Preparation of a bacterial cell aggregate

A shaped bacterial cell aggregate having increased hardness is produced by adding previously-produced dried finely-divided bacterial cell aggregate to bacterial cell aggregate subsequent to its formation but prior to its shaping.

BACKGROUND AND PRIOR ART 
Glucose isomerase is an enzyme that can be employed to catalyze the 
conversion of glucose (dextrose) to fructose (levulose). It is known that 
glucose isomerase can be produced by fermentation of certain organisms, 
such as Streptomyces flavovirens, Streptomyces echinatur, Streptomyces 
achromogenus, Streptomyces albus, Streptomyces olivaceus, Bacillus 
coagulans and the like, in appropriate nutrient media. The glucose 
isomerase is formed inside the bacterial cells which grow during its 
production. The cells can be filtered off from the fermentation beer and 
used directly as a source of glucose isomerase. Direct commercial use of 
such enzyme-containing bacterial cells had been hampered, however, by a 
major disadvantage. The enzyme activity was lost from the cells during use 
and thus the useful life of the cells was reduced. This disadvantage was 
overcome by the treatment of the bacterial cells with glutaraldehyde as 
described in U.S. Pat. No. 3,779,869. Additional techniques for 
immobilizing the enzyme activity in bacterial cells as well as for forming 
aggregates of such enzyme-containing bacterial cells are described, for 
example, in U.S. Pat. No. 3,821,086 and its U.S. Pat. Nos. Re. 29,130 and 
Re. 29,136 and in South African Pat. No. 73/5917. The above U.S. patents 
relate to use of certain anionic and cationic polyelectrolyte flocculating 
agents. The South African patent discloses various combinations of 
binders, reinforcing agents and cross-linking agents. While the above 
techniques provided bacterial cell aggregates which generally retained 
their enzyme activity during use, there was still a need to increase the 
hardness of the aggregates so that they could be commercially used in 
reactor beds of increasing depth. U.S. Pat. No. 3,935,069 describes the 
addition of certain metallic compounds in conjunction with polyelectrolyte 
flocculating agents to improve the hardness. However, this technique has 
limited utility. 
A further development to improve the hardness of bacterial cell aggregates 
is described in copending U.S. Patent application Ser. No. 890,500, filed 
Mar. 27, 1978, which issued as U.S. Pat. No. 4,212,943, July 15, 1980, and 
assigned to the same assignee of this application. In Ser. No. 890,500 the 
mass of bacterial cells having desired enzymatic activity is treated with 
a cross-linking reaction product of (1) glutaraldehyde, cyanuric halide or 
combinations thereof and (2) a water-soluble cationic polymer obtained by 
the polymerization of an epihalohydrin with an alkylene polyamine, and 
then recovering the resulting aggregate. The resulting aggregates can be 
extruded or otherwise shaped to form shaped aggregates of improved 
hardness. However, when such shaped products are used in a column to 
conduct enzymatic processes, further hardness to reduce compaction losses 
is desirable. 
In the manufacture of shaped bacterial cell aggregates, a certain amount of 
finely-divided product is produced having particle sizes too small for 
commercial use. Various techniques for utilizing these fines have been 
proposed. U.S. Pat. No. 4,060,456 discloses the recycling of product fines 
into a flocculation tank where a flocculant is employed to form bacterial 
cell aggregates. While this disclosure provides a use for recycled fines 
in the production of a flocculated aggregate, there is no indication as to 
any effect upon product hardness. Use of recycled enzyme supports is 
disclosed in U.S. Pat. Nos. 4,002,576; 4,078,970 and 4,087,330, but in no 
instance is there a disclosure or suggestion that such recycled materials 
can aid in improving shaped product hardness. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a process is provided for 
improving the hardness of a shaped bacterial cell aggregate which 
comprises adding previously produced dried finely-divided bacterial cell 
aggregate to bacterial cell aggregate subsequent to its formation but 
prior to its shaping. This invention is especially useful when the 
resulting aggregate is dried and then rehydrated for subsequent use. 
DESCRIPTION OF THE INVENTION 
The process of the present invention can be used with various 
enzyme-containing bacterial cells. The remainder of the disclosure will be 
directed at using the process with bacterial cells containing glucose 
isomerase activity. 
The bacterial cells containing glucose isomerase activity useful in the 
process of the present invention can be produced by well-known procedures. 
The preferred enzyme-containing cells are produced by growing under 
submerged aerobic conditions a culture of Streptomyces olivaceus NRRL 3583 
or mutants thereof in a medium containing appropriate nutrients. This is 
described in U.S. Pat. No. 3,625,828. The resulting bacterial cells are 
separated from the fermentation beer by filtration or centrifugation. 
The bacterial cell aggregates used as starting materials in the process of 
the present invention can be produced by various well-known techniques. 
Such aggregates are preferably obtained by treating the above bacterial 
cells with glutaraldehyde in accordance with the procedure set forth in 
U.S. Pat. No. 3,779,869. The most preferred bacterial cell aggregates are 
obtained by treating the above bacterial cells with a cross-linking 
reaction product of (1) glutaraldehyde, cyanuric halide or combinations 
thereof and (2) a water-soluble cationic polymer obtained by the 
polymerization of an epihalohydrin with an alkylene polyamine. This 
procedure is described in copending U.S. application Ser. No. 890,500, 
filed Mar. 27, 1978, which issued as U.S. Pat. No. 4,212,943 on July 15, 
1980. The following description relates to the production of bacterial 
cell aggregates employing the procedure of the above application Ser. No. 
890,500. 
The ingredients employed in the aggregation process are readily available. 
Glutaraldehyde and cyanuric halide, such as cyanuric trichloride, cyanuric 
tribromide, cyanuric triiodide and the like, are commercially available or 
can be produced by well-known techniques. The particular 
epihalohydrin-polyamine polymer used in this aggregation process is 
commercially available under the trademark BETZ 1180 from Betz 
Laboratories, Inc., Trevose, Penn. BETZ 1180 has a molecular weight less 
than one million, contains about 0.288 millimoles of amino groups per gram 
of solution (based on a ninhydrin assay) and is marketed as a solution 
containing 30 weight percent solids, based on total solution weight. This 
compound is disclosed in U.S. Pat. No. 3,915,904. The compound is 
described therein as a water-soluble cationic polymer obtained by the 
polymerization of an epihalohydrin with an alkylene polyamine having the 
formula R.sub.1 R.sub.2 NRNH.sub.2 wherein R is a lower alkylene having 
from 2 to about 6 carbon atoms, and R.sub.1 and R.sub.2 are each a lower 
alkyl of from about 1 to about 6 carbon atoms, the mole ratio of 
epihalohydrin to polyamine being from about 0.60:1 to about 2.7:1, said 
polymerization comprising reacting with the alkylene polyamine from about 
50 to about 90 percent of the amount of epihalohydrin to be polymerized, 
allowing the reaction to continue until the reaction medium attains a 
substantially uniform viscosity, and reacting the remaining portion of the 
epihalohydrin incrementally to obtain the cationic polymer, the 
temperature of polymerization being from about 60.degree. C. to about 
120.degree. C. This material will hereinafter be referred to as the 
"polyamine polymer". 
The cross-linking reaction product employed to form the bacterial cell 
aggregate can be one of three possible compositions. The polyamine polymer 
can be reacted with glutaraldehyde or cyanuric halide or with both 
glutaraldehyde and cyanuric halide. 
The glutaraldehyde and/or cyanuric halide, which is collectively identified 
as component (1), is reacted with the polyamine polymer, which is 
identified as component (2), at a pH about 6 to 10 and at about 0.degree. 
to 30.degree. C. for about 0.5 to 2.5 hours. The overall cross-linking 
reaction product contains from about 12 to about 77 weight percent of 
component (1) and from about 23 to about 88 weight percent of component 
(2) based on the total weight of the active ingredients in components (1) 
and (2). The glutaraldehyde content of the reaction product is from about 
0 to about 77 weight percent and the cyanuric halide content is from about 
0 to about 22 weight percent based on the total weight of the active 
ingredients in components (1) and (2). 
The reaction between glutaraldehyde and the polyamine polymer is preferably 
carried out at pH 8 to 9 and at about 18.degree. to 25.degree. C. for 
about 0.5 hour. The glutaraldehyde should be present in a molar ratio of 
at least one mole per mole of amino group in the polyamine polymer in 
order to avoid undesirable cross-linking of the polyamine polymer with 
glutaraldehyde. 
The reaction between cyanuric halide alone and the polyamine polymer is 
preferably carried out at pH 8 to 9 and at 0.degree. to 10.degree. C. for 
about 1 to 2 hours. The cyanuric halide should be present in a molar ratio 
of at least one mole per mole of amino group in the polyamine polymer in 
order to avoid undesirable cross-linking of the polyamine polymer with 
cyanuric halide. Cyanuric halide, such as cyanuric trichloride, has three 
halogen reactive sites. One of these sites will react at 0.degree. C. or 
higher. After reaction at the first site, the second site will react at 
30.degree. to 50.degree. C. and the final site will react at 90.degree. to 
100.degree. C. It is desirable to initially react only the first site on 
the cyanuric halide with the polyamine polymer. When the resulting 
cross-linking reaction product is subsequently reacted with the bacterial 
cells and heated to higher temperatures during drying, the remaining 
reactive sites on the cyanuric halide will then react with the polyamine 
polymer to provide additional cross-linking to the bacterial cell 
aggregate. 
The reaction between the polyamine polymer and the combination of 
glutaraldehyde and cyanuric halide is carried out in steps. First, the 
cyanuric halide is reacted with the polyamine polymer at pH 8 to 9 and at 
0.degree. to 10.degree. C. for about 1 to 2 hours. Preferably, in this 
situation the reactants have a mole ratio of one mole of cyanuric halide 
to two moles of amino groups on the polyamine polymer. An excess amount of 
glutaraldehyde is then added and the reaction is continued under the same 
pH and temperature conditions for about 0.5 hour. 
The cross-linking reaction product employed in the production of the 
preferred bacterial cell aggregate is not a cationic polyelectrolyte, 
since the amino groups on the polyamine polymer which initially provided 
the cationic characteristic have been reacted with the glutaraldehyde 
and/or cyanuric halide and are thus no longer available. 
Bacterial cell aggregates are prepared by contacting a mass of bacterial 
cells with the cross-linking reaction product prepared as described above 
at pH about 8 to 9 and at about 0.degree. to 30.degree. C. for about 0.5 
to 1.5 hours. The cross-linking reaction product is employed in such 
amount and concentration that the bacterial cells are contacted with from 
about 4.5 to about 60 weight percent of the cross-linking reaction product 
active ingredients based upon the dry weight of the cells. 
After the above reaction takes place, the resulting bacterial cell 
aggregate slurry is conveniently placed in a holding or surge tank 
upstream of filtration apparatus, such as a rotary vacuum filter. The 
slurry is then filtered to produce a filter cake of moist bacterial cell 
aggregate. This moist bacterial cell aggregate is then extruded or 
otherwise shaped into desirable shapes and then dried at about 60.degree. 
C. for several hours. 
The above-produced dried aggregate is ground and sized to produce desired 
aggregate particles having a size such as to pass through a 16 mesh screen 
and be retained on a 25 mesh screen (U.S. Screen sizes). The product 
material having a particle size larger than 16 mesh is reground while the 
product material having a particle size smaller than about 25 mesh and a 
moisture content of about 12 weight percent is recycled in accordance with 
the present invention. 
The previously-produced dried finely-divided bacterial cell aggregate or 
fines which passed through the 25 mesh screen are added to the bacterial 
cell aggregate subsequent to its formation but prior to its shaping, such 
as by extrusion. This can be conveniently accomplished in at least two 
ways. In one procedure, the fines can be mixed with the bacterial cell 
aggregate slurry in the holding or surge tank upstream of the filter. In 
another procedure, the fines can be blended with the moist bacterial cell 
aggregate before it enters the extruder. 
The dried finely-divided bacterial cell aggregate is added to the bacterial 
cell aggregate in an amount up to about 70 weight percent based on the 
total weight of the mixture solids. Preferably, the fines are added to the 
bacterial cell aggregate in an amount from about 5 to about 70 weight 
percent based on the total weight of the mixture solids. Most preferably, 
the fines are added in an amount of about 30 weight percent based on the 
total weight of the mixture solids. 
The mixture of fines and moist bacterial cell aggregate is shaped, 
preferably by extruding such mixture through a die of reduced 
cross-sectional area, the shaped mixture or extrudate is dried and the 
dried aggregate is ground and separated to produce the desired particle 
size range. Any fines produced can be recycled in accordance with this 
invention. An extrusion die having openings of about 1/8-in. (3.18 mm) to 
about 1/16-in. (1.59 mm.) is presently preferred. 
The resulting dried aggregate can be stored until subsequently needed for 
use in an enzymatic process. At that time the dried aggregate is 
rehydrated and conditioned for use. One illustrative conditioning process 
is described in U.S. Pat. No. 3,974,036. 
A principal advantage of the present invention is an increase in the 
hardness of the bacterial cell aggregate after rehydration as compared to 
prior art bacterial cell aggregates. The hardness is expressed in relation 
to resistance to compression of the bacterial cell aggregate particles. An 
Instron Universal Tester Model 1102 was employed in a manner similar to 
that described in U.S. Pat. No. 3,935,069. This instrument is available 
from Instron Corporation, Canton, Mass. 
The load or test cell employed with the above Instron Tester consists of a 
transparent acrylic plastic cylinder having an I.D. of 1.720 in. (4.37 
cm.), an O.D. of 2.520 in. (6.45 cm.) and a height of 8.5625 in. (21.8 
cm.). The bottom portion has a step 0.25 in. (0.635 cm.) thick with an 
opening of 1.5 in. (3.81 cm.) to form a support for a micro-filter. A 
convenient micro-filter is a spinnerette employed in textile spinning 
having 14,500 openings about 0.008 in. (0.2032 mm.) dia. 
A Type 304 stainless steel plunger 1.693 in. (4.3 cm.) dia. and 5.375 in. 
(13.66 cm.) long is mounted so as to move coaxially into the above 
cylinder. Appropriate indicia are located along the load cell to show a 
sample depth of 4 in. (10.17 cm.). Provisions are also made for applying a 
reduced pressure or vacuum to the bottom of the load cell and for 
collecting any liquid which passes through the micro-filter. 
If a sample of bacterial cell aggregate is placed in the above load cell 
and pressure is applied to the sample through the plunger, the sample will 
be compressed. The pressure needed to compress the sample a given amount 
is an indication of the sample hardness. 
The following is the Rehydration Hardness Assay Procedure employed in the 
examples of this specification: 
A 33 weight percent aqueous solution of glucose is adjusted to pH 8.1. A 
130 g. portion of dried bacterial cell aggregate is mixed with 1300 ml. of 
such glucose solution with gentle agitation at 24.degree. C. for one hour. 
The resulting mixture is drained over a 20 mesh screen (U.S. sieve size) 
for about 30 seconds. The solids are then resuspended in a fresh portion 
of the above glucose solution and stirred for 5 minutes at 24.degree. C. 
The resulting slurry is allowed to settle for 5 minutes and then is 
drained as above. The solids are then resuspended in a fresh portion of 
the above glucose solution and stirred for 5 minutes at 24.degree. C. 
Approximately half of the resulting slurry is then poured into the test 
cell to a height of 4 in. (10.17 cm.). A reduced pressure or vacuum of 
1-in. (2.54 cm.) of mercury is applied to the bottom of the test cell for 
three minutes to suck liquid through the micro-filter. The plunger is then 
lowered until it just touches the top of the sample. The crosshead on the 
Instron instrument is attached to the plunger and is set to move downward 
at a speed of 0.5 in./min. (1.27 cm./min.) and to stop at a penetration of 
1-in. (2.54 cm.). The recording chart speed is set at 5-in./min (12.7 
cm./min.). At the end of the penetration, the plunger is allowed to 
recover for exactly one minute. The plunger is again placed in contact 
with the top of the sample and the Instron is set to stop at a penetration 
of 1-in. (2.54 cm.). The pressure necessary to achieve the second 
penetration is the measure of the sample hardness.

The invention is described in further detail in the following illustrative 
examples. 
EXAMPLE 1 
A solution of polyamine polymer was prepared by diluting 1750 g. of BETZ 
1180 solution containing 525 g. of active material with distilled water to 
form 15 liters. The pH was adjusted to 9. A solution of glutaraldehyde was 
prepared by diluting 2670 ml. of 25 weight percent glutaraldehyde 
containing 667 g. active material with distilled water to form 15 liters. 
The pH was adjusted to 9. These two solutions were then mixed and 
distilled water was added to form a total of 42 liters. The reaction took 
place at a pH of about 9 and a temperature of about 25.degree. C. for 
about 0.5 hour. The resulting product was formed from a reaction mixture 
containing 56 weight percent glutaraldehyde and 44 weight percent 
polyamine polymer based on the total weight of the glutaraldehyde 
(Component 1) and the polyamine polymer (Component 2). 
A culture of a mutant of Streptomyces olivaceus NRRL 3583 was grown in an 
agitated aerated fermentor containing an appropriate nutrient medium 
described in U.S. Pat. No. 3,625,828. The resulting fermentor broth 
containing a mass of bacterial cells was adjusted to pH 8-9 by addition of 
appropriate buffering materials. The above-prepared solution was added to 
the fermentor broth in an amount of 6 ml. per gram of dry cell weight to 
provide 17 weight percent total reaction product based on the dry weight 
of the bacterial cells. After about 30 min. reaction time at 25.degree. C. 
and pH 8-9, the treated broth containing the bacterial cell aggregate was 
placed in a holding tank from which it then passed to a rotary vacuum 
filter. The resulting wet filter cake was cut into small pieces about 1 
sq. cm. in a chopper. The chopped moist filter cake pieces having a 
moisture content of about 76 weight percent were then divided into four 
portions. One portion was then extruded through a die having six 1/8-in. ( 
3.18 mm.) dia. openings using a non-compression extruder screw with 100 
RPM screw rotation. The resulting extruded bacterial cell aggregate was 
then dried at 60.degree. C. for 4 to 6 hours and milled. The milled 
particles were then separated to collect the desired fraction which passes 
through a 16 mesh screen but which is retained on a 25 mesh screen. The 
fines which passed through the 25 mesh screen were collected for further 
use. The -16+25 fraction was designated as the "Control". Its glucose 
isomerase activity was measured by the assay method set forth in U.S. Pat. 
No. 3,779,869 to be 430 glucose isomerase units (G.I.U.) per gm. Its 
hardness was also measured in the above-described Rehydration Hardness 
Assay Procedure. 
The remaining three portions of moist bacterial cell aggregate filter cake 
were then individually mixed in a blender with controlled amounts of 
previously-prepared dried Streptomyces olivaceus bacterial cell aggregate 
having a particle cell of less than about 25 mesh and having a moisture 
content of 12 weight percent. Each of the three mixed portions (Samples 
1B, 1C and 1D) were then individually extruded, dried, milled and 
separated as described above for the Control to produce portions of 
bacterial cell aggregate having particle sizes which pass through a 16 
mesh screen and are retained on a 25 mesh screen. The hardness and enzyme 
activity of each portion product were then obtained. The results are shown 
in the below Table I. 
The hardness of the samples is expressed as a percentage increase over the 
hardness of the Control. 
TABLE I 
______________________________________ 
Weight Percent 
Overall Based on 
Moisture Total Solids Percent 
Content Recycled Filter 
Increased 
Activity 
Sample 
Wt.Percent Fines Cake Hardness 
G.I.U./gm. 
______________________________________ 
1A 76.0 0 100 -- 430 
(Con- 
trol) 
1B 69.5 28.1 71.9 42.46 459 
1C 56.5 60.1 39.9 49.01 433 
1D 43.5 77.9 22.1 133.15 231 
______________________________________ 
The reduction in enzyme activity for Sample 1D is believed due to 
inactivation caused by elevated temperature and pressure created in the 
extruder by the lower moisture content. It appears that an addition of 
fines up to about 70 weight percent based on total solids can be used 
without impairing the enzyme activity. 
EXAMPLE 2 
The procedure of Example 1 was repeated with the following changes. Three 
portions of the moist filter cake were individually mixed with 46.8 weight 
percent recycled bacterial cell aggregate fines based on total solids. 
These three portions were then separately extruded through dies having six 
openings of 1/8-in. (3.18 mm.) dia., eight openings of 1/16-in. (1.59 mm.) 
dia. and ten openings of 3/64-in. (1.19 mm.) dia. The results are shown in 
the following Table II. 
TABLE II 
______________________________________ 
Weight Percent 
Moisture Based On 
Content Total Solids Die Percent 
Activity 
Wt. Recycled Filter 
Dia. Increased 
G.I.U./ 
Sample 
Percent Fines Cake Inch Hardness 
gm. 
______________________________________ 
2A 80.32 0 100 1/8 -- 576 
(Con- 
trol) 
2B 73.87 46.8 53.2 1/8 36.22 542 
2C 73.87 46.8 53.2 1/16 53.58 557 
2D 73.87 46.8 53.2 3/64 14.82 533 
______________________________________ 
The preferred die dia. of 1/8-in. (3.18 mm.) to 1/16-in. (1.59 mm.) 
provides a desirable high increase in hardness with no corresponding loss 
in activity. 
EXAMPLE 3 
The procedure of Example 1 was repeated with the following changes. Three 
portions of the moist filter cake were individually mixed with 60.1 weight 
percent recycled bacterial cell aggregate fines based on total solids. All 
four portions were then extruded through eight 1/16-in. (1.59 mm.) dia. 
openings. The Control portion (Sample 3A) was extruded using a 
non-compression screw rotating at 60 RPM. A first portion (Sample 3B) 
mixed with the fines was extruded under the same conditions as the 
Control. A second portion (Sample 3C) mixed with the fines was extruded 
using a single flight 3:1 compression screw at 40 RPM. A third portion 
(Sample 3D) mixed with the fines was extruded using a double flight 3:1 
compression screw at 30 RPM. The results are shown in the following Table 
III. 
TABLE III 
______________________________________ 
Weight Percent 
Based On 
Moisture Total Solids Percent 
Content Recycled Filter 
Increased 
Activity 
Sample 
Wt. Percent 
Fines Cake Hardness 
G.I.U./gm. 
______________________________________ 
3A 78.30 0 100 -- 650 
(Con- 
trol) 
3B 58.11 60.1 39.9 114.58 506 
3C 58.11 60.1 39.9 117.20 517 
3D 58.11 60.1 39.9 178.01 484 
______________________________________ 
All of the samples employing recycled fines had desirable increases in 
hardness with acceptable levels of retained activity. 
EXAMPLE 4 
The procedure of Example 1 was repeated with the following changes. One 
portion (Sample 4B) of the moist filter cake was mixed with 46.8 weight 
percent recycled bacterial cell aggregate fines based on total solids. 
Another portion (Sample 4C) was dried at 60.degree. C. for 10 min. prior 
to extrusion. A further portion (Sample 4D) was dried at 60.degree. C. for 
20 min. prior to extrusion. Still another portion (Sample 4E) was dried at 
60.degree. C. for 30 min. prior to extrusion. A final portion (Sample 4F) 
was dried at 60.degree. C. for 40 min. prior to extrusion. The untreated 
Control portion (Sample 4A) and the other treated portions were all 
extruded through eight 1/16-in. (1.59 mm.) dia. openings using a 
non-compression screw at 80 RPM. The results are shown in the following 
Table IV. 
TABLE IV 
______________________________________ 
Weight Percent 
Based On 
Moisture Total Solids Percent 
Content Recycled Filter 
Increased 
Activity 
Sample 
Wt. Percent 
Fines Cake Hardness 
G.I.U./gm. 
______________________________________ 
4A 74.36 0 100 -- 440 
(Con- 
trol) 
4B 61.69 46.8 53.2 58.14 440 
4C 68.90 0 100 -21.65 446 
4D 67.20 0 100 2.29 465 
4E 65.10 0 100 9.22 471 
4F 57.00 0 100 18.37 452 
______________________________________ 
It can be seen from the above data that merely drying the filter cake, 
without adding recycled fines, cannot achieve the increased hardness 
produced by the controlled addition of the fines. 
All the above examples added the recycled fines to the moist filter cake 
just prior to extrusion. The following example describes the effect of 
adding the recycled fines to the bacterial cell aggregate prior to 
filtration. 
EXAMPLE 5 
The procedure of Example 1 was followed to produce a slurry of bacterial 
cell aggregate in the holding tank prior to the filter. Recycled fines of 
bacterial cell aggregate were then added to one portion of the tank 
contents in an amount of 30 weight percent based on the total solids. The 
resulting mixture as well as the untreated portion were separately 
filtered, extruded through eight 1/16-in. (1.59 mm.) dia. holes using a 
non-compression screw at 80 RPM, dried, milled and separated according to 
Example 1. The treated portion having the recycled fines had hardness 
45.15 percent greater than the untreated Control. It can thus be seen that 
addition of recycled fines of bacterial cell aggregate subsequent to 
formation and prior to extrusion of a bacterial cell aggregate can 
significantly improve the hardness of the resulting shaped aggregate 
particles. 
The bacterial aggregates produced in the manner described above were all 
capable of converting glucose to fructose. The glucose isomerase activity 
was not impaired through the use of this novel process.