CH6705 Biochemical Engineering ASSIGNMENT-V 2016-2017

Part – A
1. What is meant by Centrifugation and Filtration?
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

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

2. What is meant by Sedimentation and Chromotography?

The method used to separate the water from contamination.
It is done when water consists of large sized organic material like leaves & gravel.
Chromotography.
They mainly settle down depending on their size and weight.

Tswett define chromatography as separation of coloured substances.

Common feature:

 Stationary phase
 Mobile phase

3. What is meant by Ion exchange chromatography and reverse osmosis?

 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.
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.

4. What is meant by Electrophoresis and Extraction?

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
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Immobilization of molecule at isoelectric pH

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

5. What are the 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)
6. Explain the 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.
7. Explain 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.
8. Explain Product recovery process
Mainly due to:
 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.

PART – B
1. Explain the 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 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

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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 2 . Two important features 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

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(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.

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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 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 u 0 and the volume fraction of particles p have been developed.
Possessing the general form

uh 1

u 0 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

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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 centrifuge is used first
to remove large or easily settled solids.

2. Describe the 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 103  104
Bacteria 300  103
Colloidal solids 100-1000
Macromolecules (proteins, polysaccharides) ( 2-10
104  106 MW)
Antibiotics (300-1000 MW) 0.6-1.2
Mono-, disachaccarides (200-400 MW) 0.8-1.0

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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)  L p ( p  )
N2 (solute)  C2 (1  )N1  Pc 2
Where L p 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 

Which, for  near 1.0 and small P and/ or large N1 , will be much smaller than the upstream
solute concentration.

Reflection coefficient
Substance Mol wt Molecular Dialysis Cellophane Wet gel
radius, A tubing

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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, …….. …….. 1.7 6.5 25
L p 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.

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t   i   ci RT[1  B2i [c]i B3i [c]i2 ...]

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.

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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

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 ,c f , 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:

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Variation of flux with amylase rejection (upper scale), pH and presence of β-

lactoglobulin.

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).

3. i. What are the general steps involved in downstream processing?

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ii) Give short notes on the following:

• Separation of mixtures
• Passing a mixture dissolved in a "mobile phase" through a stationary phase, which
separates the analyte to be measured from other molecules in the mixture and
allows it to be isolated.
Ion exchange chromatography
Definition of ion:
• Ion is an atom or molecule which has lost or gained one or more valence electrons,
giving it a positive or negative electrical charge.
• Anions are negatively charged ions, formed when an atom gains electrons in a
reaction. Anions are negatively charged because there are more electrons associated
with them than there are protons in their nuclei.
• Cations are positively charged ions, formed when an atom loses electrons in a
reaction, forming an 'electron hole'.
• Ion exchange chromatography
• Used charged stationary phase to separate charged compounds
• Resin that carries charged functional groups which interact with oppositely charged
groups of thecompound to be retained.
• FPLC

Ion exchange chromatography Affinity chromatography

Affinity chromatography
• Affinity chromatography separates the protein of interest on the basis of a
reversible interaction between it and its antibody coupled to a chromatography
bead (here labeled antigen) .
• With high selectivity, high resolution, and high capacity for the protein of interest,
purification levels in the order of several thousand-fold are achievable.
• The protein of interest is collected in a purified, concentrated form. Biological
interactions between the antigen and the protein of interest can result from
electrostatic interactions, van der Waals' forces and/or hydrogen bonding. To elute
the protein of interest from the affinity beads, the interaction can be reversed by
changing the pH or ionic strength.
• The concentrating effect enables large volumes to be processed. The protein of
interest can be purified from high levels of contaminating substances.
• Making antibodies to the protein of interest is expensive, so affinity chromatography
is the least economical choice for production chromatography.
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Size exclusion chromatography

• Gel permeation/filtration
• chromatography (GPC)
• Separates molecules
• according to their size
• Low resolution"polishing"
• Tertiary/Quaternary structure(native)

Reversed-phase chromatography
Reversed-phase chromatography is an elution procedure used in liquid
chromatography in which the mobile phase is significantly more polar than the
stationary phase.

Polarity:
• The dipole-dipole intermolecular forces between the slightly positively-charged end of
one molecule to the negative end of another or the same molecule.
• Molecular polarity is dependent on the difference in electronegativity between
atoms in a compound and the asymmetry of the compound's structure.
Liquid Chromatography
• Mobile phase is a liquid.
• Carried out either in a column or a plane.
• HPLC
• In the HPLC technique, the sample is forced through a column that is packed with
irregularly or spherically shaped particles or a porous monolithic layer (stationary
phase) by a liquid (mobile phase) at high pressure.

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HPLC
4. Describe the 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 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

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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

Figure:
Schematic diagram of an anaerobic digestion unit.

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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 H2S 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 some of the other options for sludge treatment, and others are discussed in the
references.

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