U.S. patent application number 12/495320 was filed with the patent office on 2009-10-22 for method of fermentation.
This patent application is currently assigned to STATOIL ASA. Invention is credited to Henrik ERIKSEN, Lars JORGENSEN, Kurt STRAND.
Application Number | 20090263877 12/495320 |
Document ID | / |
Family ID | 32178862 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090263877 |
Kind Code |
A1 |
ERIKSEN; Henrik ; et
al. |
October 22, 2009 |
METHOD OF FERMENTATION
Abstract
The invention provides a process for the production of biomass
by culturing a microorganism in an aqueous liquid culture medium
circulating in a loop reactor having an effluent gas removal zone
where from carbon dioxide-containing effluent gas is removed from
the reactor and upstream thereof a degassing zone in which a
driving gas is introduced to drive carbon dioxide in the liquid
phase into a separable effluent gas phase and having upstream of
said degassing zone a nutrient gas introduction zone in which
oxygen is introduced into the reactor and mixed with the liquid
culture medium therein, characterized in that oxygen introduction
in said nutrient gas introduction zone is effected at a plurality
of locations along the flow path through said loop reactor at a
rate such that the average dissolved oxygen content of said liquid
culture medium measured using a polarographic oxygen electrode does
not exceed 25 ppm.
Inventors: |
ERIKSEN; Henrik; (Stavanger,
NO) ; STRAND; Kurt; (Stavanger, NO) ;
JORGENSEN; Lars; (Stavanger, NO) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
STATOIL ASA
Stavanger
NO
|
Family ID: |
32178862 |
Appl. No.: |
12/495320 |
Filed: |
June 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10486694 |
Jul 9, 2004 |
7579163 |
|
|
PCT/GB02/03798 |
Aug 16, 2002 |
|
|
|
12495320 |
|
|
|
|
Current U.S.
Class: |
435/170 ;
435/297.1 |
Current CPC
Class: |
A23J 1/005 20130101;
B01J 2219/32227 20130101; C12M 29/18 20130101; B01J 2219/32237
20130101; C12M 29/20 20130101; B01J 2219/3221 20130101; B01J
2219/32213 20130101; B01J 2219/32231 20130101 |
Class at
Publication: |
435/170 ;
435/297.1 |
International
Class: |
C12P 1/04 20060101
C12P001/04; C12M 1/12 20060101 C12M001/12 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 16, 2001 |
GB |
0120025.2 |
Apr 18, 2002 |
GB |
0208906.8 |
Claims
1. A process for generating biomass comprising: culturing a
microorganism in a liquid reaction medium which circulates in a
loop reactor, wherein said reactor has an effluent gas removal
zone, and, upstream of said effluent gas removal zone, an effluent
gas-liquid reaction medium separating zone, and wherein oxygen
and/or methane is fed into the liquid reaction medium in said
effluent gas-liquid reaction medium separating zone; and harvesting
biomass from said reactor.
2. The process as claimed in claim 1 wherein said liquid reaction
medium contains a methanotrophic bacterium and wherein oxygen and
methane are introduced into said reactor and are mixed with said
liquid reaction medium.
3. The process as claimed in claim 1 further comprising separating
biomass from a liquid.
4. A process for the production of a chemical compound comprising:
culturing a microorganism in a liquid reaction medium which
circulates in a loop reactor, wherein said reactor has an effluent
gas removal zone, and, upstream of said effluent gas removal zone,
further has an effluent gas-liquid reaction medium separating zone,
and wherein oxygen and/or methane is fed into the liquid reaction
medium in said separating zone; harvesting biomass from said
reactor; and separating therefrom a chemical compound produced by
said microorganism.
5. The process as claimed in claim 1 wherein oxygen is introduced
at a controlled rate at a plurality of locations along a
loop-reactor flow path through said loop reactor so as to achieve
an average dissolved oxygen content of said liquid reaction medium
measured using a polarographic oxygen electrode of not greater than
25 parts per million (ppm) by weight.
6. The process as claimed in claim 1 wherein oxygen is introduced
at a controlled rate at a plurality of locations along a
loop-reactor flow path through said loop reactor so as to achieve
an average dissolved oxygen content of said liquid reaction medium
measured using a polarographic oxygen electrode of not greater than
15 parts per million (ppm) by weight.
7. The process as claimed in claim 1 wherein oxygen is introduced
at a controlled rate at a plurality of locations along a
loop-reactor flow path through said loop reactor so as to achieve a
dissolved oxygen content of said liquid reaction medium at each
said location of at least 0.5 parts per million (ppm) by
weight.
8. The process as claimed in claim 1 wherein a driving gas is
introduced at a driving gas inlet and wherein oxygen is introduced
at a controlled rate so as to achieve a dissolved oxygen content of
the liquid reaction medium over at least 30% of the path length of
the loop reactor of at least 10 parts per million (ppm) by weight,
a dissolved oxygen content of the liquid reaction medium
immediately prior to the driving gas inlet of at least 3 parts per
million (ppm) by weight, and an oxygen content of said effluent gas
of at least 1 mole %.
9. The process as claimed in claim 1 wherein oxygen is introduced
at a controlled rate into said liquid reaction medium in a nutrient
gas introduction zone so as to achieve between the nutrient gas
introduction zone and the effluent gas-liquid reaction medium
separating zone a dissolved oxygen content of the liquid reaction
medium of not less than 3 parts per million (ppm) by weight.
10. The process as claimed in claim 1 wherein oxygen is introduced
at a controlled rate into said liquid reaction medium so as to
achieve a dissolved oxygen content of said liquid reaction medium
of not less than 3 parts per million (ppm) by weight over a path
length of the loop reactor corresponding to more than 30
seconds.
11. The process as claimed in claim 1 wherein said fluid culture
medium is circulated through said loop reactor under the action of
a propeller having overlapping or multiple, radially curved
blades.
12. The process as claimed in claim 1 wherein nutrient gas and
liquid reaction medium are mixed in said loop reactor by passage
through a static mixer comprising a stack of parallel corrugated
flexible plates arranged with the stacking direction perpendicular
to the direction of flow of said fluid medium and with the
corrugation ridges thereof angled to said direction of flow and
with their angle to the direction of flow being substantially equal
and opposite for adjacent plates.
13. The process as claimed in claim 1 wherein oxygen is introduced
at a controlled rate into said liquid reaction medium in a nutrient
gas introduction zone so as to achieve between said nutrient gas
introduction zone and the effluent gas-liquid reaction medium
separating zone a dissolved oxygen content of the liquid culture
medium of not less than X parts per million (ppm) by weight, where
X is defined by X=1.35YB where B is the biomass content of the
culture medium in g/L and Y is from 0.75 to 1.25, and B is from 5
to 30.
14. The process as claimed in claim 1 wherein said loop reactor has
a flow path of at least 40 m.
15. A fermentation reactor comprising an effluent gas-liquid
reaction medium separating zone upstream of an effluent gas removal
zone, wherein said separating zone comprises an inlet for feeding
oxygen and/or methane into liquid reaction medium contained in said
effluent gas-liquid reaction medium separating zone.
16. A fermentor apparatus comprising a loop reactor comprising an
effluent gas-liquid reaction medium separating zone upstream of an
effluent gas removal zone, wherein said separating zone has an
inlet for feeding oxygen and/or methane into liquid reaction medium
therein.
Description
[0001] The present application is a Divisional of U.S. patent
application Ser. No. 10/486,694, filed Jul. 9, 2004, which is a
National Stage Entry of PCT/GB02/03798, filed on Aug. 16, 2002,
which claims priority from United Kingdom Patent Application Nos.
0120025.2 and 0208906.8 filed Aug. 16, 2001 and Apr. 18, 2002,
respectively. The entire disclosures of the prior applications are
hereby incorporated by reference.
[0002] This invention relates to improvements in and relating to
processes for fermenting microorganisms, e.g. for the generation of
biomass or the preparation of materials produced by microorganisms,
and to fermentor apparatus for the performance thereof, in
particular processes for culturing microorganisms in a loop reactor
using a gaseous nutrient.
[0003] Recently, much attention has been directed toward the
development of new sources of protein which may be incorporated
into foods for human and/or animal consumption. A number of
different protein-containing materials have been proposed as
substitutes for more traditional sources of protein, such as fish
meal, soya products and blood plasma, in human foods and as animal
feeds. These materials include single-cell microorganisms such as
fungi, yeasts and bacteria which contain high proportions of
proteins. These may be grown by reproduction producing biomass
through the growth of the microorganisms on hydrocarbon or other
substrates. Today, the most widely used protein-containing
microorganisms (also referred to herein as "single-cell proteins")
are those derived from fungi or yeast.
[0004] Single-cell protein materials can be used directly in foods,
e.g. as a spray dried product, or the biomass may be further
processed, e.g. by hydrolysis and/or separation, before use.
[0005] Besides being simply used as sources of biomass,
microorganisms may be grown and harvested to serve as sources of
useful chemicals, e.g. drug compounds, proteins, carotenoids, etc.
Thus for example Du Pont in WO 02/18617, WO 02/20815, WO 02/20728
and WO 02/20733 (the contents of which are hereby incorporated by
reference) describe the use of methanotrophic bacteria, and in
particular Methylomonas 16a (ATCC PTA 2402), for the preparation of
carotenoids.
[0006] In DK-B-170824 (Dansk Bioprotein A/S) and in WO01/60974
(Norferm DA), the contents of both of which are hereby incorporated
by reference, is described a loop reactor for use in cultivating
microorganisms to generate biomass, e.g. for culturing
methanotrophic bacteria to generate a material which can for
example be used in or as or as a precursor to human or animal
feed.
[0007] The loop reactor described in DK-B-170824 has a top unit
connected to a conduit having, in order, a down-flow section, a
substantially horizontal flow section, an up-flow section and a
non-vertical out-flow section. The top unit is cylindrical with its
axis vertical and with the down-flow section of the conduit
attached to its base. The out-flow section of the conduit extends
tangentially from a side of the top unit. In this way, effluent gas
and liquid culture medium entering the top unit from the out-flow
section may be separated efficiently with the gas being withdrawn
from the headspace of the top unit and the liquid being returned to
the down-flow section of the conduit. The aqueous liquid culture
medium is driven around the reactor loop, with gaseous and
non-gaseous nutrients (e.g. oxygen, methane, nitrogen sources and
minerals) being introduced into the conduit and mixed with the
liquid culture medium therein, with biomass-containing culture
medium being withdrawn from the conduit, and with effluent gas
(e.g. carbon dioxide-containing gas) being withdrawn from the top
unit. Nutrient addition and effluent gas withdrawal is generally
continuous, while biomass withdrawal may be continuous or
batchwise.
[0008] In the loop reactor described in DK-B-170824 the circulation
rate of the reaction medium was generally about 30 to 90 seconds
per circuit. Since unconsumed oxygen and methane in the effluent
gas would make this potentially explosive and would be uneconomical
since oxygen and methane would be wasted, gaseous nutrient addition
could not occur at all points along the conduit. Likewise, to
ensure efficient separation of carbon dioxide from the liquid
reaction medium for venting from the top unit, gas/liquid mixing
could not be effected at all points along the conduit either. In
practice therefore, in the loop reactor of DK-B-170824, gaseous
nutrient addition was such that the dissolved oxygen content in the
culture medium would drop to about zero by the time the culture
medium reached the top of the upflow section of the conduit.
[0009] However, while the loop reactor described in DK-B-170824 can
be successfully used for biomass production, when used at high
biomass concentrations (e.g. above 5 g/L) the production process is
susceptible to occasional sudden failure, requiring the process to
be restarted with fresh live bacteria.
[0010] We have now found that this problem may be reduced or
avoided if nutrient gases (e.g. methane and/or oxygen, particularly
oxygen) are supplied into the conduit so as to reduce the time the
culture medium is essentially free of an essential nutrient and/or
so as to avoid or minimize periods of unduly high concentrations of
nutrient gases in the culture medium.
[0011] Thus in one aspect the invention provides a process for the
production of biomass by culturing a microorganism, preferably a
methanotrophic bacterium, in an aqueous liquid culture medium
circulating in a loop reactor having an effluent gas removal zone
wherefrom carbon dioxide-containing effluent gas is removed from
the reactor and upstream thereof a degassing zone in which a
driving gas is introduced to drive carbon dioxide in the liquid
phase into a separable effluent gas phase and having upstream of
said degassing zone a nutrient gas introduction zone in which
oxygen, and preferably also methane, are introduced into the
reactor and mixed with the liquid culture medium therein,
characterized in that oxygen introduction in said nutrient gas
introduction zone is effected at a plurality of locations along the
flow path through said loop reactor at a rate such that the average
dissolved oxygen content of said liquid culture medium measured
using a polarographic oxygen electrode (e.g. in mixer-free sections
of the reactor) does not exceed 25 ppm, more preferably 20 ppm,
especially 15 ppm, more especially 10 ppm, and preferably also such
that the dissolved oxygen content of said liquid culture medium at
each said location (with the optional exception of the first said
location downstream of said degassing zone) is at least 0.5 ppm,
especially at least 1 ppm, more especially at least 3 ppm.
[0012] In this aspect of the invention, the oxygen introduction
locations are preferably so spaced that the flow time between
locations is less than 20 seconds, especially less than 16 seconds,
particularly less than 6 seconds, more preferably less than 5
seconds, still more preferably less than 4 seconds, other than
between the last and first locations (ie those immediately upstream
and downstream of the degassing zone).
[0013] By average dissolved oxygen content is meant the average
along a diameter of the cross sectional plane of the reactor. Where
the reactor is not circular in cross section, the term diameter
should be understood to mean maximum dimension between inside
walls. Such an average may be taken by measuring dissolved oxygen
content at several, e.g. 3, 5 or 7, equally spaced points along the
diameter.
[0014] The dissolved oxygen content should desirably be measured at
mixer-free sections of the loop reactor, ie places within the
reactor where the cross section through the reactor does not pass
through a mixer device. Desirably the dissolved oxygen content is
measured at mixer-free sections downstream of the oxygen
introduction zones and the subsequent mixers.
[0015] Thus the dissolved oxygen content of the culture medium is
conveniently measured using a polarographic oxygen electrode
(available commercially, e.g. from Ingold) at positions between the
oxygen introduction locations sufficiently far downstream of the
previous such location that mixing of oxygen and culture medium is
substantially complete.
[0016] In order that oxygen concentration extremes may be avoided
on oxygen introduction, introduction is preferably effected through
a plurality of apertures at each introduction location, e.g. an
array of apertures spaced apart in the plane transverse to the flow
direction and optionally also along the flow direction. This may be
achieved by dispersing in the reactor multi-armed, perforated gas
distributors, preferably with the pressure drop from inside to
outside the distributor being up to 6 bar, e.g. 0.4 to 3 bar
especially 0.6 to 1 bar. Such distributors are conveniently
disposed in the gaps between adjacent static mixer arrays in the
reactor. In one preferred embodiment, gas distributors are also
disposed within the static mixer arrays, e.g. by disposing
perforated tubes along mixer channels or by the use of perforated
corrugated panels that function both as gas distributors and as
plates within the mixer array. Hollow panel mixer plates through
which coolant (e.g. cold water) is flowed may also be used to cool
the culture medium in the reactor.
[0017] The dissolved oxygen content is preferably measured upstream
of each oxygen introduction location with the measured values being
fed to a computer arranged to control nutrient introduction rates,
cooling, biomass withdrawal, etc. In this way the dissolved oxygen
content can be maintained within the desired range throughout the
reactor and for extended operation periods.
[0018] Desirably, the oxygen content of the culture medium in the
effluent gas removal zone, or at the lowest dissolved oxygen
concentration point in the loop, is less than 0.1 ppm by weight.
This results in the biomass product having greater bioavailability
as a feedstuff.
[0019] While the dissolved oxygen content of the liquid culture
medium may drop to negligible or undetectable levels, i.e. about 0
ppm by weight, it is preferred that it should remain at detectable
levels over at least a significant proportion of the path length
through the reactor. Thus viewed from a further aspect the
invention provides a process for the production of biomass by
culturing a microorganism, preferably a methanotrophic bacterium,
in an aqueous liquid culture medium circulating in a loop reactor
having an effluent gas removal zone wherefrom carbon
dioxide-containing effluent gas is removed from the reactor and
upstream thereof a degassing zone in which a driving gas is
introduced to drive carbon dioxide in the liquid phase into a
separable effluent gas phase and having upstream of said degassing
zone a nutrient gas introduction zone in which oxygen, and
preferably also methane, are introduced into the reactor and mixed
with the liquid culture medium therein, characterized in that
oxygen introduction in said nutrient gas introduction zone is such
that between said nutrient gas introduction zone and said degassing
zone the dissolved oxygen content of the liquid culture medium does
not fall below 3 ppm by weight (preferably at least 4 ppm,
especially at least 5 ppm).
[0020] Viewed from a still further aspect the invention provides a
process for the production of biomass by culturing a microorganism,
preferably a methanotrophic bacterium, in an aqueous liquid culture
medium circulating in a loop reactor having an effluent gas removal
zone wherefrom carbon dioxide-containing effluent gas is removed
from the reactor and upstream thereof a degassing zone in which a
driving gas is introduced to drive carbon dioxide in the liquid
phase into a separable effluent gas phase and having upstream of
said degassing zone a nutrient gas introduction zone in which
oxygen, and preferably also methane, are introduced into the
reactor and mixed with the liquid culture medium therein,
characterized in that oxygen introduction into said liquid culture
medium is so effected that the dissolved oxygen content of said
liquid culture medium does not fall below 3 ppm by weight
(preferably at least 4 ppm, especially at least 5 ppm) over a path
length of the loop reactor corresponding to more than 30 seconds,
preferably 20 seconds, more preferably 10 seconds, especially 5
seconds.
[0021] Viewed from a yet still further aspect the invention
provides a process for the production of biomass by culturing a
microorganism, preferably a methanotrophic bacterium, in an aqueous
liquid culture medium circulating in a loop reactor having an
effluent gas removal zone wherefrom carbon dioxide-containing
effluent gas is removed from the reactor and upstream thereof a
degassing zone in which a driving gas is introduced to drive carbon
dioxide in the liquid phase into a separable effluent gas phase and
having upstream of said degassing zone a nutrient gas introduction
zone in which oxygen, and preferably also methane, are introduced
into the reactor and mixed with the liquid culture medium therein,
characterized in that oxygen introduction in said nutrient gas
introduction zone is such that between said nutrient gas
introduction zone and said degassing zone the dissolved oxygen
content of the liquid culture medium does not fall below X ppm by
weight, where X is defined by
X=1.35Y.B
where B is the biomass content of the culture medium in g/L and Y
is from 0.75 to 1.25, preferably 0.80 to 1.20, especially 0.85 to
1.15, more preferably 0.90 to 1.10, more especially 0.95 to 1.05,
and B is greater than 5, especially greater than 10, particularly
15 to 30, especially 18 to 25.
[0022] Viewed from a still further aspect the invention provides a
process for the production of biomass by culturing a microorganism,
preferably a methanotrophic bacterium, in an aqueous liquid culture
medium circulating in a loop reactor having an effluent gas removal
zone wherefrom carbon dioxide-containing effluent gas is removed
from the reactor and upstream thereof a degassing zone in which a
driving gas is introduced to drive carbon dioxide in the liquid
phase into a separable effluent gas phase and having upstream of
said degassing zone a nutrient gas introduction zone in which
oxygen, and preferably also methane, are introduced into the
reactor and mixed with the liquid culture medium therein,
characterized in that oxygen introduction in said nutrient gas
introduction zone is such that the dissolved oxygen content of the
liquid culture medium over at least 30% (preferably at least 50%,
more preferably at least 60%) of the path length of the loop
reactor is at least 10 ppm by weight, the dissolved oxygen content
of the liquid culture medium immediately prior to introduction of
said driving gas in said degassing zone is at least 3 ppm by weight
(preferably at least 4 ppm, especially at least 5 ppm), and the
oxygen content of said effluent gas is at least 1 mole %
(preferably at least 2 mole %, e.g. 2 to 8 mole %).
[0023] These methods described herein as further aspects of the
invention are preferably combined with the method of the invention
as first defined.
[0024] In the process of the invention, it is preferred to drive
the liquid culture medium around the loop reactor using a
propeller. It is especially preferred to use a propeller having
overlapping or multiple, radially curved blades, i.e. a
low-cavitation propeller. Such low-cavitation propellers are
well-known in the field of submarine design. By overlapping blades
it is meant that at least one line exists that is parallel to the
propeller axis and that passes through at least two blades. By a
radially curved blade it is meant that there is an angle between
the radial lines passing through the base and tip of the blade. The
use of such propellers in a loop fermentation reactor is novel and
forms a further aspect of the invention. Thus viewed from a further
aspect the invention provides a process for the production of
biomass by culturing a microorganism, preferably a methanotrophic
bacterium, in an aqueous liquid culture medium circulating in a
loop reactor (preferably one having an effluent gas removal zone
wherefrom carbon dioxide-containing effluent gas is removed from
the reactor and upstream thereof a degassing zone in which a
driving gas is introduced to drive carbon dioxide in the liquid
phase into a separable effluent gas phase and having upstream of
said degassing zone a nutrient gas introduction zone in which
oxygen and preferably also methane are introduced into the reactor
and mixed with the liquid culture medium therein), characterized in
that said liquid culture medium is circulated through said loop
reactor under the action of a propeller having overlapping or
multiple, radially curved blades.
[0025] Using such low cavitation propellers, it is possible to pump
liquids having a greater gas content than with conventional
propellers and thus it is possible to have nutrient gases
introduced into the liquid culture medium closer upstream of the
propeller than is otherwise possible. In this way, the proportion
of the reactor path length which has low dissolved gas content may
be reduced.
[0026] The mixing of the nutrient gases into the liquid culture
medium is an important aspect of the performance of the process of
the invention. We have found that the use of static mixers, each
comprising an array of mixer plates, positioned downstream of
nutrient gas injection points results in a particularly efficient
dissolution of the nutrient gases even though the gas:liquid volume
ratio is relatively small. Such use of mixer plate arrays in loop
fermentation reactors is novel and forms a further aspect of the
invention. Viewed from this aspect the invention provides a process
for the production of biomass by culturing a microorganism,
preferably a methanotrophic bacterium, in an aqueous liquid culture
medium circulating in a loop reactor (preferably having an effluent
gas removal zone wherefrom carbon dioxide-containing effluent gas
is removed from the reactor and upstream thereof a degassing zone
in which a driving gas is introduced to drive carbon dioxide in the
liquid phase into a separable effluent gas phase and having
upstream of said degassing zone a nutrient gas introduction zone in
which oxygen and preferably also methane are introduced into the
reactor and mixed with the liquid culture medium therein),
characterized in that nutrient gas and liquid culture medium are
mixed in said loop reactor by passage through a static mixer
comprising a stack of parallel corrugated flexible (and preferably
perforated) plates arranged with the stacking direction
perpendicular to the direction of flow of said fluid medium and
with the corrugation ridges thereof angled to said direction of
flow (e.g. at an angle of 20 to 70E, preferably 40 to 50E,
especially 45E, relative to the direction of flow) and with their
angle to the direction of flow being substantially equal and
opposite for adjacent plates.
[0027] The stacking direction, i.e. the normal to the surface of a
planar sheet placed over either major surface of a mixer plate, may
be anywhere between vertical and horizontal. However the stacking
direction between successive stacks is preferably rotated through
90E, more preferably 80 to 90E, most preferably about 90E, 0E, 90E,
0E etc. or +45E, -45E, -45E relative to the vertical. Especially
preferably the stacking direction for successive stacks is not 0E,
0E, 0E etc. relative to the vertical.
[0028] In these last two aspects of the invention, indeed in most
aspects of the invention, the loop reactor is preferably one having
a centre line, i.e. flow path length, of at least 40 m, more
preferably at least 80 m.
[0029] Viewed from a further aspect, the invention provides a
process for generating biomass by culturing a microorganism in a
liquid reaction medium circulating in a loop reactor having an
effluent gas-liquid reaction medium separating zone upstream of an
effluent gas removal zone, characterized in that oxygen and/or
methane is fed into the liquid reaction medium in said separating
zone.
[0030] Viewed from a further aspect, the invention provides a
fermentor apparatus comprising a loop reactor comprising an
effluent gas-liquid reaction medium separating zone upstream of an
effluent gas removal zone, characterized in that said separating
zone has an inlet for feeding oxygen and/or methane into liquid
reaction medium therein.
[0031] The loop reactor used in the processes of the present
invention preferably is one which relies on propulsion rather than
gas uplift to move the liquid culture medium around the loop. Since
it is easier to maintain nutrient gas and liquid culture medium
properly mixed in a substantially horizontal section of the loop,
and since mass transfer (i.e. transfer of nutrient gas into the
liquid phase) increases with increased pressure in the culture
medium, the loop preferably comprises a substantially vertical
down-flow zone from the effluent gas removal zone followed by a
substantially horizontal (e.g. "U" shaped) zone in turn followed by
a substantially vertical up-flow zone leading back to the effluent
gas removal zone. In order that degassing and effluent gas/liquid
culture medium should be particularly effective, between the
substantially vertical up-flow zone and the effluent gas removal
zone, it is desirable to have a substantially non-vertical, e.g.
horizontal, out-flow zone between the two. This, in which most or
all effluent gas/liquid culture medium separation occurs, may have
a uniform gradient, may gradually become more horizontal or may
change stepwise towards horizontal. The driving gas, i.e. the gas
used to displace carbon dioxide from the dissolved phase (usually
nitrogen but optionally another inert non-flammable gas) may for
example be introduced at one or more points from the beginning of
the substantially vertical up-flow zone to the entry into the
effluent gas removal zone, however particularly preferably it will
be introduced at one or more points between the upper portion (e.g.
the upper 20%, more preferably the upper 10%) of the vertical
portion of the up-flow zone and the beginning of the flattest (i.e.
most horizontal) portion of the out-flow zone. Especially
preferably, the driving gas is introduced in the upper portion of
the vertical portion of the up-flow zone. In this context it will
be understood that the "point" at which a gas may be introduced may
have an extended length within the liquid path within the loop,
e.g. where gas is introduced through a series of inlet ports or
through a multiply perforated inlet port.
[0032] In general, where the centre line of the loop, outside the
gas effluent removal zone, changes direction, this will be by
curvature of the loop rather than by a sharp angled bend so as to
improve the flow properties of the liquid culture medium.
[0033] The gas pressure in the headspace in the gas effluent
removal zone will preferably be from -0.5 to +1.0 atmosphere
relative to ambient pressure, especially +0.2 to +0.6. The vertical
drop between the gas-liquid surface at the end of the outflow zone
and the centre line of the loop in the horizontal zone is
preferably at least 10 m, especially at least 18 m, e.g. 18 to 30
m.
[0034] The cross sectional area of the loop reactor (outside the
gas effluent removal zone) may be constant but preferably is
increased at least in the outflow zone, e.g. to a maximum value of
at least 2.0 times, more preferably at least 4.0 times that in the
minimum cross sectional area part of the down flow and horizontal
flow zones (which may typically be before the propeller).
Typically, the inner diameters of the loop, outside the gas
effluent removal zone may be in the range 30 cm to 3.0 m,
especially 1.0 to 2.5 m. Typically also the centre line of the
loop, excluding the gas effluent removal zone, has a length in the
range 40 to 200 m, preferably 80 to 150 m, preferably with at least
50%, more preferably at least 60%, especially at least 70%, of this
in the horizontal zone. The outflow zone preferably has a centre
line length of 0.5 to 10 m, especially 1.5 to 8 m.
[0035] The loop reactor will generally be circular in cross-section
outside the gas removal zone; however in the separation zone other
configurations, e.g. rectangular, elliptical or ovoid
cross-sections, may be adopted to enhance gas/liquid
separation.
[0036] Nutrient gas introduction in the loop is preferably effected
at least three, more preferably at least six, positions along the
length of the loop, preferably with at least 60% being introduced
in the horizontal zone. While it is preferred that nutrient gases
be introduced in the downflow section, this may require the use of
a low cavitation propeller for subsequent propulsion of the liquid
culture medium. Such propeller designs are well-known in the field
of submarine construction. Typical low-cavitation propellers may
have over-lapping blades, or multiple (e.g. at least 6)
radially-curved blades (i.e. blades where the base and tip are
radially displaced from each other). Unlike in prior reactor
designs where nutrient gas input was designed to give a
substantially zero dissolved oxygen content in the culture medium
by the beginning of the degassing zone, it is desirable to
introduce nutrient gases, in particular oxygen, in such amounts
that dissolved oxygen content at the beginning of the degassing
zone, is at least 3 ppm. To this end, some of the nutrient gas,
e.g. up to about 25% of the nutrient gas, is desirably introduced
in the upflow zone.
[0037] As excess nutrient gas can give rise to toxicity problem for
the microorganisms being cultured in the reactor, oxygen
introduction is preferably such as to achieve a maximum dissolved
oxygen content in the liquid culture medium of no more than 25 ppm,
especially no more than 20 ppm, more especially no more than 15
ppm. Likewise to maximize process efficiency in terms of biomass
production, methane is preferably introduced in a 1:1 to 1:3,
especially 1:1.2 to 1:2.5, more especially about 1:1.8, mole ratio
relative to oxygen.
[0038] Methane may be used in purified form or in a gas mixture,
e.g. natural gas or methane-enriched natural gas. Likewise purified
oxygen or oxygen in a gas mixture (e.g. air or oxygen-enriched air)
may be used. Where air is used, it is preferably filtered to avoid
introduction of toxic impurities.
[0039] The methane and oxygen used may likewise be in gaseous (e.g.
compressed) or liquefied form; in the latter case however
pre-heating will generally be required to prevent the nutrient gas
cooling the liquid culture medium unduly.
[0040] Natural gas mainly consists of methane, although its
composition will vary for different gas fields. Typically, natural
gas may be expected to contain about 90% methane, about 5% ethane,
about 2% propane and some higher hydrocarbons. During the
fermentation of natural gas, methane is oxidized by methanotrophic
bacteria to biomass and carbon dioxide. Methanol, formaldehyde and
formic acid are metabolic intermediates. Formaldehyde and to some
extent carbon dioxide are assimilated into biomass. However,
methanotrophic bacteria are unable to use substrates comprising
carbon-carbon bonds for growth and the remaining components of
natural gas, i.e. ethane, propane and to some extent higher
hydrocarbons, are oxidized by methanotrophic bacteria to produce
the corresponding carboxylic acids (e.g. ethane is oxidized to
acetic acid). Such products can be inhibitory to methanotrophic
bacteria and it is therefore important that their concentrations
remain low, preferably below 50 mg/l, during the production of the
biomass. One solution to this problem is the combined use of one or
more heterotrophic bacteria which are able to utilize the
metabolites produced by the methanotrophic bacteria. Such bacteria
are also capable of utilizing organic material released to the
fermentation broth by cell lysis. This is important in order to
avoid foam formation and also serves to minimize the risk of the
culture being contaminated with undesirable bacteria. A combination
of methanotrophic and heterotrophic bacteria results in a stable
and high yielding culture.
[0041] Besides oxygen and methane, other nutrients, e.g. minerals
and a nitrogen source (e.g. ammonia, nitrates, urea, etc.) will
generally be added to the liquid culture medium. Unlike oxygen and
methane however the degassing operation does not critically affect
their concentration and thus their addition can generally each be
at only one, two or three points along the loop. For certain
minerals, in particular copper, it may however be desirable to
effect introduction at a higher number of points along the loop.
For copper this is relevant as increased copper concentration
serves to increase methane consumption.
[0042] Air or pure oxygen may be used for oxygenation and ammonia
is preferably used as the nitrogen source. In addition to these
nutrients, the bacterial culture will typically require water,
phosphate (e.g. as phosphoric acid) and several minerals which may
include magnesium, calcium, potassium, iron, copper, zinc,
manganese, nickel, cobalt and molybdenum, typically used as
sulphates, chlorides or nitrates. All minerals used in the
production of the single-cell material should be of food-grade
quality.
[0043] In the process of the invention it is desirable to monitor
carbon dioxide, oxygen and methane content in the effluent gas, and
the biomass, nitrogen, dissolved oxygen, phosphate and mineral
contents of the culture medium. Biomass content may be measured
using samples of the biomass-containing medium extracted for
further processing; e.g. by separating biomass from liquid by
centrifugation and weighing. Nitrogen, phosphate and mineral
contents may also be measured in this extracted material, e.g.
using standard procedures, e.g. atomic absorption, etc. Dissolved
oxygen content, again measured by standard procedures, is
preferably monitored at two or more points about the loop. Effluent
gas is preferably sampled and cooled to about 5EC whereafter oxygen
is preferably determined by measuring the paramagnetism of the gas
and methane and carbon dioxide by infra-red spectrometry. Typically
oxygen content is preferably about 7.5% (v/v), methane content
about 4.0% (v/v) and carbon dioxide content about 35% (v/v) when
the process is running smoothly.
[0044] Ammonia concentration is preferably up to 200 ppm,
especially 0.1 to 5 ppm by weight.
[0045] Biomass concentration is preferably up to 30 g/L, e.g. 5 to
20 g/L.
[0046] Phosphate content in the culture medium is preferably at
least 10 ppm by weight so as to minimize foaming at the top of the
reactor, especially about 100 to 200 ppm.
[0047] Potassium, magnesium and calcium contents in the culture
medium are preferably at least 5, 0.5 and 0.5 ppm by weight,
especially about 100-200, 20-50 and 20-50 ppm respectively. Copper
and iron contents may conveniently be measured in the extracted
biomass; preferably their minimum contents are 5 and 200 mg/kg
respectively.
[0048] During production of the single-cell material, the pH of the
fermentation mixture will generally be regulated to between about
5.5 and 7.5, e.g. to 6.5 .A-inverted. 0.3. Suitable acids/bases for
pH regulation may be readily selected by those skilled in the art.
Particularly suitable for use in this regard are sodium hydroxide
and sulphuric acid. During fermentation the temperature within the
fermentor should preferably be maintained to within the range of
from 40EC to 50EC, most preferably 45EC .A-inverted. 2EC.
[0049] In operation of the process of the invention, liquid (e.g.
some or all of the liquid nutrients, the liquid added to compensate
for biomass removal, a fraction of the liquid withdrawn from the
reactor, cooled and returned to the reactor to control the
temperature, etc.), optionally containing antifoam, is preferably
sprayed onto the surface of the liquid culture medium in the
degassing zone to reduce foam build up. Likewise, the horizontal
flow section of the degassing zone is preferably provided in its
upper section with transversely extending baffles to combat foam
build up. As a further antifoaming measure, the reactor can be
provided in the degassing zone with steam inlets arranged to inject
steam into the headspace above the liquid surface.
[0050] Any single-cell protein material may be produced in
accordance with the processes of the invention. However, preferred
microorganisms include bacteria and yeasts. Any bacteria or yeast
approved for use in food products may be used and suitable species
may be readily selected by those skilled in the art. Particularly
preferably, the single-cells for use in the invention will be a
microbial culture which consists of methanotrophic bacteria
optionally in combination with one or more species of heterotrophic
bacteria, especially preferably a combination of methanotrophic and
heterotrophic bacteria. As used herein, the term "methanotrophic"
encompasses any bacterium which utilizes methane or methanol for
growth. The term "heterotrophic" is used for bacteria that utilize
organic substrates other than methane or methanol for growth.
[0051] While the process of the invention is especially suited to
the production of biomass which can be used with relatively little
further processing as a feedstuff or food additive, the process may
also be used for the preparation of specific chemicals which are
generated by the microorganism(s) in the liquid culture medium. In
this event, the post-fermentation treatment of the culture medium
will involve separation out of the particular chemicals of
interest, e.g. by conventional chemical techniques, optionally
following lysis of the microorganism cells to release the chemicals
of interest. In this embodiment of the invention, microorganisms
which naturally produce the chemicals of interest (e.g. proteins,
drugs, carotenoids, etc) or which have been genetically modified to
produce the chemicals of interest, may be used. Many such
microorganisms are known from the literature; however it is
particularly preferred to use methanotrophic bacteria.
[0052] Preferred bacteria for use in the invention include
Methylococcus capsulatus (Bath), a thermophilic bacterium
originally isolated from the hot springs in Bath, England and
deposited as NCIMB 11132 at The National Collections of Industrial
and Marine Bacteria, Aberdeen, Scotland. M. capsulatus (Bath) has
optimum growth at about 45EC, although growth can occur between
37EC and 52EC. It is a gram-negative, non-motile spherical cell,
usually occurring in pairs. The intracellular membranes are
arranged as bundles of vesicular discs characteristic of Type I
methanotrophs. M. capsulatus (Bath) is genetically a very stable
organism without known plasmids. It can utilize methane or methanol
for growth and ammonia, nitrate or molecular nitrogen as a source
of nitrogen for protein synthesis.
[0053] Other bacteria suitable for use in the invention include the
heterotrophic bacteria Ralstonia sp. formerly Alcaligenes
acidovorans) DB3 (strain NCIMB 13287), Brevibacillus agri formerly
Bacillus firmus) DB5 (strain NCIMB 13289) and Aneurinibacillus sp.
formerly Bacillus brevis) DB4 (strain NCIMB 13288) which each have
optimum growth at a temperature of about 45EC.
[0054] Ralstonia sp. DB3 is a gram-negative, aerobic, motile rod
belonging to the family Pseudomonadaceae which can use ethanol,
acetate, propionate and butyrate for growth. Aneurinibacillus sp.
DB4 is a gram-negative, endospore-forming, aerobic rod belonging to
the genus Bacillus which can utilize acetate, D-fructose,
D-mannose, ribose and D-tagatose. Brevibacillus agri DB5 is a
gram-negative, endospore-forming, motile, aerobic rod of the genus
Bacillus which can utilize acetate, N-acetyl-glucosamine, citrate,
gluconate, D-glucose, glycerol and mannitol.
[0055] Especially preferred for use in the invention is a microbial
culture comprising a combination of the methanotrophic bacterium
Methylococcus capsulatus (Bath) (strain NCIMB 11132), and the
heterotrophic bacteria Ralstonia sp. DB3 (strain NCIMB 13287) and
Brevibacillus agri DB5 (strain NCIMB 13289), optionally in
combination with Aneurinibacillus sp. DB4 (strain NCIMB 13288). The
role of Ralstonia sp. DB3 is to utilize acetate and propionate
produced by M. capsulatus (Bath) from ethane and propane in the
natural gas. Ralstonia sp. DB3 may account for up to 10%, e.g.
about 6 to 8%, of the total cell count of the resulting biomass.
The role of Aneurinibacillus sp. DB4 and Brevibacillus agri DB5 is
to utilize lysis products and metabolites in the medium. Typically,
Aneurinibacillus sp. DB4 and Brevibacillus agri DB5 will each
account for less than 1% of the cell count during continuous
fermentation.
[0056] Suitable yeasts for use in the processes of the invention
may be selected from the group consisting of Saccharomyces and
Candida.
[0057] If desired, the process of the invention may be performed
using bacteria (or yeasts) genetically modified so as to generate a
desired chemical compound which can then be extracted from the
intercellular fluid or the biomass harvested from the reactor. The
scientific and patent literature contains numerous examples of such
genetically modified microorganisms including, inter alia,
methanotrophic bacteria.
[0058] In one especially preferred embodiment of the invention, the
process is performed using methanotrophic bacteria of the type
described in WO 02/18617 to produce carotenoids, e.g.
antheraxanthin, adonixanthin, astaxanthin, canthaxanthin,
zeaxanthin and the other carotenoids mentioned on pages 39 and 40
of WO 02/18617. To this end, the methanotrophic bacterium
Methylomonas 16a (ATCC PTA 2402) may particularly suitably be used.
Carotenoids produced in this way may be separated out from the
liquid culture medium as described in WO 02/18617, WO 02/20728 and
WO 02/20733.
[0059] Ideally, the biomass produced from fermentation of natural
gas will comprise from 60 to 80% by weight crude protein; from 5 to
20% by weight crude fat; from 3 to 15% by weight ash; from 3 to 15%
by weight nucleic acids (RNA and DNA); from 10 to 30 g/kg
phosphorus; up to 500 mg/kg iron; and up to 250 mg/kg copper.
Particularly preferably, the biomass will comprise from 68 to 73%,
e.g. about 70% by weight crude protein; from 9 to 11%, e.g. about
10% by weight crude fat; from 5 to 10%, e.g. about 7% by weight
ash; from 8 to 12%, e.g. about 10% by weight nucleic acids (RNA and
DNA); from 10 to 25 g/kg phosphorus; up to 310 mg/kg iron; and up
to 110 mg/kg copper. The amino acid profile of the protein content
should be nutritionally favorable with a high proportion of the
more important amino acids cysteine, methionine, threonine, lysine,
tryptophan and arginine. Typically these may be present in amounts
of about 0.7%, 3.1%, 5.2%, 7.2%, 2.5% and 6.9%, respectively
(expressed as a per cent of the total amount of amino acids).
Generally the fatty acids will comprise mainly the saturated
palmitic acid (approx. 50%) and the monounsaturated palmitoleic
acid (approx. 36%). The mineral content of the product will
typically comprise high amounts of phosphorus (about 1.5% by
weight), potassium (about 0.8% by weight) and magnesium (about 0.2%
by weight).
[0060] The biomass product of the invention is especially useful as
a component or precursor in food products, particularly when used
as a substitute for natural plasma in animal feeds and in pet
foods. When used in pet foods, additional ingredients may be added
to the product such as fats, sugars, salt, flavorings, minerals,
etc. The product may then be formed into chunks resembling natural
meat chunks in appearance and texture. The product of the invention
has the further advantages that this is readily formulated to
contain necessary nutrients, is easily digested by the animals and
is palatable to the animals.
[0061] The product of the invention may find further use as a
texturant in meat products (e.g. meat balls), as a replacement for
plasma proteins conventionally used as extenders in fresh meat to
increase weight and volume, as an emulsifier (e.g. in dressings,
etc.), and in bakery products to enhance dough properties.
[0062] When used in food products, the biomass, or processed
biomass material will typically be used in an amount of from 1 to
10% by weight, preferably up to 5% by weight. The exact proportion
will depend on the desired function of the material and can be
readily determined by those skilled in the art. Typically, when
used as a gelling agent this may be present in an amount of up to
20% by weight, e.g. 5 to 10% by weight (based on dry matter content
of the product).
[0063] When the reactor was run without meeting the dissolved
oxygen content conditions specified above (in particular when
overly high dissolved oxygen contents occurred), and when a certain
biomass content was reached, although the reactor continued to
perform satisfactorily for many hours, relatively suddenly ammonia
usage dropped and within a period of only two or three hours the
live bacterial population (and hence the biomass content of the
material, if it continued to be extracted from the reactor) dropped
to virtually zero. While in some cases, immediate cessation of
ammonia feed, reduction or cessation of biomass extraction from the
reactor and increase or decrease of oxygen feed could "cure" the
microorganism population and restore the biomass yield, in many
cases this was not sufficient. Investigation of the dead bacteria
showed that while these had not lysed the internal organelle
structure had been significantly disrupted. This indicated that the
bacterial genome was host to hitherto unrecognised viral nucleic
acid sequences which under the extraordinary conditions experienced
in the reactor were activated to express products which served to
shut down normal operation of the cell. It is believed that such
genomic prophage rather than plasmid viral infection of
monocellular microorganisms has hitherto not been identified and
may likewise prove to be problematic in high biomass operation of
aerobic loop reactor fermentors, e.g. with bacteria, fungi (e.g.
fusarium), yeast or genetically modified cells (e.g. bacterial,
mammalian (e.g. human, hamster, mouse, etc.) and it is a further
aspect of the invention to use the solutions of the invention
relating to the reduction of the dissolved oxygen depleted path
length of the reactor in aerobic loop reactor fermentations of
bacteria, fungi, yeast or genetically modified cells, e.g. in the
production of alcohols (especially methanol or ethanol), foods or
food additives or precursors therefore, pharmaceuticals,
antibodies, etc.
[0064] The rearrangement of the microorganism organelles observed
on process malfunction may also be used as a control parameter for
feedback control of fermentation reactors in general, and loop
reactors in particular, e.g. with observation of organelle
rearrangement at all, or above a threshold level, serving to
trigger increase in nutrient supply, in particular oxygen and/or
methane supply, or to reduce or halt ammonia supply, etc. Such
observation could for example be made using flow cytometry on cells
removed from an extraction or sampling port in the reactor. This
form of feedback control of fermentation reactors forms a further
aspect of the invention.
[0065] A primary indicator of malfunction in the operation of the
processes of the invention is when a depressed pH in the culture
medium occurs which recurs despite pH adjustment by base (e.g.
NaOH) addition. This appears to be due to formic acid build up or
inadequate formic acid to carbon dioxide conversion by the
methanotrophic bacteria. An advance warning of this problem could
be obtained by monitoring the intracellular formic acid
concentration of the bacteria, e.g. by sampling the culture medium,
lysing the cells and spectrometrically assaying for formic acid.
Where the detected formic acid concentration rises above a preset
threshold, preventative action may be taken, e.g. by reducing
oxygen and/or ammonia feed rates, etc. Besides intracellular formic
acid, extracellular formic acid concentration may be monitored and
used as a process control parameter. Likewise dissolved oxygen
concentration, dissolved methane concentration and dissolved
ammonia concentration may be monitored and used as a process
control parameters. Desirably such monitoring is performed on line,
i.e. at the reactor, and conventional monitoring apparatus (e.g.
spectrometers and the like) may be used. Such monitoring and
control forms a further aspect of the invention.
[0066] Viewed from a further aspect the invention provides a
fermentation reactor adapted for use in one or more processes
according to the invention, e.g. by the inclusion of appropriately
positioned nutrient gas inlets, low cavitation propellers, stacked
plate static mixers, etc.
[0067] Viewed from a yet further aspect the invention provides
biologically generated products (e.g. biomass, drugs, antibodies,
alcohols, etc.) produced by the processes of the invention and
their uses, e.g. as, in or as precursors for foodstuffs, as
pharmaceuticals, as chemical raw materials, etc.
[0068] Where a loop reactor is used for cell culturing, the removal
of dissolved carbon dioxide from the culture medium is important
and nitrogen is particularly suitable for use in this regard (i.e.
as a driving gas as mentioned above).
[0069] As a result it is especially beneficial to locate the
reactor in the vicinity of (e.g. within 1 km of) an apparatus with
a high demand for oxygen, e.g. an alcohol or ethanol production
plant, in this way the nitrogen produced in air separation (e.g.
liquefication) to produce oxygen for one plant may be used in the
fermentor apparatus, together if desired with a small proportion of
the oxygen (e.g. as a nutrient gas in the fermentor). Moreover,
where the fermentor product is to be dried and, especially where it
is oxygen sensitive on drying as is the case with biomass
production for bioproteins, the nitrogen may also be used as the
drying gas in the spray drier. Such synergistic co-locations of
nitrogen/oxygen separator, fermentor and oxygen-demanding plants
form a further aspect of the invention.
[0070] Where larger scale operation of the process of the invention
is desired, it is of course possible simply to increase the
dimensions of the reactor and the power of the drive motor. An
alternative however is to operate a set of reactors, e.g. arranged
radiating out from a central unit housing the degassing zones for
all the reactors. One advantage of using a set (e.g. 2, 3, 4 or 5)
of reactors is that reaction start-up in one reactor may be
effected by inoculation with liquid culture medium from a reactor
which is already in operation. Since the reactors generally do have
to have down time for cleaning or repair, such an arrangement
ensures that operation can be continuous and that the start-up time
for the individual reactors may be significantly reduced.
[0071] Embodiments of the processes and apparatus of the invention
will now be described further with reference to the accompanying
drawings, in which:
[0072] FIG. 1 is a schematic drawing of a loop reactor according to
the invention;
[0073] FIG. 2 is a cross section through a horizonal section of the
reactor of FIG. 1 showing schematically the arrangement of the
plates of the static mixer therein;
[0074] FIG. 3 is a schematic view of a further loop reactor
according to the invention; and
[0075] FIG. 4 is a cross section through a horizontal section of
the reactor of FIG. 1 or FIG. 3 showing schematically the
arrangement of the plates of the static mixer therein.
[0076] Referring to FIG. 1 there is a shown a loop reactor 1, the
loop whereof comprises a top unit 2 for effluent gas removal, a
vertical down-flow section 3, a horizontal flow section 4, a
vertical up-flow section 5, and a horizontal effluent gas/liquid
reaction medium separation section 6. Down-flow section 3 is
attached to the conical base of cylindrical top unit 2 while
separation section 6 enters top unit 2 tangentially at a side so as
to achieve cyclonic gas/liquid separation. From the top of top unit
2, effluent gas is removed through outlet port 7. The top of the
top unit is also provided with emergency vent 8 which opens
automatically if the effluent gas pressure in the top unit exceeds
a pre-set maximum, e.g. 0.5 atmosphere above ambient.
[0077] The liquid culture medium 9 is circulated round the loop by
propeller 10 driven by motor 11 (e.g. a 650 kW motor). Upstream of
propeller 10 is exit port 12 from which biomass is removed for
dewatering and further processing, e.g. hydrolysis, spray drying
etc.
[0078] Within the horizontal section 4 of the loop are disposed a
series of nutrient gas (e.g. O.sub.2 and CH.sub.4) inlets 13 (e.g.
diffuser plates) and static mixers 14. Nutrient gas inlets 15 and
16 are preferably also provided in the downflow and upflow sections
3 and 5. Ammonia and mineral inlets 17 and 18 and sampling ports
are preferably provided around the loop. Monitoring and sampling
ports (not shown) are also preferably provided around the loop and
in the head space of top unit 2.
[0079] Towards the top of upflow section 5 is provided a drive gas
(e.g. nitrogen) inlet 19, for example a diffuser plate.
[0080] In the base of separation section 6, there is preferably
provided a porous diffuser 20 for nutrient gas (especially oxygen)
which is elongated along the flow direction so as to supply
nutrient gas into the liquid separated out in this section.
[0081] Referring to FIG. 2, the loop reactor is shown containing a
stack of parallel horizontal corrugated perforated plates 21
disposed one above the other with the corrugations in registry but
alternating in direction so as to provide flow channels 22 for the
culture medium.
[0082] Referring to FIG. 3 there is a shown a loop reactor 1, the
loop whereof comprises a top unit 2 for effluent gas removal, a
vertical down-flow section 3, a horizontal flow section 4, a
vertical up-flow section 5, and a horizontal effluent gas/liquid
reaction medium separation section 6. Down-flow section 3 is
attached to the conical base of cylindrical top unit 2 while
separation section 6 enters top unit 2 at a side. From the top of
top unit 2, effluent gas is removed through outlet port 7. The top
of the top unit is also provided with emergency vent which opens
automatically if the effluent gas pressure in the top unit exceeds
a pre-set maximum, e.g. 0.5 atmosphere above ambient.
[0083] The liquid culture medium 9 is circulated round the loop by
a propeller driven by a vertically dispersed drive shaft driven by
motor (e.g. a 650 kW motor). Upstream of a propeller driven by a
vertically disposed drive shaft is exit port 12 from which biomass
is removed for dewatering and further processing, e.g. hydrolysis,
spray drying etc.
[0084] Within the horizontal section 4 of the loop are disposed a
series of nutrient gas (e.g. O.sub.2 and CH.sub.4) inlets 13 (e.g.
diffuser plates) and static mixers. Ammonia and mineral inlets 17
and 18 and sampling ports are preferably provided around the loop.
Monitoring and sampling ports (not shown) are also preferably
provided around the loop and in the head space of top unit 2.
[0085] Towards the top of upflow section 5 is provided a drive gas
(e.g. nitrogen) inlet 19, for example a diffuser plate.
[0086] Dissolved oxygen content is measured by probes 23 within the
reactor. Ammonia concentration and cell density are measured by
probes 24 and 25 in the harvesting outlet to the reactor, gas
composition (CO.sub.2, O.sub.2, CH.sub.4) is measured by probe 26
in the off gas venting line. Temperature and pH are measured within
the reactor by probes 27 and 28. The information from these probes
is used as a feedback control whereby a yield-optimum gas and
ammonia distribution is calculated based on a mechanistic model of
the fermentation process providing, repeatedly, optimal settings
for the O.sub.2, CH.sub.4 and NH.sub.3 introduction valves, for the
diverted flow of culture medium from the reactor into heat
exchangers and back into the reactor, and for acid/base dosing to
optimize pH.
[0087] In FIG. 4 a more preferred mixer array is shown. In this
embodiment, the parallel vertical corrugated perforated plates 21
are disposed with their corrugations angled with respect to the
flow direction and alternating in orientation.
[0088] The mixer plates are preferably flexible as in this way they
are self-cleaning. To this end they are conveniently formed from
stainless steel of at least 0.2 mm thickness, e.g. about 0.5 to 1.5
mm, especially 0.8 to 1.2 mm thickness. The maximum interplate
spacing, i.e. the channel height between corrugations, is
preferably at least 25 mm, e.g. 50 to 250 mm, more preferably 80 to
150 mm, especially 90 to 110 mm.
[0089] The following non-limiting Example serves further to
illustrate the invention.
EXAMPLE 1
Preparation of Homogenized Biomass
[0090] A microbial culture comprising Methylococcus capsulatus
(Bath) (strain NCIMB 11132), Ralstonia sp. (formerly Alcaligenes
acidovorans) DB3 (strain NCIMB 13287) and Brevibacillus agri
(formerly Bacillus firmus) DB5 (strain NCIMB 13289), and optionally
and preferably Aneurinibacillus sp. DB4 (strain NCIMB 13288) is
produced in a loop-type fermentor by continuous aerobic
fermentation of natural gas in an ammonium/mineral salts medium
(AMS) at 45EC, pH 6.5. The AMS medium contains the following per
litre: 10 mg NH.sub.3, 75 mg H.sub.3PO.sub.4, 380 mg
MgSO.sub.4.7H.sub.2O, 100 mg CaCl.sub.2.2H.sub.2O, 200 mg
K.sub.2SO.sub.4, 75 mg FeSO.sub.4.7H.sub.2O, 1.0 mg
CuSO.sub.4.5H.sub.2O, 0.96 mg ZnSO.sub.4.7H.sub.2O, 120 .mu.g
CoCl.sub.2.6H.sub.2O, 48 .mu.g MnCl.sub.2.4H.sub.2O, 36 .mu.g
H.sub.3BO.sub.3, 24 .mu.g NiCl.sub.2.6H.sub.2O and 1.20 .mu.g
NaMoO.sub.4.2H.sub.2O.
[0091] The fermentor is filled with water which has been
heat-sterilized at 125EC for 10 secs. Addition of the different
nutrients is regulated according to their consumption. With gradual
build-up over time, continuous fermentation is operated with 1-3%
biomass (on a dry weight basis).
[0092] The biomass is subjected to centrifugation in an industrial
continuous centrifuge at 3,000 rpm, optionally followed by
homogenization in an industrial homogenizer (pressure drop: 1000
bar (100 MPa); inlet temperature: 15EC to produce a homogenized
biomass), followed by ultrafiltration using membranes having an
exclusion size of 200,000 Daltons.
* * * * *