CA1288076C - Rhizobial growth enhancement - Google Patents
Rhizobial growth enhancementInfo
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- CA1288076C CA1288076C CA000531064A CA531064A CA1288076C CA 1288076 C CA1288076 C CA 1288076C CA 000531064 A CA000531064 A CA 000531064A CA 531064 A CA531064 A CA 531064A CA 1288076 C CA1288076 C CA 1288076C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
ABSTRACT
The growth characteristics of rhizobial bacteria, and the secretion products of fermentation thereof such as exopolysaccharides are controlled and enhanced by adjustments to the dissolved oxygen concentration in the fermentation broth, the concentration of sugar (e.g. sucrose) in the fermentation broth, the sugar:yeast extract ratio and the pH of the broth, either individually or in combinations of two or more of such features.
The growth characteristics of rhizobial bacteria, and the secretion products of fermentation thereof such as exopolysaccharides are controlled and enhanced by adjustments to the dissolved oxygen concentration in the fermentation broth, the concentration of sugar (e.g. sucrose) in the fermentation broth, the sugar:yeast extract ratio and the pH of the broth, either individually or in combinations of two or more of such features.
Description
~ 1288~6 This inven~ion relates to fermentation processes and proàucts, and more particularly to the fermentation of rhizobial microorganisms .
Rhizobial microorganisms (rhizobia) are soil bacteria whicn play an important role in the nodulation and nitrogen fixation the roots of growing plants. Specific rhizobia have a symbiotic relationship with specific leguminous plants.
They are grown anà used commercially as soil inoculants to create a favourable ~rhizosphere" in the soil in the vicinity of a leguminous plant root, to promote growth of the plant.
~ hizobia are grown in large batch fermentations for the production of commercial inoculants for use on leguminous plants. There is, therefore, an incentive to maximize the rate of growth and production of rhizobia. The cells produced during the fermentation are often mixed directly with a carrier such as neutrallzed peat before packaging. There are times, however, when it might be aesirable to harvest the cells from the fermentation broth for storage before processing for inoculant proauction. Harvesting can be complicated because many rhizobia proauce large amounts of soluble extracellular polysaccharide (~PS) which increases the viscosity of the medium and interferes with large-scale centrifugation or filtration. Increased viscosity of the medium can also limit efficient production of cell biomass duriny the fermentation. In addition to soluble EPS, variable amounts of insoluble capsular polysaccharides are also produce~ that are tightly associated with the cell.
The extracellular polysaccharides produced as a by-product of rhizobia fermentation do, however, have certain interesting properties and utilities. For example, they can be usea in the preparation of seed dressing compositions whereby rhizobia are adhered to seeds of leguminous plants so that the rhizobia are maintained in the vicinity of the roots of the plant growing from such seeds, to enhance the rhizosphere.
,;~ .
Rhizobial microorganisms (rhizobia) are soil bacteria whicn play an important role in the nodulation and nitrogen fixation the roots of growing plants. Specific rhizobia have a symbiotic relationship with specific leguminous plants.
They are grown anà used commercially as soil inoculants to create a favourable ~rhizosphere" in the soil in the vicinity of a leguminous plant root, to promote growth of the plant.
~ hizobia are grown in large batch fermentations for the production of commercial inoculants for use on leguminous plants. There is, therefore, an incentive to maximize the rate of growth and production of rhizobia. The cells produced during the fermentation are often mixed directly with a carrier such as neutrallzed peat before packaging. There are times, however, when it might be aesirable to harvest the cells from the fermentation broth for storage before processing for inoculant proauction. Harvesting can be complicated because many rhizobia proauce large amounts of soluble extracellular polysaccharide (~PS) which increases the viscosity of the medium and interferes with large-scale centrifugation or filtration. Increased viscosity of the medium can also limit efficient production of cell biomass duriny the fermentation. In addition to soluble EPS, variable amounts of insoluble capsular polysaccharides are also produce~ that are tightly associated with the cell.
The extracellular polysaccharides produced as a by-product of rhizobia fermentation do, however, have certain interesting properties and utilities. For example, they can be usea in the preparation of seed dressing compositions whereby rhizobia are adhered to seeds of leguminous plants so that the rhizobia are maintained in the vicinity of the roots of the plant growing from such seeds, to enhance the rhizosphere.
,;~ .
- 2 - ~
Accoràingly, since EPS produc~io8~1~n ~ rmentation of rhizobia has both disadvantages and advantages, it is desirable to be able to control the extent of EPS production, so as to be able to minimize its formation or maximize its formation, as desired in specific circumstances.
~ t is, therefore, an object o. the present invention to provide a rhizobia fermentation process, whereby the extent of proauction or formation of EPS may be controlled.
The present invention is based upon the discovery that certain features of the composition and nature of the fermentation media in which rhizobia are grown and multiplied have a drastic effect on the production of such EPS, and moreover that these features are controllable at will, to adjust the extent of EPS production.
Accoràing to a first aspect of the present invention, the dissolved oxygen concentration in the fermentation broth has been founa to exert a significant influence over the EPS
production and the rate of growth of the rhizobial cells. More specifically, if dissolved oxygen in the broth is maintained between about 4 and 20~ of saturation cell growth is exponential. At oxygen transfer rates less than the minimum required for this exponential growth, the dissolved oxygen falls to below 1%. Whilst this has the effect that growth becomes linear, and in direct relation to the oxygen transfer rate, it also has the effect of increasing the EPS production and ~ecreasing the biomass production under oxygen-limited con~itions.
According to a second aspect of the present invention, the concentration of sugar (sucrose, glucose, fructose, maltose or the like) in the fermentation medium affects the EPS
production an~ the cell biomass production, whilst, within a relatively wiae range, it does not significantly affect cell ` - 3 -~ ~288~
growth rate. More specifically, at sugar concentrations in the medium of from about one gram/litre to near saturation, and especially from about one gram/litre up to about 30-50 g/L, rhizobial cell growth rate remains substantially constant, whilst EPS production is maximized at sugar concentrations from about 5.0 to about 30-50 g/L. Meanwhile, cell biomass production remains relatively constant over a range of sugar concentrations in the fermentation medium of from about 0.75 g/L
to about 30-50 g/L.
According to a third aspect of the present invention, yeast extract present in the fermentation medium is required for good cell growth, and there is an optimum range of sugar: yeast extract in the medium for production of cell biomass with respect to EPS production.
A fourth aspect of the present invention pertains to pH control. It has been found that excess acidity in the fermentation medium causes increased production of EPS. Cell biomass production, EPS production and specific cell growth rate all have optima at pH 6.5. Whilst complete enhancement of the production of either biomass or EPS at the expense of the other does not appear to be practical at the expense of the other by varying pH, nevertheless EPS production compared to the amount of biomass yield is high between pH 5.5 and 6Ø
Thus the main constituents of one of the standard fermentation media used to grow rhizobia for inoculants can be manipulated for more efficient and cost effective production.
l`he use of simple process controls such as dissolved oxygen and pH control, combined with judicious medium selection, can maximize cell yields and growth rates and minimize EPS
production, all key factors in industrial production of these organisms.
~21~80~
One of the conditions that promotes exopolysaccharide production in media containing excess carbon sources, therefore, is low DO levels and growth-limiting oxygen transfer rates.
These conditions are found in batch culture and inadequately aerated fermentation production systems. It is known that viable cell numbers of R. trifolii are affected by aeration rate. Simple DO control systems applied to commercial Rhizobium fermentation performed in economically advantageous media can reduce exopolymer production and increase apparent specific growth rates and biomass yields by maintaining DO above approximately 1-4% saturation.
In the accompanying drawings:
FIGURE 1 is a diagrammatic illustration of the apparatus used to conduct the experiments described in Examples 1 and 2 hereof;
FIGURES 2, 3 and 4 are graphical presentations of the results obtained from the experiments described in Example 1 hereof;
FIGURES 5-10 are graphical presentations of the results obtained from the experiments described in Example 2 hereof. Specifically:
Figure 5 shows the effect of increasing sucrose concentrations on apparent specific growth rates (~), exopolysaccharides (~) and biomass production (o) during fermentations of _ trifolii 0403;
Figure 6 shows exopolysaccharide (a) and biomass (o) yields per unit of sucrose from fermentations of R. trifolii 0403;
lZ88076 Figure 7 shows the effect of increasing yeast extract concentrations on apparent specific growth rates ( ), expolysaccharide ( ) and biomass (o) production during fermentations of R. trifolii 0403;
Figure 8 shows exopolysaccharide ( ) and biomass ~o) yields per unit of yeast extract from fermentations of R.
trifolii 0403;
Figure 9 shows the pH during a fermentation of R.
trifolii 0403 in medium containing 10 g/L sucrose and 1 g/L
yeast extract.
Figure 10 shows the effect of pH control on apparent specific growth rates ( ), exopolysaccharide ( ) and biomass (o) production during fermentations of R. trifolii 0403.
The invention is further illustrated in the following specific, non-limiting examples.
Ihis example illustrates the effects of dissolved oxygen on the production of soluble exopolysaccharides, biomass and specific growth rate o-f Rhizobium trifolii 0403 grown in a medium approximating a commonly used commercial medium.
The organism used was R. trifolii 0403. This organism was selected because of its capability to produce large amounts of soluble exopolysaccharide and small amounts ( lOg of cell biomass) of insoluble capsular polysaccharide. The medium used in this study had the following composition (g/l): yeast extract, 1.0; NaCl, 0.2; MgSO4.7H2O, 0.2; K2HPO4, 0.5;
FeC13.6H2O, 6.7xlO 3; and unless specified otherwise, sucrose, 10Ø The pH of the medium before autoclaving was 7.4.
1288~7fi Shake-flask studies were performed to determine the sucrose concentration that promoted the highest soluble exopolysaccharide formation. Batch cultures containing 0.0, 1.0, 2.5, 5.0, 7.5, and 10.0 9/1 sucrose were grown at 30C with shaking at 150 rpm until early stationary phase was reached.
Growth was followed by measuring the optical ~ensity at 620 nm.
The cells were separated from the medium by centrifugation at 12,000xg for 20 min. at 4C. The cell pellets were washed once in distilled water, dried at 70C, and weighed to determine biomass production ~9/1). The supernatants from the cell pellets were made to 70% (v/v) ethanol and cooled to 4C to induce soluble exopolysaccharide precipitation. The precipitate was separated from the supernatant by filtration, dried to a constant weight and weighed.
Fermentations were performed in the apparatus shown in Figure 1. A Multigen fermentor (New Brunswick Corp.) with a 2-1 vessel containing 1.6 1 of medium with 10 g/l sucrose was used.
The temperature was controlled at 30C. Agitation was provided with two 4-bladed flat impellers rotating at 400 rpm. The pH
(A, Fig. 1), redox potential (B, Fig. 1), and DO (C, Fig. 1), were monitored continuously with Chemcadet pH controllers (Cole Parmer) with data output to a Vax 11-730 computer (Digital Corp.). Data points were stored every 10 seconds. The reference electrode in the pH probe served as the reference for the redox electrode. The redox electrode was set to 0 mV when the system was saturated with 100% DO. The DO was measured with an oxygen electrode [Instrumentation Laboratories].
Two oxygen control systems were utilizea. Constant DO
was maintained by direct feedback control through a Chemcadet pH
controller. The on-off action of the controller provided 0.4 1 air/min through a surgeless air delivery system (1, Fig. 1).
The DO setpoint was varied between 4 and 20% using this system.
Constant air input rates insufficient to support exponential growth were supplied by flushing the heaaspace of the fermentor * Trademark with variable rates of air from source 2. Oxygen transfer rates from the headspace were determined by flushing the culture medium with oxygen-free nitrogen, then measuring the rate of reoxygenation at different air input rates.
Growth in the fermentor was followed by taking periodic samples and measuring their OD620 by use of a standard curve of OD620 vs dry weight constructed for this organism. Fermentations where DO was controlled and the cells grew exponentially were terminated when oxygen consumption began to decline. Under conditions of low oxygen transfer rates where the cells did not grow exponentially, the fermentations were terminated when biomass stopped increasing. Total soluble exopolysaccharide was determined from samples in which biomass accumulation had ceased.
Results The production of rhizobia for commercial legume inoculants is performed in media often selected on the basis of cost and availability rather than for optimum production. Thus it was chosen to examine the effects of dissolved oxygen and oxygen transfer rates on the production of soluble exopolysaccharide under medium conditions that favour maximum exopolysaccharide production over biomass production.
The initial concentration of sucrose in the medium was found to affect both biomass and exopolysaccharide yields in batch culture (Fig. 2). Maximum biomass yields were found at 7.5 g/l sucrose. Below this concentration, sucrose limited both biomass and exopolysaccharide production. At levels of sucrose above 7.5 g/l, biomass production declined and exopolysaccharide production increased slightly. Therefore, we used 10.0 g/l sucrose in fermentations as a representative medium that favours biopolymer production at the expense of biomass production.
1~88~6 In fermentations where DO was controlled between 4 to 20%, exponential growth occurred. Over this DO range, the apparent specific growth rate, ~, and biomass yields did not change significantly (Table 1). However, exopolysaccharide production increased by 19% as the DO was decreased from 20 to 4%. A continuous output of DO, redox potential and pH is shown in Fig. 3 for a fermentation controlled at a DO of 10%. The DO
fluctuated by approximately +1.5% of the set point in this and all the other fermentations. Attempting to control the DO at less than 4% resulted in fluctuations of up to 10%.
Fermentations performed at oxygen transfer rates less than those capable of supporting exponential growth exhibited markedly different growth characteristics from fermentations where growth was not oxygen-limited (Table 2). Under these conditions growth was linear and in direct proportion to oxygen transfer rates in contrast to the exponential growth found in fermentations not limited by oxygen transfer rates (Fig. 4).
Biomass production declined during linear growth with decreasing oxygen transfer rates while exopolysaccharide production increased.
128~3~76 Tablc t. The cffect o~ di~ol~d a~rg~ concentra-t~ (DO) 011 ~pp~t paific gro~ te, bi~ u~d opol~ccharidc produ~on.
Ava~c Bi~ Exopol~accharidc ~
,, ~) ~/l) ~owth r~tc 20 . 1. 18 0. aB9 0. 165 10 1. IS 0.09B 0. 165 1. IQ 0. 106 0, 16 T~blc 2. The cffect ol' ~en tr~fcr r~tc~ not ~
p-blc ol wpport~ng c~ponc~ti l ~nh ~t DO
Ic~ bcbw 1% on lu~c~r ~ ratc, biom~, and c~opol~cch-ridc produc~
Line r t~gCrn~t; Biomau E~opol~cch~
9.S9 1.0~ 0.2S2 o.on 6. ~1 0. 91 0. ~01 0. 018 O.BO 0.~67 O.011 ~ .~8~
EXAMP~E 2 This example reports the effects of constant pH, and variations in the concentrations of yeast extract or sucrose on the production of soluble EPS, biomass, anà specific growth rates of R. trifolii 0403 grown in a medium approximating a commonly used commercial medium. The bacterial strain, culture medium and conditions were as described in Example 1.
Fermentations were performed in a 2L New Brunswick Multigen*SCTR. Oxygen was continually supplied at levels sufficient to maintain exponential growth of this organism, given no other limitations in nutrients. For experiments when either yeast extract or sucrose levels were varied, the pH of the medium was not controlled during the fermentation. During the pH control experiments, a Chemcaàet pH-MV controller (Cole-Parmer) was used to feed 2 N NaOH or HCl to the medium as needed. ~
Growth of the cells was monitored by taking periodic sample~ and measuring their OD620. At the end of 48 h, the cells had entered the stationary phase of growth and the fermentations were terminated and sampled to determine cell biomass and EPS production. Samples of 100 ml were centrifuged at 12,000 xg for 20 minutes at 4C to pellet the cells. The cell pellets were washed once in distilled water, dried at 70C, and weighed to aetermine biomass production (-g/L). The supernatents from the cell pellets were made to 70% (v/v) with ethanol an~ cooled overnight at 4C to precipitate soluble EPS. The precipitated EPS was collected by centrifugation, dried and weighe~ to determine EPS production (g/L).
Changing the sucrose concentration in the medium (Fig.
5) affected the apparent specific growth rate (u), EPS and cell biomass production. With no sucrose, some growth occurred at a slow rate indicating the R. trifolii 0403 can utilize yeast * Trade-marks 128~
extract as a sole carbon and nitrogen source as has been reported for other Rhizoblum sp. As sucrose was added, the growth rate rose immediately and then remained constant. The cell biomass obtained at the end of the fermentation increased up to a sucrose concentration of approximately 5 g/L and then declined slightly. Cell biomass and EPS production were linked unt:il the sucrose concentration reached 10 g/L and then excess EPS was produced.
The yields of EPS and cell biomass per unit of sucrose both declined as sucrose concentrations were increased up to 5-10 g/L (Fig. 6). This is a further indication that R.
trifolii 0403 is using other carbon compounds present in the yeast extract for growth and is not using the sucrose efficiently. There are indications that only approximately 3 g/L of sucrose are used during the fermentation when excess yeast extract is present.
Changing the yeast extract concentration in the medium (Fig. 7) affected the apparent specific grwoth rate (u), EPS and cell biomass production. The addition of yeast extract to the medium was necessary as growth was limited without it. The apparent specific growth rate was constant up to 2 g/L yeast extract and then began to decline. However, the maximum cell biomass yields at the end of the fermentation were found when the yeast extract concentration was 3 g/L. Above 3 g/L of yeast extract, the biomass yield declined. High yeast extract levels can inhibit growth and distort cells of R. trifolii and this can be overcome by addition of calcium to the medium. There were two peaks of EPS production as the yeast extract levels were increased. One peak occurred at yeast extract concentrations below 1 g/L, when the sucrose in the medium was in a large excess and cell growth was limited. Above 1 g/L of yeast extract, EPS and biomass production were linked with maximum EPS
production also beins reached at 3 g/L of yeast extract.
These results ~ ~a~e7 ~hat there is an optimum ratio for sucrose to yeast extract in this medium for production of cell biomass with respect to EPS. AT high sucrose levels excess EPS is produced. More cell biomass production can be achieved by increasing the yeast extract concentration but only up to a certain point, after which cell yields (g cell produced/g yeast extract) are adversely affected (Fig. 8).
The standard medium containing 10 g/L sucrose and 1 g/L yeast extract was used to investigate the effect of controlling the pH throughout the fermentation. It had been found that the pH of this medium remained near pH 7.0 during the first 24 hours but then slowly became acidic (E~ig. 9), probably due to the production of the EPS which contains glucuronic and pyruvic acids.
Figure lO shows that during pH controlled fermentations, EPS and cell biomass production, and the specific growth rate (p) all had optima at pH 6.5. It was not possible to completely enhance the production of either biomass or EPS at the expense of the other by varying pH. However, EPS production was high between pH 5.5 to 6.0 compared to the amount of biomass yield. During an uncontrolled fermentation, if the pH of the medium became too acidic, EPS production could become problematic. Controlling the pH at 6.5 also increased the cell biomass and EPS yield compared to that of an uncontrolled fermentation using the standard medium (Fig. 5).
. . .
Accoràingly, since EPS produc~io8~1~n ~ rmentation of rhizobia has both disadvantages and advantages, it is desirable to be able to control the extent of EPS production, so as to be able to minimize its formation or maximize its formation, as desired in specific circumstances.
~ t is, therefore, an object o. the present invention to provide a rhizobia fermentation process, whereby the extent of proauction or formation of EPS may be controlled.
The present invention is based upon the discovery that certain features of the composition and nature of the fermentation media in which rhizobia are grown and multiplied have a drastic effect on the production of such EPS, and moreover that these features are controllable at will, to adjust the extent of EPS production.
Accoràing to a first aspect of the present invention, the dissolved oxygen concentration in the fermentation broth has been founa to exert a significant influence over the EPS
production and the rate of growth of the rhizobial cells. More specifically, if dissolved oxygen in the broth is maintained between about 4 and 20~ of saturation cell growth is exponential. At oxygen transfer rates less than the minimum required for this exponential growth, the dissolved oxygen falls to below 1%. Whilst this has the effect that growth becomes linear, and in direct relation to the oxygen transfer rate, it also has the effect of increasing the EPS production and ~ecreasing the biomass production under oxygen-limited con~itions.
According to a second aspect of the present invention, the concentration of sugar (sucrose, glucose, fructose, maltose or the like) in the fermentation medium affects the EPS
production an~ the cell biomass production, whilst, within a relatively wiae range, it does not significantly affect cell ` - 3 -~ ~288~
growth rate. More specifically, at sugar concentrations in the medium of from about one gram/litre to near saturation, and especially from about one gram/litre up to about 30-50 g/L, rhizobial cell growth rate remains substantially constant, whilst EPS production is maximized at sugar concentrations from about 5.0 to about 30-50 g/L. Meanwhile, cell biomass production remains relatively constant over a range of sugar concentrations in the fermentation medium of from about 0.75 g/L
to about 30-50 g/L.
According to a third aspect of the present invention, yeast extract present in the fermentation medium is required for good cell growth, and there is an optimum range of sugar: yeast extract in the medium for production of cell biomass with respect to EPS production.
A fourth aspect of the present invention pertains to pH control. It has been found that excess acidity in the fermentation medium causes increased production of EPS. Cell biomass production, EPS production and specific cell growth rate all have optima at pH 6.5. Whilst complete enhancement of the production of either biomass or EPS at the expense of the other does not appear to be practical at the expense of the other by varying pH, nevertheless EPS production compared to the amount of biomass yield is high between pH 5.5 and 6Ø
Thus the main constituents of one of the standard fermentation media used to grow rhizobia for inoculants can be manipulated for more efficient and cost effective production.
l`he use of simple process controls such as dissolved oxygen and pH control, combined with judicious medium selection, can maximize cell yields and growth rates and minimize EPS
production, all key factors in industrial production of these organisms.
~21~80~
One of the conditions that promotes exopolysaccharide production in media containing excess carbon sources, therefore, is low DO levels and growth-limiting oxygen transfer rates.
These conditions are found in batch culture and inadequately aerated fermentation production systems. It is known that viable cell numbers of R. trifolii are affected by aeration rate. Simple DO control systems applied to commercial Rhizobium fermentation performed in economically advantageous media can reduce exopolymer production and increase apparent specific growth rates and biomass yields by maintaining DO above approximately 1-4% saturation.
In the accompanying drawings:
FIGURE 1 is a diagrammatic illustration of the apparatus used to conduct the experiments described in Examples 1 and 2 hereof;
FIGURES 2, 3 and 4 are graphical presentations of the results obtained from the experiments described in Example 1 hereof;
FIGURES 5-10 are graphical presentations of the results obtained from the experiments described in Example 2 hereof. Specifically:
Figure 5 shows the effect of increasing sucrose concentrations on apparent specific growth rates (~), exopolysaccharides (~) and biomass production (o) during fermentations of _ trifolii 0403;
Figure 6 shows exopolysaccharide (a) and biomass (o) yields per unit of sucrose from fermentations of R. trifolii 0403;
lZ88076 Figure 7 shows the effect of increasing yeast extract concentrations on apparent specific growth rates ( ), expolysaccharide ( ) and biomass (o) production during fermentations of R. trifolii 0403;
Figure 8 shows exopolysaccharide ( ) and biomass ~o) yields per unit of yeast extract from fermentations of R.
trifolii 0403;
Figure 9 shows the pH during a fermentation of R.
trifolii 0403 in medium containing 10 g/L sucrose and 1 g/L
yeast extract.
Figure 10 shows the effect of pH control on apparent specific growth rates ( ), exopolysaccharide ( ) and biomass (o) production during fermentations of R. trifolii 0403.
The invention is further illustrated in the following specific, non-limiting examples.
Ihis example illustrates the effects of dissolved oxygen on the production of soluble exopolysaccharides, biomass and specific growth rate o-f Rhizobium trifolii 0403 grown in a medium approximating a commonly used commercial medium.
The organism used was R. trifolii 0403. This organism was selected because of its capability to produce large amounts of soluble exopolysaccharide and small amounts ( lOg of cell biomass) of insoluble capsular polysaccharide. The medium used in this study had the following composition (g/l): yeast extract, 1.0; NaCl, 0.2; MgSO4.7H2O, 0.2; K2HPO4, 0.5;
FeC13.6H2O, 6.7xlO 3; and unless specified otherwise, sucrose, 10Ø The pH of the medium before autoclaving was 7.4.
1288~7fi Shake-flask studies were performed to determine the sucrose concentration that promoted the highest soluble exopolysaccharide formation. Batch cultures containing 0.0, 1.0, 2.5, 5.0, 7.5, and 10.0 9/1 sucrose were grown at 30C with shaking at 150 rpm until early stationary phase was reached.
Growth was followed by measuring the optical ~ensity at 620 nm.
The cells were separated from the medium by centrifugation at 12,000xg for 20 min. at 4C. The cell pellets were washed once in distilled water, dried at 70C, and weighed to determine biomass production ~9/1). The supernatants from the cell pellets were made to 70% (v/v) ethanol and cooled to 4C to induce soluble exopolysaccharide precipitation. The precipitate was separated from the supernatant by filtration, dried to a constant weight and weighed.
Fermentations were performed in the apparatus shown in Figure 1. A Multigen fermentor (New Brunswick Corp.) with a 2-1 vessel containing 1.6 1 of medium with 10 g/l sucrose was used.
The temperature was controlled at 30C. Agitation was provided with two 4-bladed flat impellers rotating at 400 rpm. The pH
(A, Fig. 1), redox potential (B, Fig. 1), and DO (C, Fig. 1), were monitored continuously with Chemcadet pH controllers (Cole Parmer) with data output to a Vax 11-730 computer (Digital Corp.). Data points were stored every 10 seconds. The reference electrode in the pH probe served as the reference for the redox electrode. The redox electrode was set to 0 mV when the system was saturated with 100% DO. The DO was measured with an oxygen electrode [Instrumentation Laboratories].
Two oxygen control systems were utilizea. Constant DO
was maintained by direct feedback control through a Chemcadet pH
controller. The on-off action of the controller provided 0.4 1 air/min through a surgeless air delivery system (1, Fig. 1).
The DO setpoint was varied between 4 and 20% using this system.
Constant air input rates insufficient to support exponential growth were supplied by flushing the heaaspace of the fermentor * Trademark with variable rates of air from source 2. Oxygen transfer rates from the headspace were determined by flushing the culture medium with oxygen-free nitrogen, then measuring the rate of reoxygenation at different air input rates.
Growth in the fermentor was followed by taking periodic samples and measuring their OD620 by use of a standard curve of OD620 vs dry weight constructed for this organism. Fermentations where DO was controlled and the cells grew exponentially were terminated when oxygen consumption began to decline. Under conditions of low oxygen transfer rates where the cells did not grow exponentially, the fermentations were terminated when biomass stopped increasing. Total soluble exopolysaccharide was determined from samples in which biomass accumulation had ceased.
Results The production of rhizobia for commercial legume inoculants is performed in media often selected on the basis of cost and availability rather than for optimum production. Thus it was chosen to examine the effects of dissolved oxygen and oxygen transfer rates on the production of soluble exopolysaccharide under medium conditions that favour maximum exopolysaccharide production over biomass production.
The initial concentration of sucrose in the medium was found to affect both biomass and exopolysaccharide yields in batch culture (Fig. 2). Maximum biomass yields were found at 7.5 g/l sucrose. Below this concentration, sucrose limited both biomass and exopolysaccharide production. At levels of sucrose above 7.5 g/l, biomass production declined and exopolysaccharide production increased slightly. Therefore, we used 10.0 g/l sucrose in fermentations as a representative medium that favours biopolymer production at the expense of biomass production.
1~88~6 In fermentations where DO was controlled between 4 to 20%, exponential growth occurred. Over this DO range, the apparent specific growth rate, ~, and biomass yields did not change significantly (Table 1). However, exopolysaccharide production increased by 19% as the DO was decreased from 20 to 4%. A continuous output of DO, redox potential and pH is shown in Fig. 3 for a fermentation controlled at a DO of 10%. The DO
fluctuated by approximately +1.5% of the set point in this and all the other fermentations. Attempting to control the DO at less than 4% resulted in fluctuations of up to 10%.
Fermentations performed at oxygen transfer rates less than those capable of supporting exponential growth exhibited markedly different growth characteristics from fermentations where growth was not oxygen-limited (Table 2). Under these conditions growth was linear and in direct proportion to oxygen transfer rates in contrast to the exponential growth found in fermentations not limited by oxygen transfer rates (Fig. 4).
Biomass production declined during linear growth with decreasing oxygen transfer rates while exopolysaccharide production increased.
128~3~76 Tablc t. The cffect o~ di~ol~d a~rg~ concentra-t~ (DO) 011 ~pp~t paific gro~ te, bi~ u~d opol~ccharidc produ~on.
Ava~c Bi~ Exopol~accharidc ~
,, ~) ~/l) ~owth r~tc 20 . 1. 18 0. aB9 0. 165 10 1. IS 0.09B 0. 165 1. IQ 0. 106 0, 16 T~blc 2. The cffect ol' ~en tr~fcr r~tc~ not ~
p-blc ol wpport~ng c~ponc~ti l ~nh ~t DO
Ic~ bcbw 1% on lu~c~r ~ ratc, biom~, and c~opol~cch-ridc produc~
Line r t~gCrn~t; Biomau E~opol~cch~
9.S9 1.0~ 0.2S2 o.on 6. ~1 0. 91 0. ~01 0. 018 O.BO 0.~67 O.011 ~ .~8~
EXAMP~E 2 This example reports the effects of constant pH, and variations in the concentrations of yeast extract or sucrose on the production of soluble EPS, biomass, anà specific growth rates of R. trifolii 0403 grown in a medium approximating a commonly used commercial medium. The bacterial strain, culture medium and conditions were as described in Example 1.
Fermentations were performed in a 2L New Brunswick Multigen*SCTR. Oxygen was continually supplied at levels sufficient to maintain exponential growth of this organism, given no other limitations in nutrients. For experiments when either yeast extract or sucrose levels were varied, the pH of the medium was not controlled during the fermentation. During the pH control experiments, a Chemcaàet pH-MV controller (Cole-Parmer) was used to feed 2 N NaOH or HCl to the medium as needed. ~
Growth of the cells was monitored by taking periodic sample~ and measuring their OD620. At the end of 48 h, the cells had entered the stationary phase of growth and the fermentations were terminated and sampled to determine cell biomass and EPS production. Samples of 100 ml were centrifuged at 12,000 xg for 20 minutes at 4C to pellet the cells. The cell pellets were washed once in distilled water, dried at 70C, and weighed to aetermine biomass production (-g/L). The supernatents from the cell pellets were made to 70% (v/v) with ethanol an~ cooled overnight at 4C to precipitate soluble EPS. The precipitated EPS was collected by centrifugation, dried and weighe~ to determine EPS production (g/L).
Changing the sucrose concentration in the medium (Fig.
5) affected the apparent specific growth rate (u), EPS and cell biomass production. With no sucrose, some growth occurred at a slow rate indicating the R. trifolii 0403 can utilize yeast * Trade-marks 128~
extract as a sole carbon and nitrogen source as has been reported for other Rhizoblum sp. As sucrose was added, the growth rate rose immediately and then remained constant. The cell biomass obtained at the end of the fermentation increased up to a sucrose concentration of approximately 5 g/L and then declined slightly. Cell biomass and EPS production were linked unt:il the sucrose concentration reached 10 g/L and then excess EPS was produced.
The yields of EPS and cell biomass per unit of sucrose both declined as sucrose concentrations were increased up to 5-10 g/L (Fig. 6). This is a further indication that R.
trifolii 0403 is using other carbon compounds present in the yeast extract for growth and is not using the sucrose efficiently. There are indications that only approximately 3 g/L of sucrose are used during the fermentation when excess yeast extract is present.
Changing the yeast extract concentration in the medium (Fig. 7) affected the apparent specific grwoth rate (u), EPS and cell biomass production. The addition of yeast extract to the medium was necessary as growth was limited without it. The apparent specific growth rate was constant up to 2 g/L yeast extract and then began to decline. However, the maximum cell biomass yields at the end of the fermentation were found when the yeast extract concentration was 3 g/L. Above 3 g/L of yeast extract, the biomass yield declined. High yeast extract levels can inhibit growth and distort cells of R. trifolii and this can be overcome by addition of calcium to the medium. There were two peaks of EPS production as the yeast extract levels were increased. One peak occurred at yeast extract concentrations below 1 g/L, when the sucrose in the medium was in a large excess and cell growth was limited. Above 1 g/L of yeast extract, EPS and biomass production were linked with maximum EPS
production also beins reached at 3 g/L of yeast extract.
These results ~ ~a~e7 ~hat there is an optimum ratio for sucrose to yeast extract in this medium for production of cell biomass with respect to EPS. AT high sucrose levels excess EPS is produced. More cell biomass production can be achieved by increasing the yeast extract concentration but only up to a certain point, after which cell yields (g cell produced/g yeast extract) are adversely affected (Fig. 8).
The standard medium containing 10 g/L sucrose and 1 g/L yeast extract was used to investigate the effect of controlling the pH throughout the fermentation. It had been found that the pH of this medium remained near pH 7.0 during the first 24 hours but then slowly became acidic (E~ig. 9), probably due to the production of the EPS which contains glucuronic and pyruvic acids.
Figure lO shows that during pH controlled fermentations, EPS and cell biomass production, and the specific growth rate (p) all had optima at pH 6.5. It was not possible to completely enhance the production of either biomass or EPS at the expense of the other by varying pH. However, EPS production was high between pH 5.5 to 6.0 compared to the amount of biomass yield. During an uncontrolled fermentation, if the pH of the medium became too acidic, EPS production could become problematic. Controlling the pH at 6.5 also increased the cell biomass and EPS yield compared to that of an uncontrolled fermentation using the standard medium (Fig. 5).
. . .
Claims (21)
1. A process for maximizing the production of exopolysaccharide during a fermentation process which comprises:
(a) providing a fermentation medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is from about 5 g/L to about 50 g/L; and (c) growing the rhizobial microorganisms in said medium, said sugar concentration being effective to maximize exopolysaccharide production.
(a) providing a fermentation medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is from about 5 g/L to about 50 g/L; and (c) growing the rhizobial microorganisms in said medium, said sugar concentration being effective to maximize exopolysaccharide production.
2. A process for minimizing the production of exopolysaccharide during a fermentation process which comprises:
(a) providing a fermentation medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is less than 5 g/L; and (c) growing the rhizobial microorganisms in said medium, said sugar concentration being effective to minimize exopolysaccharide production.
(a) providing a fermentation medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is less than 5 g/L; and (c) growing the rhizobial microorganisms in said medium, said sugar concentration being effective to minimize exopolysaccharide production.
3. The process of claim 1 wherein the fermentation medium also contains less than 4% of saturation value of dissolved oxygen content.
4. The process of claim 3 wherein the pH of the fermentation medium is in the range of 5.5 to 6.5.
5. The process of claim 4 wherein the fermentation medium also contains from about 1 g/L to about 3 g/L of yeast extract.
6. The process of claim 5 wherein the initial sugar concentration in the fermentation medium is from about 5 g/L to about 30 g/L and the dissolved oxygen content is from about 1% -4% of saturation.
7. The process of any one of claims 1 or 2 wherein the sugar is sucrose.
8. The process of any one of claims 1 or 2 wherein the rhizobial microorganism is Rhizobium trifolii.
9. A process for enhancing the cell growth rate of rhizobial microorganisms during a fermentation process, said process yielding a significant amount of exopolysaccharide, which comprises:
(a) providing a fermentatin medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is from about 1 g/L to saturation and the dissolved oxygen concentration is from about 4% - 20% of saturation; and (c) growing rhizobial microorganisms in said medium.
(a) providing a fermentatin medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is from about 1 g/L to saturation and the dissolved oxygen concentration is from about 4% - 20% of saturation; and (c) growing rhizobial microorganisms in said medium.
10. The process of claim 9 wherein the initial sugar concentration is from about 1 g/L to about 30 g/L.
11. The process of claim 10 wherein the initial sugar concentration is from about 1 g/L to about 5 g/L.
12. The process of claim 11 wherein the pH of the fermentation medium is about 6.0 to 6.5.
13. The process of claim 12 wherein the fermentation medium additionally contains yeast extract in amounts up to about 2 g/L.
14. The process of claim 13 wherein the sugar is sucrose.
15. The process of claim 14 wherein the rhizobial microorganism is Rhizobium trifolii.
16. A process for increasing the biomass production of rhizobial microorganisms during a fermentation process, said process yielding a significant amount of exopolysaccharide, which comprises:
(a) providing a fermentation medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is from about 0.75 g/L to about 30 g/L and the dissolved oxygen concentration is at least 1% of saturation; and (c) growing rhizobial microorganisms is said medium.
(a) providing a fermentation medium suitable to support the growth of rhizobial microorganisms;
(b) controlling the medium so that the initial sugar concentration is from about 0.75 g/L to about 30 g/L and the dissolved oxygen concentration is at least 1% of saturation; and (c) growing rhizobial microorganisms is said medium.
17. The process of claim 16 wherein the pH of the fermentation medium is about 6.5.
18. The proces of claim 17 wherein the fermentation medium also contains from about 1 g/L to 3 g/L of yeast extract.
19. The process of claim 18 wherein the initial sugar concentration is from 0.75 g/L to about 10 g/L.
20. The process of claim 19 wherein the sugar is sucrose.
21. The process of claim 20 wherein the rhizobial microorganism is Rhizobium trifolii.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000531064A CA1288076C (en) | 1987-03-03 | 1987-03-03 | Rhizobial growth enhancement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000531064A CA1288076C (en) | 1987-03-03 | 1987-03-03 | Rhizobial growth enhancement |
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| Publication Number | Publication Date |
|---|---|
| CA1288076C true CA1288076C (en) | 1991-08-27 |
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ID=4135087
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000531064A Expired - Fee Related CA1288076C (en) | 1987-03-03 | 1987-03-03 | Rhizobial growth enhancement |
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| Country | Link |
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| CA (1) | CA1288076C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100390295C (en) * | 2005-11-26 | 2008-05-28 | 山西大学 | Microorganism polysaccharide and its preparation method and application |
| EP3702440A4 (en) * | 2017-10-27 | 2020-12-02 | Kumiai Chemical Industry Co., Ltd. | Culturing method for genus rhizobium bacteria |
-
1987
- 1987-03-03 CA CA000531064A patent/CA1288076C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100390295C (en) * | 2005-11-26 | 2008-05-28 | 山西大学 | Microorganism polysaccharide and its preparation method and application |
| EP3702440A4 (en) * | 2017-10-27 | 2020-12-02 | Kumiai Chemical Industry Co., Ltd. | Culturing method for genus rhizobium bacteria |
| JPWO2019082906A1 (en) * | 2017-10-27 | 2021-02-18 | クミアイ化学工業株式会社 | Rhizobium bacterium culture method |
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