Biological Systems - A Special Case

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Suspensions Processing Guide:
GBHE SPG PEG 308

Biological Systems: A Special Case
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Process Engineering Guide:

Biological Systems:
A Special Case

CONTENTS

0

INTRODUCTION

1

TYPES OF BIOLOGICAL SEPARATION

1.1
1.2
1.3

Whole-Organism Case
Part-Cell Separations
Isolation of Individual Molecular Species

2

SETTING ABOUT DEVISING AN EFFECTIVE
PROCESS FOR SEPARATION OF A BIOLOGICAL MATERIAL

2.1

Whole-Organism Case
2.1.1 Characterization of Biopolymers in the Liquor
2.1.2 Release of Internal Water

2.2

Part -Cell Separations
2.2.1 Selectivity
2.2.2 Cost

2.3


3

Examples

3.1

3.5

Effective Design and Operation of a Process for Harvesting of Single Cell
Protein
Harvesting of Mycoprotein for Human Consumption
Thickening of a Filamentous Organism Suspension
Separation of Poly-3-hydroxybutyrate Polymer (PHB) from Alcaligenes
Eutrophus Biomass
Isolation of Organic Acid Produced by an Enzymatic Process

4

REFERENCES

3.2
3.3
3.4


TABLES
1

PROCEDURES, KEY VARIABLES AND TECHNIQUES
FORIDENTIFYING PROCESS OPTIONS FOR
WHOLE-ORGANISM SEPARTATION

2

DIFFICULTIES WITH POSSIBLE METHODS FOR PART
SEPARATION OF CELL CONTENTS

3

PRINCIPAL FLOCCULANTS REGIMES ENCOUNTERED FOR
AGGREGATION OF ASI SUSPENSIONS THROUGH THE USE OF
HEAT SHOCK AND ACID

4

SEDIMENT VOLUMES VERSUS PROPORTION OF SUSPENSION
HEAT-TREATED

FIGURES

1

A SCHEME FOR ISOLATION OF AN INTRACELLULAR PROTEIN

2

SOME OF THE PHENOMENA OCCURING WITH THE CHANGING PH
IN THE FORWARD FLOCCULATION OF THE “PROTEIN” ORGANISM

3

SEDIMENT VOLUME AS PROPORTION OF ORIGINAL SUSPENSION
VOLUME

4

MODULUS VERSUS SOLIDS CONTENT FOR FLOCCULATED, PILOT
PLANT MATERIAL WITH VARYING FINAL PH

5

PRECIPITATION BEHAVIOR OF FLOCCULATING FRACTION IN
PROTEIN SYSTEM

6

A SCHEMATIC REPRESENTATION OF TRENDS IN FLOC SIZE, WITH
PH AND DEGREE OF CELL BREAKAGE, FOR FORWARD
FLOCCULATIONOF “CLEAN” AS1 SUSPENSIONS

7

A COMPARISON OF TREND LINES FOR THE MODULUS OF
FORWARD FLOCCULATED, MAIN-PLANT MATERIAL


8

MYCO-PROTEIN PROCESS: FLOW DIAGRAM

9

PROCESS EMPLOYED FOR EXTRACTION OF AN ORGANIC ACID


0

INTRODUCTION

Up till now we have discussed various aspects of the separation and processing
of fine solids without too much reference (except in the examples) to the specifics
of the properties of the materials concerned. Though the material properties are
the dominant influence on efficient process design and operation, it has been
postulated that the necessary characteristics for process selection and
optimization can be found fairly readily using easily-applicable rheological and
other techniques. This underlying assumption also seems to hold good for
biological suspensions; however, certain aspects of the behavior of these
systems are sufficiently specialized for them to merit a separate discussion viz:
(i)

According to the system involved, either complete harvesting or a very
selective isolation of certain biological fragments may be required.

(ii)

Due to the size of the species involved in biological suspensions some
kind of flocculation procedure is usually needed to allow effective
separation. However the possible choice of aggregation method/agent is
often very constrained because:
(a)

Flocculants will often interact strongly with the biopolymers in
solution, giving an excessive consumption of agent;

(b)

The normal end-uses for the isolated products, e.g. in
pharmacology or in (human or animal) food, limit the range of
flocculants which could possibly be used.

(iii)

Biological suspensions are often particularly cohesive owing to strong
attractions between the cells and released biopolymers. The filamentous
nature of many organisms of commercial interest is a factor which
increases this tendency (Section 3).

(iv)

Biological systems, particularly those in which the cells have been lysed,
contain a vast range of different polymers; very careful tailoring of
conditions is required to avoid unwanted flocculation or dispersion effects.

(v)

The amount of internal water retained In biological cell systems is often an
important influence on the suspension mechanical properties. There are
only limited analogues for this type of behavior with inanimate dispersions.
An additional, associated factor is that the bulk density of microbial
species (e.g. bacteria and fungi) is often close to that of the suspension
medium.


The above constraints superficially make the design of biological separation
processes a daunting prospect: In practice the major factors involved are largely
those governing any fine particle separation though due regard must be paid to
special aspects such as internal water release.

1 TYPES OF BIOLOGICAL SEPARATION
It is convenient to divide these types of processes into three classes as given
below:

1.1

Whole-Organism Separations [21]

In these operations it is desired to remove all of the cell contents from the
fermenter broth. Examples of such processes include harvesting of single cell
protein and isolation of Mycoprotein filaments (discussed, as an example, in
Section 3) for human consumption [11, 12]. This type of solids recovery forms, In
general, the least complicated class of biological separation to design and
operate and factors requiring consideration in the development phase are mainly
obvious ones such as the manipulation of conditions to obtain suitable floc sizes
and strengths. However, the avoidance or minimization of supernatant haze
(caused by stabilization of cell fragments by adsorbed biopolymers) is a
particular problem needing attention.

1.2

Part-Cell Separations [21, 22]

In this case one is considering recovery processes in which it is desired to isolate
a portion of the cell contents. An archetypal example (see Section 3) is recovery
of granules of the biological polymer poly-3-hydroxybutyrate (PHB) from a
suspension of the organism alcaligenes eutrophus [13]. The main difficulty in
developing such processes is the Identification of a “handle” by which the desired
species can be distinguished, and isolated, from the (unwanted) remainder of the
cell fragments.


1.3

Isolation of Individual Molecular Species [7, 23, 24]

This may be considered as a particular version of (b) but in this instance, one will
generally be concerned with isolating, in a relatively pure form, only a small
fraction of the biologically produced material. Examples of the types of compound
involved include polypeptide fractions, pharmacologically-active secondary
metabolites and benzene-cis-glycol, a precursor to a novel specialty polymer,
made by a fermentation process [14].
Again the key problem in separation is devising some means of distinguishing
the desired material from the rest of the biomass. Often constraints are greater
than for part-cell separations (b), but in some cases, where recovery of relatively
low molecular weight species Is needed, the task is comparatively
straightforward due to the high solubility of such species compared to that of
most of the components of a typical biological system.

2

SETTING ABOUT DEVISING AN EFFECTIVE
PROCESS FOR SEPARATION OF A BIOLOGICAL MATERIAL

2.1

Whole-Organism Case [21]

The separation of whole organisms is, in many ways, not that different in
character and complexity from the isolation of inanimate fine particles from
suspension; parameters such as particle size and density tend to dominate the
efficiency of the process in both instances. Accordingly, we would recommend
that attempts to identify viable separation options be based upon the general
scheme, given in Section 3, for synthesis of a fine particle separation process.
However, due allowance must be made for the "extra" biological factors noted in
the Introduction, 3, Table 1 lists the procedures, key variables and techniques
which are appropriate to the elucidation of pathways for effective Isolation of
whole organisms. For the most part the items in the Table are self-explanatory
but we will briefly comment upon two points:


2.1.1 Characterization of Biopolymers in the Liquor
Biopolymers in fermenter broth or process liquor have an enormous
influence on the course of bioseparation. Depending on the system,
these species can result in dispersion or flocculation of particulates.
Also, such effects will be dependent upon metal ion concentrations
(such as Ca2+) pH or temperature [19, 20]. For example, high
temperatures tend to denature proteins, resulting in their
precipitation from solution, often with accompanying aggregation of
the suspended cells. Relative concentrations of biopolymers
fluctuate with factors such as fermenter conditions, pH or
temperature. In the latter two cases concentrations can either
Increase (through cell lysis) or decrease (through
adsorption/precipitation) depending on circumstances. It is thus
essential in any Investigations to have to hand methods which will
allow characterization of the concentrations and types of species in
solution, and of their solubility and adsorption properties.
We refer readers Interested in this area to references [2,8-10] for
details of some methods which are employed In this field.
One final point to be remembered with regard to the solution
species, is that any flocculating action observed need not be
dependent upon a single type of polymer. As will be seen in the first
example of Section 3, aggregation may Involve interactions
between two or more kinds of semi-soluble species.
2.1.2 Release of Internal Water
Biological cells typically contain about 3 to 4 times as much water
as solids. Because this "internal water” greatly increases the
effective volume fraction of the cells it drastically modifies the
rheological and dewatering characteristics of the material. For
example, a liquor containing 100 g/l of cells (ostensibly "dilute"
compared with many fine particle suspensions) may well have 50%
of its volume occupied by the organisms. With an inanimate
material if a thickening process had yielded 50% v/v solids it would
generally be considered as highly successful whereas with the
biological system we are still left with a major dewatering/thermal
drying problem.


Moreover, biological suspensions at circa 100 g/l can often be fairly
viscous, due to the volume effect of the particles, giving handling
difficulties despite the nominally high water concentration [20].
Two methods of estimating proportions of internal water are briefly
discussed In reference [5]. It should be apparent that the very
definition and measurement of particle volume fraction in biological
suspensions are particularly complex [19].

2.2

Part -Cell Separations

The key need in devising a separation strategy in this kind of system is the
identification of a suitable "handle" which allows the target material to be
distinguished from the reminder of the cell debris. Table 2 lists some possible
methods of Isolation together with a few of the potential difficulties in their
application. At the time of writing there is no rapid procedure for focusing upon
the optimum separation technique: potential methods have first to be screened
"on paper” against the known physico-chemical properties of the cell components
then surviving candidates must be assessed in laboratory experimental
programs. Two particular difficulties with part-cell separations should also be
strongly borne in mind when attempting to "work up" a possible process:
2.2.1 Selectivity
The complexity of biological systems often conspires to defeat elegant
schemes for isolation of particular components. For example, particulates
which we would expect to differ radically in surface properties often do not
behave very differently due to the adsorption of species from solution.
Proteins, say, will, often adsorb strongly on the surfaces of hydrophobic
particles rendering the latter hydrophilic [24].

2.2.2 Cost
It is essential this be considered at an early stage in screening as
technical difficulties in part-cell separations can give rise to crippling
production costs. A good Illustration is the cash hemorrhage consequent
to formation of intractable emulsions in solvent extraction of solids.


2.3


Again, it is not possible to offer a general prescription for isolation of particular
molecular species from a fermentation broth except to state the obvious: one
must identify some characteristic of the compound which distinguishes It from all
the other (often chemically closely-related) substances in the system.
Parameters used include precipitation behavior on change of pH or ionic
strength, adsorption characteristics on specialized substrates, and partitioning
properties between two liquid phases. Much of this part of the field is beyond the
immediate scope of this manual whose range is limited to processing of
particulate suspensions and accordingly we refer the interested reader to reviews
such as references [6] and [7] which give an idea of what can be achieved in the
area.
Because of its importance in medical science, protein separation has been the
subject of the greatest amount of study to date, and methodology is most
advanced and systematic in this part of the field. Figure 1 shows a typical
sequence of operations for extraction of a particular protein fraction. Other
techniques, particularly liquid-liquid extraction, are also becoming available and
are likely to have specific advantages in certain cases. Reference [7] describes
the state-of-the-art in protein recovery.

3

Examples

Below are a number of examples which illustrate a number of points made in 2
concerning the methodology of design of separation processes for biological
systems. For reasons of commercial secrecy, certain details have been omitted
in two or three of the "case histories". It is intended that more complete pictures
of key examples may be obtained In fullness of time when confidentiality can be
relaxed to a certain degree.


3.1

Effective Design and Operation of a Process for Harvesting of Single
Cell Protein (“pruteen’) (See also Section 3.4)

The commercial process to produce single cell protein for animal feed ('Pruteen")
provides the archetypal example of a large-scale, whole-cell separation. The
general approach taken to design and operation of potential harvesting
processes is described in detail in Section 3.10 (Process Synthesis). In this part
we will concentrate upon the work necessary to identify key variables governing
the Important flocculation step.
Due to the small size (~ 1 micron), and low density (~ 1.05 g/cm3) of the
“Pruteen” organism (AS1) it was inevitable that an aggregation step would be
needed to allow a satisfactory rate of mechanical (i.e. gravity-based) dewatering.
The end-use of the product limited the additives which could be employed at this
stage of the process. Luckily it was observed that a combination of heat shock
and acidification flocculated the fermenter broth; simplistically It may be
considered that heat shock induced breakage of some of the cells whilst
acidification promoted Interaction between the newly-released solution
biopolymers and the various cell fragments. However, the characteristics of the
flocs, and of associated important parameters such as supernatant clarity, varied
considerably with the pH of acidification. In addition, floc and suspension
behavior was observed to depend not only upon pH but upon factors such as the
composition of liquor recycled from dewatering to the upstream part of the
process. Figure 2 gives an idea of some of the phenomena occurring merely In
the range pH 3.5 - 5.0. Thus before performing work to identify and quantify,
options for process design and operation it was necessary to:
(i) Understand the mechanism of flocculation;
(ii) Identify the key variables governing the floc behavior;

(iii) Characterize the effects of such parameters on the material's flotation,
thickening and other significant technical properties.
Figure 1 of Section 3.10 shows a schematic diagram of the "Pruteen" process.


Initial Work: “Once–Through” Culture
To avoid probable complexities imposed by perturbing species in recycle liquor,
initial work was performed with "once-through" culture. Measurements were
made of floc size and structure (using optical and electron microscopy), of simple
properties such as sedimentation behavior, and of the absorption/precipitation
characteristics of the soluble materials released on heat shock. pH was quickly
identified as the most important variable: sedimentation and shear modulus data
(Figures 3 and 4) indicated that, in general, progressively stronger structures
were built up as conditions become more acid. The microscopical studies,
furthermore, were consistent with the tentative mechanism for flocculation using
acid, or heat and acid (Table 3).
The critical evidence in support of this hypothesis was supplied by careful
fractionation of the soluble materials in the supernatant after heat shock. One
particular "cut" - the so-called Fraction A -proved to have a precipitation /
adsorption profile (Figure 5) implying that it was the key agent involved In the
flocculation of the cell fragments. Detailed study demonstrated [2] that
aggregation was not due to a single species, rather to an interacting mixture of a
basic protein and high molecular weight nucleic acid, a fact of great importance
in understanding the flocculation behavior of recycle containing culture (see
later).
Extent of cell lysis (promoted by, for example, more vigorous heat shock) was
almost as important a variable as pH. However, examination of floe properties
showed that it was manifest in two, partly compensating, ways: Firstly, greater
cell breakage yielded higher concentrations of the species required for
flocculation, this resulting in larger, stronger flocs and cohesive networks (Figure
6). Secondly, higher cell lysis apparently gave a larger loss of internal water.
Despite the stronger particle-particle attraction, this loss of phase volume
occupied by the cells often allowed easier thickening than would be achieved
under conditions where cell breakage was less severe (Table 4).


Culture Containing Recycled Supernatant
The second, and more important, section of the work involved cell cultures
derived from fermenters (e.g. the "Pruteen" Works Unit) into which supernatant
had been recycled from the flocculation stage. (This Is essential for economic
running of a single-cell protein process.) Techniques used were as in the Initial
stage of the investigations: floc structure characterization methods;
measurements of floc mechanical properties and procedures for identification of
the nature and behavior of the solution biopolymers.
Though general trends, for example of extent of flocculation with pH, were
observed to be as for "once-through" culture, values for specific properties were
significantly modified from those seen previously: Network strengths were
generally higher and the material was more difficult to dewater (Figure 6).
Optimum conditions (principally pH) for thickening and supernatant clarification
differed from those found for the simpler system and It was apparent that these
also varied a good deal with fermenter batch. Analysis of supernatant content
gave the answer to the riddle: due to recycle, soluble protein and nucleic acid
concentrations were very much higher than before. Indeed the major proportion
of these species In the system arose from the standing concentration in the
cycling supernatant, new material coming from heat shock of *live" cells only
acting as a perturbation. Owing to the higher "flocculant" concentrations, floc
strengths were usually higher and the suspensions were more difficult to thicken.
The rather different flocculation mechanism, compared with “once-through"
culture did, however, give rise to a number of novel phenomena. For example,
with the “clean" system increasing heat shock gave a more easily concentrated
material (presumably due to internal water release). In contrast with broths with
high background levels of protein greater heating seemed to yield a less easily
thickened sludge, owing it is believed to coagulation of soluble polymers by the
temperature shock. In addition, as the flocculant profile arose in a complex way
from a mix of existing species in the supernatant plus those released by (partial)
cell breakage, it was easy for the system to drift from the optimum mix of protein:
nucleic acid for aggregation, eventually resulting in very inadequate flocculation.
Study of the relationships between aggregation and different levels and mixes of
supernatant species allowed identification of operational “windows”, and
procedures for avoidance of the degenerative addition in which aggregation
became progressively more unsatisfactory as a consequence of lack of balance
in the concentrations of the various supernatant polymers.
Description of the way in which options for design and operation of single cell
protein harvesting were elucidated, is provided in Section 3.10. Discussion of the
methods employed in the study of this system are given In references [l-5, 8-10].

3.2

Harvesting of Mycoprotein for Human Consumption [11, 16 – 20]

A second, somewhat more complex, instance of whole-organism harvesting is
provided by the process of isolation of Mycoprotein for human consumption. The
latter venture arose from a collaborative arrangement between two European
Biological Products companies. In the 1960s staff from one of the companies had
discovered a filamentous organism, Fusarium graminearum, which could be
harvested and processed to give meat analogues of excellent texture and
nutritional characteristics. Over a number of years a pilot-scale (~50 tonnes/year)
operation had been developed for fermentation, isolation and texturizlng of the
material [12, 15].
In 1984 agreement was reached between the two aforementioned companies to
attempt production (~20,000 tonnes/year) of Mycoprotein using fermentation
facilities in Europe, However, before this could be done modifications had to be
sought in the downstream process, Figure 7, for cost and other reasons.
Particular problems to be solved included:
(i)

Replacement of the (open) horizontal belt-filter operation by
another process which would be less costly and which would
present fewer difficulties with regard to contamination.

(ii)

Effective characterization of the rheological characteristics of the
various biological materials to enable accurate scaleup of pumping
etc [20].

(iii)

(If possible) Introduction of a primary thickening step prior to RNA
reduction to allow clean liquor to be recycled to the fermenter.

(iv)

Determination of techniques for manipulating process variables to
allow maintenance of a controlled texture in the final product.

Many of the constraints on possible process options were rather similar to those
encountered for “Pruteen” harvesting (Example (a), this section) ; for example
toxicological considerations greatly curtailed potential use of flocculants and
similar additives. Moreover, the problem was given another “twist” by certain
factors proper to this type of system viz:


(i)

Maintenance of texture requires avoidance of degradation of
Organism length (hyphal length, on the order of 100’s of pm long)
during any production step. Thus high Intensity mixing regimes,
such as might be encountered in equipment such as centrifuges,
have to be treated with caution.

(ii)

The material, to meet a specification for nucleic acids prescribed in
law, has to be put through an RNA-reduction stage. This Involves
heating and consequent lysis of the organisms completely changing
the character of the suspension due to loss of internal water and
other similar phenomena (hyphal turgor pressure>.

Very little useful “prior art” existed with regard to understanding the structure and
rheology of suspensions of this kind having such a high aspect ratio (~ few
microns diameter by ~ hundreds of microns length).
At the time of writing, work on this project is still in progress, and much remains
to be done to resolve the various problems. Nevertheless, systematic application
of the principles embodied in this manual has begun to show the way forward, at
least in particular areas:

(i)

With regard to the main thickening operation, optimum design
seems to be being achieved by a relatively straightforward
matching of material mechanical properties (as determined by
techniques such as shearometry) with equipment characteristics
and with constraints such as the need for maintenance of hyphal
length. The manner in which a satisfactory solution is being
obtained is very analogous to that employed in the investigations of
“Pruteen” harvesting [11, 16,183] the relationship between
Fusarium suspension properties and dewatering characteristics
may be found in [19].

(ii)

Rheological characterization and flow equipment scale-up
procedures have been developed. Great difficulty was encountered
in developing appropriate measurement techniques for the exfermenter suspension [16, 18]. A detailed description of the factors
involved and best means of conducting rheological characterization
of filamentous broths may be found in [20].


3.3

Thickening of a Filamentous Organism Suspension

In the previous two case histories involving whole cell harvesting, cost and
toxicological considerations prohibited use of added flocculants. It is Important to
emphasize that this situation certainly does not prevail for all biological
separations and in many instances judicious selection of a suitable floc agent
greatly enhances potential process efficiency. A good Illustration of this approach
is provided by Example 2, Section 3.7.3 (see appropriate pages of the manual for
full details): The problem was to thicken a fermenter broth of filamentous
organisms to provide a paste from which a pharmacologically-active compound
could be eluted by solvent. In this Instance it was found that use of the
hydrolyzing coagulant Fe3+, combined with a small amount of high molecular
weight flocculant, gave a flocculated mass of sufficient, but not excessive,
strength that the material could be thickened In a filter press to the needed solids
content without significant “bleeding” of fines.

3.4

Separation of Poly-3-hydroxybutyrate Polymer (PHB) from
Alcaligenes Eutrophus Biomass

This is an interesting example of a part-cell separation. PHB is a storage polymer
accumulated by a large range of micro-organisms but it is yielded in particularly
high concentrations by the bacterium alcaligenes eutrophus under a suitable
fermentation regime. The material has useful properties and has been launched
as a specialty product (“Biopol”) by the Biological Products Business of a
European company. Within the organism the polymer builds up in granular form
(see Section 3.4) and thus one can visualize three possible routes (at least) to
separation:
(i)

Cell lysis to release the granules followed by an operation to
separate the two kinds of species which exploits some simple
difference In physico-chemical characteristics between the granules
and the remainder of the material. Properties which one could
consider using include density, electrophoretic mobility, and
flotation or flocculation behavior.


(ii)

Solvent extraction of the PHB which is relatively hydrophobic (NB:
the remainder of the components are comparatively hydrophilic).

(iii)

Digestion of the biomass by processes which do not modify the
PHB but which convert the rest of the material to water soluble
residues.

All of the above were considered in some detail with a procedure within the
general class (iii) being eventually selected. Attempts at “whole granule” isolation
by route (i) proved unsuccessful : though hydrophobic the granules are coated
with hydrophilic species making their surfaces negligibly different from that of
other debris. Use of vigorous techniques to isolate components failed, in part due
to the comparative mechanical “softness” of the individual granules (see p8,
reference [13] ), Solvent extraction to give polymer of acceptable purity was
found to be technically feasible even for a significant scale of production, but was
eventually rejected on grounds of cost. Even with optimum “tuning” of the
system, solvent losses were insupportable.

3.5

Isolation of Organic Acid Produced by an Enzymatic Process

An organic acid, soluble in water, was produced from given feedstock by the
action of enzymes contained in a whole cell slurry. The object of the separation
process was to isolate product, as a salt of the acid, free from suspended solids
(e.g. cell debris) and contaminating solubles. A schematic outline of the
separation procedure is provided in Figure 9. It should be noted that the key
physico-chemical factors which enabled purification to take place were:
(i)

The material, due to its chemical structure, remains water soluble
even at very low pHs which induces precipitation of many
undesired components.

(ii)

Though water-soluble at low pHs, under these conditions it is
preferentially soluble in a semi-polar organic liquid whereas
contaminating species, such as salts of strong acids, remain in the
aqueous phase. (At neutral to alkaline pHs, however, the acid, In its
fully ionized form, partitions almost exclusively into the aqueous
layer.)


Thus in Step 1 (Figure 9)) the characteristic of water solubility at low pHs allows
separation of suspended solids and much of the solubles, which precipitate out in
this regime. However, at Stage II, the property of selective partitioning ((Ii) above)
from acid solution to organic solvent, enables Isolation from the remainder of the
water soluble species. This effect is then reversed in (III) by back extraction of
the solvent with alkaline medium.


TABLES
1

PROCEDURES, KEY VARIABLES AND TECHNIQUES
FORIDENTIFYING PROCESS OPTIONS FOR WHOLE-ORGANISM
SEPARTATION

Procedures:
As per the approach related in Section 3.10 but taking account of the variables (i)
- (iv), present in biological systems but rarely in suspensions involving inanimate
particulates.
Process Variables to be considered:
Particle size, solids content, pH and so on as for inanimate materials plus:
(i)

Factors which influence internal water release; effects of internal
water release on suspension properties. Relationship between
weight and volume fraction.

(ii)

Effects of operations such as lysis in increasing ambient
concentrations of biological molecules (especially polymers) which
may have a flocculating or dispersing action. It is also necessary to
consider the effects of ageing of the suspension, particularly on the
concentrations and distributions of biopolymer species.

(iii)

Influence of heat and other factors (presence or otherwise of ions
such as Ca2+) on the precipitation/desaturation of species such as
Ca2+.

(iv)

Possible interactions (e.g. co-precipitation) between different
biological species in solution.

It is also most important to take detailed account of toxicological factors not only
regarding end-use of the product but also with respect to the influence of any
additives on the state of fermentation, for example by recycle of process liquor to
the fermenter .


Techniques for Investigating Process Options
As for standard investigations of the separation of (inanimate) fine particulates
(see e.g. Sections 3.2 - 3.6, 3.10) plus
(i)

Effective techniques for identifying the nature and concentrations of
biologically-derived molecules (particularly polymers) in solution.

(ii)

A methodology for estimating proportions of internal water in
biological organisms or fragments. In essence this involves
constructing a relation between weight and volume fraction.


2

DIFFICULTIES WITH POSSIBLE METHODS FOR PART
SEPARATION OF CELL CONTENTS

Method

Main Potential Properties

Solvent Extraction [7]

Emulsion formation

Enzymatic
Digestion

Identification of systems of
suitable selectivity

Precipitation

Selectivity; separation of
precipitates from residue

Selective
Flocculation

Selectivity; separation of
flocs from residue

Adsorption [24]
(e.g. on support matrix)

Selectivity; cost

Electrical Methods [23]
(e.g. electrophoresis)
Magnetic methods)

Selectivity; cost;
difficulties of scale-up
to intensive process

Membrane Methods
[Sections 3.5.2 and 3.9]

Selectivity; cost; fouling


2 PRINCIPAL FLOCCULANTS REGIMES ENCOUNTERED FOR
AGGREGATION OF ASI SUSPENSIONS THROUGH THE USE OF
HEAT SHOCK AND ACID

1. Acid Only
pH~ 3-4

Weak aggregation due to natural attraction between
cell surfaces after neutralization of electrical
repulsions.

2. Heat and Acid
pH > 5-6

No flocculation due to repulsion between natural
polymers and particulate matter.

pH ~ 4-5

"Bridging" type of mechanism?

pH < ~ 4

"Sticky" particles; random aggregation of cells or cell
fragments to give open structures; evidence for
precipitation of solubles as spherical masses under
conditions of high cell lysis.


3 SEDIMENT VOLUMES VERSUS PROPORTION OF SUSPENSION
HEAT-TREATED (The experiment involved acidification to pH 3)

The size of the aggregates also increased visibly with proportion of suspension
heat-treated.


FIGURES

FIGURE 1. A SCHEME FOR ISOLATION OF AN INTRACELLULAR PROTEIN


FIGURE 2.

SOME OF THE PHENOMENA OCCURING WITH THE CHANGING PH IN THE FORWARD
FLOCCULATION OF THE “PROTEIN” ORGANISM


FIGURE 3. SEDIMENT VOLUME AS PROPORTION OF ORIGINAL
SUSPENSION VOLUME

Sediment volume as a function of pH, and of proportion of cells heat treated
before pH reduction.


FIGURE 4. MODULUS VERSUS SOLIDS CONTENT FOR FLOCCULATED,
PILOT PLANT MATERIAL WITH VARYING FINAL PH


FIGURE 5. PRECIPITATION BEHAVIOR OF FLOCCULATING FRACTION IN
PROTEIN SYSTEM


FIGURE 6. A SCHEMATIC REPRESENTATION OF TRENDS IN FLOC
SIZE, WITH pH AND DEGREE OF CELL BREAKAGE, FOR FORWARD
FLOCCULATIONOF “CLEAN” AS1 SUSPENSIONS


FIGURE 7.

A COMPARISON OF TREND LINES FOR THE MODULUS OF
FORWARD FLOCCULATED, MAIN-PLANT MATERIAL


FIGURE 8. MYCO-PROTEIN PROCESS: FLOW DIAGRAM


FIGURE 9. PROCESS EMPLOYED FOR EXTRACTION OF AN ORGANIC
ACID


Biological Systems - A Special Case

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