Fermentation process

Fermentation process, e.g. for the production of an oxidation product such as vinegar, which comprises continuously cultivating a bacterial strain, preferably in a vessel of elongated shape, in a medium containing the substrate to be fermented in the presence of a hydrous oxide of a metal selected from the group consisting of titanium, vanadium, zirconium, iron and tin and a chelated polyhydroxy support. The bacterial strain used in the process is preferably chelated or complexed with the hydrous metal oxide and the polyhydroxy support.

STATE OF THE ART 
Vinegar production is an important fermentation process in which vinegar is 
produced from dilute aqueous alcoholic solutions such as charging wort. 
Vinegar is made by a variety of processes and the most widely used modern 
apparatus for the production of vinegar is the Fring's generator (British 
Pat. No. 731,804 and No. 1,101,560) which is a semi-continuously operated, 
vortex-stirred tank giving volumetric efficiencies of up to 0.5 (the 
volumetric efficiency is the ratio between the volume produced daily and 
the effective volume of the fermentation vessel) with 96 to 98% conversion 
of ethanol into acetic acid, thus permitting performances of up to 0.48 to 
0.49 (the performance is obtained by multiplying the volumetric efficiency 
and the percentage of conversion divided by a factor 100). This system has 
still not entirely replaced the much older and far less efficient, 
so-called "Quick" process (cf. British Pat. No. 781,584), involving the 
continuous recirculation and sparging over birch twigs and other fillings 
in large wooden vats. 
A recent development in the production of vinegar is that of Greenshields 
(cf. British Pat. No. 1,263,059), using an elongated vessel similar to 
that of the Fring's generator, but using an upwardly moving fermentation 
medium, whereas the Fring's process uses a downward stream. According to 
the Greenshields process, volumetric efficiencies of up to about 1.0 are 
achieved, with up to 88% conversion, thus permitting a performance of 
about 0.88. 
In the vinegar process special kinds of bacteria are used, generally 
Acetobacter species, and for a high efficiency it is advantageous that the 
bacteria are well suspended in the culture medium. On the other hand, it 
is preferable that the bacteria settle quickly to achieve high volumetric 
efficiencies without the risk that the bacteria, or a substantial part 
thereof, are taken along with the final product, in which they remain 
suspended. Therefore, it is important to use elongated vessels, as 
indicated in British Pat. No. 1,263,059. Loss of bacteria can also be 
reduced by including a separate sedimentation vessel having a larger 
cross-section than the fermentation vessel to achieve sedimentation of the 
bacteria. Thus, an important limitation of the volumetric efficiency of 
known processes is the limited capability of the bacteria to settle out 
before removal of the final product. 
To achieve a higher volumetric efficiency, it could be considered desirable 
to use a flocculant strain of bacterium, as for example is known for yeast 
(cf. for example Greenshields and Smith, The Chemical Engineer, May 1971, 
pages 182-190). Unfortunately, however, bacteria are generally not 
flocculant but, as indicated in U.S. Pat. No. 3,912,593, cells of 
Escherichia coli and yeast can be immobilized by certain hydrous metal 
oxides, i.e. hydrous metal oxides of Ti, V, Fe, Zr and Sn and it was shown 
in the said patent that E. Coli cells immobilized with hydrous zirconium 
oxide at pH 7 still respired. 
OBJECTS OF THE INVENTION 
It is an object of the invention to provide a novel continuous fermentation 
process in a medium containing a hydrous oxide of titanium, vanadium, 
zirconium, iron or tin and a chelated polyhydroxy support. 
It is a further object of the invention to provide a continuous 
fermentation process with a bacterial strain chelated or complexed with a 
hydrous metal oxide and a polyhydroxy support. 
It is an additional object of the invention to provide a novel chelate of a 
polyhydroxy support and a hydrous oxide of a metal selected from the group 
consisting of titanium, vanadium, zirconium, iron and tin. 
These and other objects and advantages of the invention will become obvious 
from the following detailed description. 
THE INVENTION 
The novel fermentation process of the invention comprises continuously 
cultivating a bacterial strain, preferably in a vessel of elongated shape, 
in a medium containing the substrate to be fermented in the presence of a 
hydrous oxide of a metal selected from the group consisting of titanium, 
vanadium, zirconium, iron and tin and a chelated polyhydroxy support. 
We have found surprisingly that bacteria cells, like Serratia marcescens 
could grow out of the previously immobilized cells obtained by addition of 
a hydrous metal oxide (e.g. the hydrous oxides of zirconium and titanium). 
On close examination, it was found that the above hydrous metal oxides 
formed a wide spectrum of metal chelates with bacterial cells and the most 
effective hydrous metal oxide at the lowest pH range was hydrous titanium 
oxide, which was quite effective at around pH 3. Therefore, it was chosen 
to assess if it could cause aggregation and subsequent flocculation of 
Acetobacter cells during vinegar production. The object was to increase 
the rate of production of the oxidation product, i.e. acetic acid, within 
the one continuously operated fermenter. Hopefully, it would effect this 
by aggregation and flocculation of the cells, thus permitting increased 
flow-rates without excessive wash-out of the bacteria in the fermenter. 
Such increased flow-rates were achieved but by themselves would not 
necessarily have increased the rate of production. The increased flow rate 
could have been accompanied by a reduction in the conversion of ethanol to 
acetic acid. Surprisingly, the sum total of catalytic activity of the 
cells was retained and even increased at the increased flow-rates probably 
by the retention of increased numbers of cells within the fermenter which, 
when aggregated and flocculated together, had the same or even an 
increased catalytic surface still accessible to substrate. 
When carrying out these experiments it appeared, however, that only some 
kinds of Acetobacter showed a positively increased performance in the 
continuously operated fermenter, especially Acetobacter derived from the 
above-indicated "Quick" process, whereas other species, particularly those 
currently found in industrially operated tower fermenters, did not. 
Surprisingly it has been found, according to the invention, that for the 
effective chelation of some strains of Acetobacter with hydrous metal 
oxides, e.g. hydrous titanium oxide, another component to which the 
hydrous metal oxide should be chelated should be present, i.e. a 
polyhydroxy support such as cellulose. Some kinds of Acetobacter, 
especially those used in the "Quick" process such as Acetobacter xylinum, 
produce extra-cellular cellulose by themselves, whereas others, especially 
those used in industrial tower fermenters, do not so that a hydrous metal 
chelated polyhydroxy support such as hydrous titanium oxide chelated 
cellulose should be added in the latter case to achieve effective 
immobilization of the bacteria. Also in other fermentation processes, such 
as the oxidation of glucose to 5-glucono-lactone and gluconic acid by 
Pseudomonas, use can be made of this technique. 
The present invention accordingly provides a fermentation process which 
comprises continuously cultivating a bacterial strain, preferably in a 
vessel of elongated shape, in a medium containing the substrate to be 
fermented in the presence of a hydrous oxide of titanium, vanadium, 
zirconium, iron or tin and a chelated polyhyroxy support. The chelated 
polyhydroxy support may be derived from the bacterial strain (cf. 
Acetobacter xylinum referred to above). 
According to a feature of the present invention, a bacterial strain is 
provided for use in a fermentation process, chelated or complexed with a 
hydrous metal oxide of titanium, vanadium, zirconium, iron or tin and a 
polyhydroxy support chelated to the hydrous metal oxide. According to a 
further feature of the present invention, there is provided a hydrous 
metal oxide of titanium, vanadium, zirconium, iron or tin chelated with a 
polyhydroxy support suitable for use in the fermentation process of the 
invention. Performances of up to about twice those of known processes for 
producing vinegar may be achieved by the invention. If the bacterial 
strain does not produce the polyhydroxy support by itself, a sufficient 
amount of that compound, chelated to the hydrous metal oxide, should be 
added to the fermentation medium. The polyhydroxy support is preferably 
cellulose, and the preferred hydrous oxide is that of tetravalent 
titanium. 
The hydrous metal oxide and the polyhydroxy support are not effective if 
added to the fermentation medium separately (if a non-aggregating 
bacterium strain is used) and it is desirable to chelate the hydrous metal 
oxide to the polyhydroxy support before addition to the fermentation 
medium. 
In a further feature of the invention, the hydrous metal oxide-chelated 
polyhydroxy support is prepared by contacting a hydrolyzable compound of 
titanium, vanadium, zirconium, iron or tin, optionally in solution, with 
the polyhydroxy support, optionally drying the mixture obtained and 
subsequently washing the mixture with water. This may be done, for 
example, by mixing cellulose powder and titanium tetrachloride in 
solution, drying the mixture and washing the dried mixture with distilled 
water until the washings are neutral. The hydrous metal oxide-chelated 
polyhyroxy support thus obtained should not be heat-dried prior to use. 
The titanium tetrachloride used in the procedure described above may be 
replaced by other hydrolyzable metal compounds, for example titanium 
lactose, titanium citrate, titanium urea or titanium acrylamide. The pH at 
which the polyhydroxy support is initially treated with the titanium 
compound will vary with the particular compound used. The metal compound 
should be substantially non-hydrolyzed before contact with the polyhydroxy 
support. 
An aqueous solution of the metal compound may be used if the pH of the 
solution is adjusted substantially to prevent hydrolysis. Alternatively, a 
solution of the metal compound in an organic solvent may be used. The 
organic solvent must then be removed prior to addition of the hydrous 
metal oxide-chelated polyhydroxy support to the fermentation medium. 
When a hydrous metal oxide is used with a bacterium strain which produces 
its own polyhydroxy support, the hydrous metal oxide may be formed in situ 
by the addition to the fermentation medium of a metal compound 
hydrolyzable at the pH of the fermentation medium, e.g. titanium chelated 
lactose, titanium citrate or titanium urea. The use of titanium acrylamide 
to produce hydrous titanium oxide in situ for a food product fermentation 
would be undesirable. It will be appreciated that the hydrolyzable metal 
compound, e.g. titanium chelated lactose, used to produced hydrous metal 
oxide in situ does not have to be non-biodegradable at the pH of the 
medium. 
Other chelates in which hydrous titanium oxide is chelated to a polyhydroxy 
support which are suitable for use in the process of the invention and 
made in an analogous manner to the hydrous titanium-cellulose chelate 
include those based on wood, sawdust, water-insoluble polysaccharides, 
cross-linked polysaccharides, glass, siliaceous materials such as Celite 
and Neosyl, in addition to a wide range of polymers containing C--OH 
groups or Si--OH groups in the form of powders, beads or fibers. 
The polyhydroxy support must form with the hydrous metal oxide a chelate 
which is substantially insoluble, non-hydrolyzable and non-biodegradable 
in the fermentation medium at the pH, temperature and ionic concentration 
employed. The disposition of hydroxy groups on the polyhyroxy support must 
be such to permit chelation with the metal. The polyhydroxy support itself 
need not be water insoluble. If the pH varies during the fermentation, the 
chelate must be insoluble during part of the fermentaion, preferably the 
part associated with maximum production of the desired fermentation 
product. 
Different hydrous metal oxides and hydrous metal oxide-chelated polyhydroxy 
supports will chelate or complex with the bacteria used in a fermentation 
process to an extent which will vary with the pH of the medium. 
When a hydrous metal oxide alone is used in the fermentation process of the 
invention, the amount of hydrous metal oxide used is suitably from about 
0.01 to about 2.2 g/liter of fermentation fluid per day. When a hydrous 
metal oxide-chelated polyhyroxy support is used, the ratio of hydrous 
metal oxide (expressed in terms of metal) to polyhydroxy support is 
preferably about 1:2 to about 1:25 (w/w), the amount of hydrous metal 
oxide (expressed in terms of metal) preferably being from about 0.02 to 
about 2.0 g/liter of fermentation fluid per day, the preferred amount of 
polyhydroxy support being from about 0.045 to about 4.5 g/liter of 
fermentation fluid per day. 
The invention is particularly advantageous for the production of a food 
product such as vinegar since although titanium dioxide is considered 
non-toxic and (in several countries) a permitted food additive, the 
average level remaining after filtration through Kieselguhr is only 5 ppm 
at most. 
Although the invention is preferably carried out in a fermenter such as 
described in British Pat. No. 1,263,059, it is also possible to use, for 
example, Fring's generator for carrying out the invention. 
In any of the fermentations of the invention, the substrate giving rise to 
an oxidation product need not be the immediate precursor of the oxidation 
product, but could be a metabolic precursor several steps removed from the 
oxidation product, e.g. glucose or sucrose for lactic acid production. 
Further, although the fermentations are preferably those taking place in 
the presence of a gas containing oxygen such as air, the oxidation product 
need not arise by actual reaction of the substrate or its metabolite with 
oxygen, but can arise by some other oxidation process. Furthermore, the 
invention can be used in non-oxidative fermentations such as the 
fermentation of glucose to e.g. butylene glycol. 
The addition of hydrous metal oxide or hydrous metal-polyhydroxy support 
confers further unforeseen advantages on the fermentation, for example (a) 
it confers greater resistance to disturbance by frothing so that, where 
desired, greater aeration rates can be employed. Where oxygen is a direct 
participant in the reaction as in the conversion of ethanol to acetic 
acid, this is particularly advantageous; (b) better consistency of the 
production can be attained in the presence of the additive since in 
addition to (a) it becomes less sensitive to wash-out.

In the following examples there are described several preferred embodiments 
to illustrate the invention. However, it should be understood that the 
invention is not intended to be limited to the specific embodiments. 
EXAMPLE 1A 
Hydrous titanium oxide 
To given amounts of titanium tetrachloride (present as 15% w/v solution in 
15% w/v hydrochloric acid), there was added 2N ammonium hydroxide solution 
with stirring to precipitate the hydrous titanium oxide and to obtain a pH 
equal to that in the fermenter (approximately pH 3.0). The suspension of 
hydrous titanium oxide was diluted to about 100 ml with distilled water, 
was stirred vigorously and was added to the fermenter contents. Mixing was 
accomplished very rapidly (10 to 20 sec) by the efficient aeration 
pattern. 
EXAMPLE 1B 
Hydrous titanium-cellulose chelate 
1.2 g of chromatographic cellulose powder (Whatman CF 11) and 1.2 g of 
titanium tetrachloride (in solution, as above) were mixed and stirred for 
2 hours. The mixture was then dried at 45.degree. C., ground to a powder, 
washed with distilled water until the washings were neutral to pH 
indicator paper and then added to the fermenter as an aqueous suspension. 
EXAMPLE 1 
Production of malt vinegar by a non-aggregating strain of Acetobacter in 
the presence of titanium-chelated cellulose 
A glass tower fermenter of a cylindrical shape was used which had a high 
aspect ratio (height to diameter ratio) containing microorganisms 
suspended in the medium and through which there is a uni-directional flow 
of medium and/or gases. The fermenter used in this experiment had a volume 
of 2.6 liters, a height of 57 cm and an average diameter of 7.32 cm so 
that its aspect ratio was 7.8:1. In operating the fermenter, compressed 
air was passed through a pressure regulator and filter, then through a 
flowmeter to measure its flow rate and finally into the fermenter medium 
via a sintered glass disc fused into the base of the fermenter. 
The effluent air from the fermenter was passed through two water-cooled 
(4.degree. C.) condensers to ensure that any volatile components in it was 
returned to the bulk of the liquid. The temperature of the fermenter was 
maintained at 32.degree. C. by a water jacket surrounding the fermenter 
which was fed by thermostatically controlled water. The rate of medium 
flow in the fermenter was controlled by a variable speed peristaltic pump 
and the flow rate through the fermenter was measured by collecting the 
vinegar produced over a period of approximately 24 hours and expressed as 
volumetric efficiency (V.E.) defined as 
##EQU1## 
The medium used in the experiments is known commercially as "charging wort" 
and is a dilute aqueous ethanol solution containing about 6% w/v of 
ethanol and is produced by a process similar to that used for beer and 
spirit fermentation from malted barley wort. In this experiment, a culture 
of a non-aggregating strain of Acetobacter was used which was obtained 
from a tower fermenter of a commercial malt vinegar manufacture and was 
known not to aggregate or produce extra-cellular polysaccharide material. 
At the beginning of each experiment, the fermenter was half-filled with 
charging wort, an inoculum (50-100 ml) of the culture was added and 
aeration was commenced at a low rate (0.1 to 0.2 v/v/m=volumes 
air/fermenter volume/minute). Samples were taken at intervals and tested 
for acetic acid and ethanol content. When the acidity reached about 4% 
w/v, the peristaltic pump was turned on and the medium was metered into 
the fermenter. The fermentation was then allowed to proceed continuously 
with the medium flow rate and aeration rate being manipulated to maximize 
the acetic acid content of the fermenter (7% w/v approximately) and 
minimize ethanol content (less than 0.57%). Measurements of pH, optical 
density (600 nm) and titanium content (if present) were also made. 
The fermentation was started without addition of the titanium hydroxide or 
the chelated titanium-cellulose complex. Addition of hydrous titanium 
hydroxide was started only after the fermenter operated continuously at 
optimal conditions of medium flow rate and aeration for several 
consecutive days. Some typical results are given below: 
______________________________________ 
Number of 
Average Hydrous titanium 
consecutive 
aeration oxide added daily 
days (v/v/m) Ti (g) cellulose (g) 
Performance 
______________________________________ 
2 0.58 -- -- 0.95 
2 0.58 0.26 -- 1.00 
2 0.57 0.53 -- 1.08 
2 0.63 0.53 1.2 1.47 
2 0.79 0.53 1.2 1.44 
______________________________________ 
The table shows that the addition of hydrous titanium oxide, either from 
0.26 g. Ti or from 0.53 g. Ti does hardly improve the performance of the 
tower fermenter, but that a marked increase of the performance is achieved 
after the addition of the hydrous titanium-cellulose chelate (expressed in 
the table in terms of its equivalent hydrous titanium oxide and cellulose 
content). 
EXAMPLE 2 
An 8 liter tower fermenter (aspect ratio 14:1) was constructed and 
continuous production of acetic acid initiated in a manner similar to and 
under the conditions of Example 1 using the non-aggregating strain of 
Acetobacter. After a period of time during which the optimum operating 
parameters and highest operating efficiencies were established, additions 
of hydrous titanium oxide and subsequently a mixture of hydrous titanium 
oxide and cellulose powder (Whatman CF 11) were made. The results of these 
additions are given below: 
______________________________________ 
Number of 
Average Hydrous titanium 
Cellulose 
consecutive 
aeration oxide added added Perform- 
days (v/v/m) daily (Ti g) daily (g) 
ance 
______________________________________ 
2 1.10 -- -- 1.23 
2 1.12 0.51 -- 1.24 
2 0.45 1.52 -- 1.22 
2 0.43 1.52 6 0.88 
______________________________________ 
This Example shows that the effect of Example 1 is not produced by a mere 
mixture of hydrous titanium oxide and cellulose and, in fact, this mixture 
is even somewhat deleterious to the process. Only the titanium-cellulose 
combinations in which the titanium oxide is chelated to the cellulose 
works with the non-aggregating species. 
EXAMPLE 3 
Production of malt vinegar by a polysaccharide producing species of 
Acetobacter in the presence of hydrous titanium oxide 
The experiment was carried out in the fermenter of Example 2 and an 
aggregating strain of Acetobacter capable of producing polysaccharide 
which was obtained from a commercial malt vinegar manufacturer employing 
the "Quick" process in which such strains predominate was used. After a 
period of time in which the highest level at which the fermenter would 
operate was established, hydrous titanium oxide was added to the fermenter 
and the results obtained after this treatment, compared with those before, 
are as below: 
______________________________________ 
Number of 
Average Hydrous titanium 
consecutive 
aeration oxide added daily 
days (v/v/m) Ti (g) Performance 
______________________________________ 
2 0.31 -- 1.10 
2 0.31 -- 1.10 
8 0.31 -- 1.08 
8 0.39 1.76 1.50 
______________________________________ 
The table shows that addition of hydrous titanium oxide alone, i.e. not 
chelated to a polyhydroxy support, is sufficient to show a marked increase 
of the performance when an aggregating strain of Acetobacter is used. 
EXAMPLE 4 
Production of malt vinegar by a polysaccharide producing species of 
Acetobacter in the presence of hydrous titanium oxide. 
The 2.6 liter tower fermenter of Example 1 was prepared for the continuous 
production of malt vinegar as in that Example and then the fermenter was 
inoculated with the aggregating species of Acetobacter capable of 
producing polysaccharide from the "Quick" process with the fermentation 
allowed to run for 29 days before addition of hydrous titanium oxide was 
commenced. Additions were continued over a long period of time (about 50 
days) during which time the volumetric efficiency of the fermenter was 
slowly increased. Some of the results of this treatment are given below. 
______________________________________ 
Number of 
Average Hydrous titanium 
consecutive 
aeration oxide added daily 
days (v/v/m) Ti (g) Performance 
______________________________________ 
2 0.49 -- 0.63 
2 1.50 0.33 1.48 
2 1.50 0.33 1.53 
______________________________________ 
Example 4 illustrates that increase in operating performance is not limited 
to just one fermenter. In an experiment almost identical to that of 
Example 3, very similar results were obtained by the addition of hydrous 
titanium oxide to an aggregating species of Acetobacter. 
EXAMPLE 5 
Production of malt vinegar by a polysaccharide producing species of 
Acetobacter in the presence of hydrous titanium oxide 
In this experiment, the tower fermenter of Example 1 was used and the 
vinegar production was carried out with the aggregating species of 
Acetobacter of Examples 3 and 4. The flow rate of the medium was 
controlled to attempt to keep the conversion of ethanol to acetic acid 
above about 90%. Samples were taken from the fermenter and from the 
effluent receiver and assayed for dissolved titanium colorimetrically at 
600 nm. The results were as follows: 
__________________________________________________________________________ 
Hydrous titanium Amount of Ti 
oxide added in solution 
Number of 
Average 
daily Volumetric (ppm) 
consecutive 
aeration cellulose 
efficiency 
Percentage 
Perform- 
Ferm- 
days (v/v/m) 
Ti (g) 
(g) (V.E.) conversion 
ance enter 
Effluent 
__________________________________________________________________________ 
2 0.50 -- -- 0.77 82 0.63 -- -- 
5 1.50 0.32 -- 1.49 91 1.36 1.8 2.1 
5 1.50 0.32 -- 1.79 94 1.68 
__________________________________________________________________________ 
The table shows again that an important increase in performance is achieved 
after the addition of hydrous titanium oxide to an aggregating strain of 
Acetobacter. The table shows further that the amounts of titanium in 
solution in the fermenter and in the effluent are extemely low. 
EXAMPLE 6 
Production of malt vinegar by a non-aggregating species of Acetobacter in 
the presence of titanium chelated cellulose 
In this Example, the fermenter of Example 1 was used as well as the 
non-aggregating strain of Acetobacter of Example 1 and additions of 
hydrous titanium oxide and titanium-chelated cellulose were studied. The 
results are shown in the following table: 
__________________________________________________________________________ 
Hydrous titanium 
oxide added 
Number of 
Average 
daily Volumetric 
consecutive 
aeration 
Ti cellulose 
efficiency 
Conversion 
days (v/v/m) 
(g) (g) (V.E.) % Performance 
__________________________________________________________________________ 
5 0.58 -- -- 1.15 91 1.04 
3 0.58 0.26 -- 1.14 86 0.99 
5 0.69 0.53 -- 1.18 88 1.05 
5 0.85 0.53 1.2 1.63 80 1.30 
__________________________________________________________________________ 
As in Example 1, the table shows that the addition of hydrous titanium 
oxide alone, i.e. not chelated to cellulose, is insufficient to provoke a 
significant increase of the performance, but after addition of the hydrous 
titanium-cellulose chelate, a marked increase of the performance is shown. 
Various modifications of the process and products of the invention may be 
made without departing from the spirit or scope thereof and it is to be 
understood that the invention is to be limited only as defined in the 
appended claims.