Biological Systems - A Special Case

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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 Whole-Organism Case 1.2 Part-Cell Separations 1.3 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 Isolation of Individual Molecular Species 3 Examples

3.1 Effective Design and Operation of a Process for Harvesting of Single Cell

Protein 3.2 Harvesting of Mycoprotein for Human Consumption 3.3 Thickening of a Filamentous Organism Suspension 3.4 Separation of Poly-3-hydroxybutyrate Polymer (PHB) from Alcaligenes

Eutrophus Biomass 3.5 Isolation of Organic Acid Produced by an Enzymatic Process 4 REFERENCES



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 Isolation of Individual Molecular Species 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 ex-fermenter 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 Identification of systems of Digestion suitable selectivity Precipitation Selectivity; separation of

precipitates from residue Selective Selectivity; separation of Flocculation flocs from residue Adsorption [24] Selectivity; cost (e.g. on support matrix) Electrical Methods [23] Selectivity; cost; (e.g. electrophoresis) difficulties of scale-up Magnetic methods) to intensive process Membrane Methods Selectivity; cost; fouling [Sections 3.5.2 and 3.9]



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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Transcript of Biological Systems - A Special Case