit-V 2016-2017

An important enzyme application occurs with protease (and other hydrolases) in synthetic detergents.
Bacillus proteases alone accounts for 40 percent of the world industrial enzyme sales, yielding roughly 500 tons
of pure protein in 1978-1979. These proteases were first produced as dusty powders, including particles of less
than 10µm diameter. The resulting dust levels caused allergic reactions among plant workers, and led to
temporary removal of enzyme detergents from the U.S. Market. Subsequent development of a granulated, dust-
free enzyme process allowed resumption of U.S. usage; the protease-containing granules are produced either as a
wax-coated 50-µm sphere or, alternately, are directly embedded in a waxy particle matrix.

Intracellular Foreign Proteins From recombinant E.coli

Proteins synthesized in genetically engineered organisms and intended fro injection into animals must be
stringently purified. Pyrogens from E.coli including the outer envelope lip polysaccharide (LPS) must be removed
or inactivated. (LPS causes fever response in humans at levels  0.5 ng/kg body weight. ) The product must be
protected from E.coli proteases. Often in purification, phenyl methyl sulfanyl fluoride, a serine protease inhibitor,
is added to buffers for this purpose.
Figure summarized the sequence of processing steps which have been used to purify human insulin,
human growth hormone , and human leukocyte
CH6705 Biochemical Engineering Unit-V 2016-2017
Figure:
Extracellular enzyme (protease) recovery. Basis: 200 m3 batch, 10-h operation with two hours’ clean-
down. (Reprinted by permission from B. Atkinson and F. Mavituna, Biochemical Engineering and
Biotechnology Handbook, Macmillan publishers Ltd., Surrey , England, 1983, p 918.

Figure:
Interferon made in recombinant E. coli. A series of precipitation and chromatography steps is applied in each case
to achieve the required purity [22].

Proteins expressed at high levels in recombinant E. coli often accumulate in intracellular refractile bodies
While easily separated from lysed cell solution by centrifugation, these crystalline agglomerates of highly cross-
linked protein must be dissolved and renatured to obtain useful product. Summarized in Figure are an oxidative
sulfitolysis protocol for dissolving refractile bodies and a refolding and disulfide bond formation procedure for
renaturing soluble denatured protein. These methods were reported by scientists at Genencor, Inc. in connection
with cloning and expression of rennin in E.coli
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure:

Summary of purification processes for three human proteins synthesized in recombinant E. coli.
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure:
Oxidative sulfitolysis and disulfide bond formation procedures used for conversion of refractile bodies to
soluble, active protein. GSH and GSSG denote the reduced and oxidized forms, respectively, of glutathione.
(Courtesy of Bryan Lawlis and Kirk Hayenga, Genencor, Ic).

Polysaccharide and Biogum Recovery

Polysaccharides and closely related derivatives are precipitable by alcohol addition, forming the basis for
recovery. Dextrans provide a high value polysaccharide products with used which may include food or medicinal
applications, hence the distinctive used in its purification of alcohol precipitation and use of washes with pyrogen-
free water. Partial hydrolysis of initial precipitate is followed by (unhydrolyzable) solids removal, and repeated
fractional crystallizations, with final impurities removed by ion exchange. Xanthan gum is precipitated with
isopropanol after broth pasteurization (to kill all cells), then spray-dried and milled for a food-grade product.

Antibiotics

Antibiotic production yields either a bulk salt form, e.g., sodium penicillin, or a more purified precipitate
(procaine penicillin) for clinical use. Note the additional use of adsorbent columns and line filters to remove
dissolved and particulate contaminants, respectively. Final spray drying of the procaine-antibiotic precipitate is
accomplished in a low temperature freeze dryer. Biomass may be fully recovered and sold as animal food
supplement.
Anaerobic treatment of affiants

Anaerobic Digestion:

Wastes containing substantial amounts of fermentable organic components can be treated biologically under
anaerobic conditions. Although anaerobic treatment has broader applications, a major use arises in treatment of
excess sludge solids figure produced in sewage-treatment processes. As we have discussed earlier, concentrated
sludge of produced at several stages of these processes, including waste particulates removed in the screening and
primary sedimentation units and also the sludge grown in the secondary biological oxidation process. This
material is further concentr4ated, or thickened, often merely by settling, before disposal, frequently with anaerobic
biological digestion as one of the steps in the process.

A simplified schematic of the overall mechanism of anaerobic digestion, which involves a multitude of
CH6705 Biochemical Engineering Unit-V 2016-2017
microbial species, is

Figure:

In the first step, large solid-sludge material is solubilized or dispersed by extracellular enzymes synthesized by a
broad spectrum of bacteria. Among the enzymes found in anaerobic digesters are proteolytic, lipolytic, and several
celluloytic enzymes. Since solids do not build up in anaerobic digesters, these solubilization reactions apparently
proceed fast enough to prevent this step from liming the rate of the overall reaction sequence.

Experimental studies of the next portion of the digestion reaction, namely bacterial synthesis of short-
chain fatty and volatile acids for soluble organic material, reveal that these steps also occur at a relatively rapid
rate. The organisms responsible for these transformations, called acid formers for obvious reasons, are facultative
anaerobic heterotrophs which function best in a range of pH form 4.0 to 6.5. While the major product of this step
is acetic acid, propionic and butyric acid are also produced.
Acetic acid is the most important substrate for the final reaction of the sequence, since about 70 percent
of the methane produced has been shown to derive from that component. This gasification step of the process
involves methane bacteria, which are strict anaerobes. A narrower range of pH, from 7.0 to 7.8, is optimal for these
organisms, which although difficult to isolate in pure culture, thrive in mixed culture in a properly operated
digester. Existing evidence suggests that this conversion of volatile acids to CH4 and CO2 is rate-limiting step
in the series of reactions shown in Equation.

Figure is a schematic diagram of an anaerobic digestion unit. Mixing is provided to prevent high local
concentrations of acids from developing. In order to maintain a sttisfactory environment for both acid formers
and the methane bacteria, digesters are operated at a pH around 7. Also indicated in figure is an external heat
exchanger, which provides an above-ambient temperature in the vessel. At present, the usual practice is operation
at the temperature in the mesophilic range which maximizes the rate of sludge digestion, about 90 to 1000F
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure:
Schematic diagram of an anaerobic digestion unit.

There is limited evidence that more rapid digestion is possible in the thermophilic range, with largest rates at
about 1300F. Operation at this temperature level is relatively rare: higher energy cost is one factor which weighs
in favor of the mesophilic range of temperatures. The solids-residence time required for anaerobic sludge
digestion at mesophilic temperatures is 10 to 30 days in a well-agitated unit.
Fortunately, the anaerobic digestion process produces a fuel which can be used to reduce energy cost for
the wastewater-treatment plant. In some instances, the methane produced by anaerobic waste treatment is used
outside the plant for heating and power. The gas mixture produced by anaerobic digestion, which is collected from
the top of the unit as indicated in figure, is roughly 65 to 70 percent methane, with CO 2 comprising most of the
remainder. Hydrogen sulfide, produced by sulfate-reducing bacteria, is present in small amounts, as are
H2 and CO . The digester off-gas has a heating value of 650 to 750 Btu/ft3 and is produced with a yield of 12 to
18 std ft3 per pound of organic matter decomposed. Since this gas has a substantially lower Btu value than natural
gas (about 1000), it has not been such an attractive product in areas where natural-gas supplies are plentiful. With
rising energy costs, however, increasing attention is being devoted to anaerobic digestion as a potential fuel source,
albeit after the necessary H 2 S removal.

Table: Effects of anaerobic digestion on sewage sludge

Fraction Raw sludge Digested sludge

Ether-soluble 34.4 8.2
Water-soluble 9.5 5.5
Alcohol-soluble 2.5 1.6
Hemicellulose 3.2 1.6
Cellulose 3.8 0.6
Lignin 5.8 8.4
Protein 27.1 19.7
Ash 24.1 56.0

As a result of anaerobic digestion the sludge is in much better condition for further treatment. First, the
organic sludge solids are reduced by as much as 50 to 60 percent. Moreover, the composition of the sludge is
profoundly changed (see Table). Because of these alterations, digested sludge is much less putrefactive than raw
sludge, and it is also easier to dewater. After dewatering, which is often accomplished with rotary-drum vacuum
filtration, the sludge is dried further, then spread of land as fertilizer, dumped, or incinerated. Figure indicates
CH6705 Biochemical Engineering Unit-V 2016-2017
some of the other options for sludge treatment, and others are discussed in the references.

BIOLOGICAL WASTEWATER TREATMENT

Wastewaters contain a complex mixture of solids and dissolved components, with the latter usually
present in very small concentrations. In treatment plants, all these contaminants must be reduced to acceptably
low concentrations or chemically transformed into inoffensive compounds. The overall system design used to
accomplish this varies depending upon the type and amount of wastewater to be treated and economic and
environmental considerations. Most of the alternatives, however, share enough common features to allow them
to be shown schematically on a single diagram like Figure. There, the parallel pathways shown for sludge handling
and removal of various contaminants represent different options for accomplishing one treatment objective. A
typical plant would employ only a few of the many possible pathways from wastewater to final effluent.
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure:
Available unit operations for primary, secondary, and tertiary wastewater treatment

We consider next the overall purpose of each of the major process trains. In primary treatment, the most
easily separated contaminants are removed. Taken out here are readily settleable solids (see figure), oil films, and
other “light” components. Suspended particles as well as soluble components are removed in secondary treatment.
In many situations these waste materials are organic, and in these cases use of a biological oxidation process is
common. We shall consider these biological processes in further detail later. Tertiary treatment is directed at
removal of all or some of the remaining contaminants. Among the processes used at this stage are electrodialysis,
reverse osmosis, deep-bed filtration, and adsorption.

Wet, concentrated solid wastes, called sludge, are removed in primary treatment; cell sludge is generated
in secondary biological treatment. We have already mentioned the interplay between substrate utilization and
biomass production. Although the secondary biological treatment processes, which involve many microbial
species, are very efficient in attacking a dilute mixture of organic wastes, they also create biomass. Thus, very small
particles and soluble components in
CH6705 Biochemical Engineering Unit-V 2016-2017

Figure:
Different levels of treatment remove characteristic ranges of particulate sizes form the wastewater.

Liquid wastes are transformed in part into a sludge waste material which is easier to separate out than the original
waste. Sludge handling and treatment is consequently an important part of the water-treatment plant. One
popular process for sludge-volume reduction in sewage plants is anaerobic digestion, where organic material is
biologically decomposed in an anaerobic environment.

We should not conclude from this discussion that all three levels of treatment and sludge digestion are
always used. Some wastewaters are discharged into receiving waters such as streams, rivers, ponds, and oceans
with no treatment whatsoever. In other situations, only primary treatment is applied. However, some form of
secondary treatment and sludge processing in the norm for most municipal sewage-treatment plants in the United
States, with tertiary treatment only infrequently used at present.

In keeping with the major theme of this text, we shall emphasize the biological components of wastewater-
treatment processes. After reviewing the characteristics of typical wastewaters in Sec., we shall consider analysis
of activated sludge and related secondary biological treatment methods. Our rapid overview of these important
applications of biochemical engineering will conclude with a discussion of anaerobic digestion. Additional
information on these processes as well as the physical and chemical operations encountered in treatment plants
is provided in the references.

Anaerobic Denitrification and phosphate removed

Nitrogen reduction is accomplished under anaerobic circumstances by a variety of bacteria which can consume
organics and utilize nitrate and nitrite as electron acceptors. Ultimately, two separate paths occur:

(a) In assimilatory nitrate reduction, some nitrogen become3s ammonia and is incorporated into cell biomass.
(b) In dissimilating reduction, molecular nitrogen is the final product.

As not all bacterial can effect both conversions, two independent reactions can be written:

NO3  organic  cells + NO2  CO2

NO2  organic  cells + N2  CO2
CH6705 Biochemical Engineering Unit-V 2016-2017

Nitrite levels observed are very low, so a single combined expression yielding cells and N 2 may often suffice.

The organic may be added to give a level sufficient of effect the desired conversion of nitrate. Methanol
has previously been used as a carbon and energy source with a relatively low cell yield, but cost mcreases have
made this organic substrate less desirable.

Packed-bed lab denitrification studies have shown that the dissolved NO3 and NO2 levels vary with
distance according to a simple sequential reaction pattern, leading to a high N removal efficiency as characterizes
a plug flow operation. Cell growth eventually lead to reactor plugging; thus process design improvements are
needed in this emerging area of denitrification conversions.

Phosphate Removal :

Phosphorus is typically present in raw wastewaters at concentrations near 10 mgP/L including

orthophosphate, dehydrated orthophosphate (polyphosphate) and organic phosphorus. Biological treatment
 
process result in conversion of most P to the orthophosphate forms H2 PO4 , HPO 42- , PO 43- . These latter forms
may be removed by precipitation if effluent guidelines on phosphorus require low phosphate values relative
relative to the influent. Precipitating agents may include calcium or aluminum :

3HPO24  5Ca 2  4OH  Ca 5 (OH)(PO4 )3  3H2 O 

HPO24  Al 3  AlPO4  H

When lime is used as a Ca source, precipitation normally follows biological treatment. With alum (or iron) as
precipitant, treatment may be effected in the activated sludge operation itself or in a primary settling basin Figure.
ANTIBODY PRODUCTION
The term "antibody production" has both general and specific meanings. In the broad sense, it refers to the
entire process of creating a usable specific antibody, including steps of immunogen preparation,
immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in
a particular method. In the more restricted sense, antibody production refers to the steps leading up to
antibody generation but does not include various forms of purifying and labeling the antibody for particular
uses.

Antibody production involves preparation of antigen samples and their safe injection into laboratory or farm
animals so as to evoke high expression levels of antigen-specific antibodies in the serum, which can then be
recovered from the animal. Polyclonal antibodies are recovered directly from serum (bleeds). Monoclonal
antibodies are produced by fusing antibody-secreting spleen cells from immunized mice with immortal
myeloma cell to create monoclonal hybridoma cell lines that express the specific antibody in cell culture
supernatant.

Successful antibody production depends upon careful planning and implementation with respect to several
important steps and considerations:

 Synthesize or purify the target antigen (e.g., peptide or hapten)

 Choose an appropriate immunogenic carrier protein
 Conjugate the antigen and carrier protein to create the immunogen
 Immunize animals using appropriate schedule and adjuvant formula
 Screen serum (or hybridoma) for antibody titer and isotype

ANTIBODY PURIFICATION
CH6705 Biochemical Engineering Unit-V 2016-2017
Antibody purification involves isolation of antibody from serum (polyclonal antibody), ascites fluid or culture
supernatant of a hybridoma cell line (monoclonal antibody). Purification methods range from very crude to
highly specific:

 Crude—precipitation of a subset of total serum proteins that includes immunoglobulins

 General—affinity purification of certain antibody classes (e.g., IgG) without regard to antigen specificity
 Specific—affinity purification of only those antibodies in a sample that bind to a particular antigen
molecule

Which level of purification is necessary to obtain usable antibody depends upon the intended application(s)
for the antibody.

ANTIBODY CHARACTERIZATION
Antibody characterization involves three kinds of activities that are usually performed at various stages
throughout an entire antibody production and purification project

 Screening—identifying antibody samples having antigen-binding specificity

 Titering—measuring antibody concentration and functional assay titer
 Isotyping—determining a monoclonal antibody class and subclass identity

Screening is first required during production to identify which animals and hybridoma clones are producing a
high level of antigen-specific antibody. This is usually accomplished using ELISA techniques.

Antibody concentration can be estimated using either a general protein assay or a species- and
immunoglobulin-specific method, such as with specialized microagglutination assay kits. Antibody titer is
related to concentration but refers more specifically to the effective potency of a given antibody sample.
Measuring titer usually means determining the functional dilution of an antibody sample necessary for
detection in a given assay, such as ELISA.

Isotyping involves determining the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG2a) of a
monoclonal antibody. This is a critical step in antibody production, as it is necessary for choosing an
appropriate purification and modification method for the molecule. Isotyping is most easily accomplished
with commercial, ready-to-use antibody isotyping kits.

ANTIBODY FRAGMENTATION
Purified antibodies can be modified for particular uses by several methods including fragmentation into
smaller antigen-binding units, conjugation with enzyme or other detectable markers, and immobilization to
solid supports. Most often antibodies are used in whole-molecule form. However, the performance of some
techniques and experiments can be improved by using antibodies whose nonessential portions have been
removed.

Antibody Fragmentation refers to procedures for cleaving apart whole antibody molecules and removing
portions that are not necessary for binding antigen. Fab and F(ab)'2 are antibody fragments of IgG that are
most frequently created and utilized by researchers.

Antibody labeling and immobilization

Antibodies are produced and purified for use as antigen-specific probes. However, their utility in any given
technique (ELISA, Western blotting, cellular imaging, immunohistochemistry) depends upon having a
mechanism to secondarily detect the antibody.
CH6705 Biochemical Engineering Unit-V 2016-2017
Techniques that utilize antibodies for immunoprecipitation or other form of affinity purification depend upon
mechanisms for attaching or immobilizing them to chromatography media (e.g., beaded agarose resin).
Strategies for accomplishing this involve the same considerations and chemical methods as antibody labeling.