CA1160171A - Recirculation tower bioreactor for solid-state fermentation - Google Patents
Recirculation tower bioreactor for solid-state fermentationInfo
- Publication number
- CA1160171A CA1160171A CA000378900A CA378900A CA1160171A CA 1160171 A CA1160171 A CA 1160171A CA 000378900 A CA000378900 A CA 000378900A CA 378900 A CA378900 A CA 378900A CA 1160171 A CA1160171 A CA 1160171A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/24—Gas permeable parts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/16—Solid state fermenters, e.g. for koji production
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/22—Perforated plates, discs or walls
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/02—Percolation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/24—Recirculation of gas
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
ABSTRACT
A method and apparatus for producing single cell proteins or other products from a substrate made up of solid lignocellulosic materials, such as wood chips, wherein a plurality of perforated trays each containing a layer of sub-strate are loaded one above another in a recirculating tower reactor. After innoculating the layers of substrate with a white rot fungi, such as Polyporus anceps, a nutrient solution is recirculated downwardly through the reactor, at a rate sufficient to keep the microorganism active and the layers of substrate moist, and air is injected upwardly through the reactor at a rate sufficient to maintain aerobic fermentation and about 2-3% carbon dioxide in the exhaust gases.
A method and apparatus for producing single cell proteins or other products from a substrate made up of solid lignocellulosic materials, such as wood chips, wherein a plurality of perforated trays each containing a layer of sub-strate are loaded one above another in a recirculating tower reactor. After innoculating the layers of substrate with a white rot fungi, such as Polyporus anceps, a nutrient solution is recirculated downwardly through the reactor, at a rate sufficient to keep the microorganism active and the layers of substrate moist, and air is injected upwardly through the reactor at a rate sufficient to maintain aerobic fermentation and about 2-3% carbon dioxide in the exhaust gases.
Description
This invention relates to solid state aerobic fermentation processes and apparatus for the production of single cell proteins (SCPs) or other economically valuable products from solid materials, such as lignocellulosics, including such waste products as wood chips, bark, straw and other forestry or agricultural materials.
Heretofo.re, most large scale aerobic fermentation processes for the produc-tion of S.C.P. and the like have been in a substantially liquid medium or at best in a slurry medium which contains up to about 10% solids. Such liquid mediums generally preclude or, at least, limit the use of such abundant waste cellulosic materials as wood, baqasse, straw and the like all of which have been shown to be eminently suitable source materials for the production of S.C.P. While solid state fer-mentation, i.e. Eermentation in mediums containing greater than about 10% by volume solids, has several advantages compared to submerged cultivation including smaller working volumes, less technology and less waste water, it is not without its diffi-culties and disadvanta~es. Firstly, i.t is relatively difficult to control fermenter operating conditions, secondly deep tray fermenters give rise to the possibility of anae:robiosis, and thirdly use of a tumble fermenter frequently inhibits growth as a result of shearing of the filamentous mycelia of the organisms employed.
It is therefore an object of the present invention to provide a process for solid state fermentation, as de.Eined hereinabove, of lignocellulosic materials to produce relatively high yields of single cell proteins and other economically valuable conversion products.
Another o~ject of the invention is to provide a novel tower-type bioreactor in which to carry out solid state fermentation processes of the present invention.
Thus by one aspect of this invention there is provided an aerobic fermentation process for conversion of solid organic materials in a tower bioreactor containing a plurality of horizontal perforated trays stacked in vertical spaced relationship therein comprising: (a) providing each of said trays with an air pervious layer of particulate organic material; (b) inoculating at least one said layer with a selected microorganism for a desired conversion; and (c) recirculating a liquid nutrient medium for said microorganism through said re-actor in an amount sufficient tOmoi5ten said particulate material and maintain said microorganism in viable form and passing air through said reactor in an amount sufficient to maintain aerobic fermentation and ensure the presence of carbon dioxide in the exhaust gas for a sufficient time to effect said desired conver-sion.
By another aspect of this invention there is provided an apparatus for solid state aerobic fermentation of particulate organic materials comprising: (a) a reactor shell; (b) a plurality of horizontally disposed and vertically spaced per-forated trays contained within said shell and each adapted to receive a layer of said particulate material thereon; (c) means to circulate air through said reactor shell and said layers of particulate material; (d) means to inoculate at least one of said layers with a microorganism; and (e) means to recirculate a liquid nutrient medium through said layers in said reactor shell.
The invention will be described in more detail hereinafter with reference to the accompanying drawings in which:-Figure 1 is a vertical section through a tower reactoraccording to one embodiment of the present invention;
Figure 2 is a graph illustrating protein production versus time in a jar fermenter;
Figure 3 is a ~raph illustrating lignin content versus time in a jar fermenter; and Figure 4 is a graph illustrating cellulase production versus time in a recirculating tower reactor of the present invention.
Referring to Figure 1 there is shown an oriented, vertically cylindrical tower 1 having a plurality of perforated horizontal.ly disposed decks or trays 2 contained therein to support the charged mass of cellulosic particles. An air inlet port 3 is provided adjacent the bottom of the tower and a gas outlet port 4 is provided at the top. Nutrient medium intro-duction and culture inoculation ports 5 are provided above each of the perforated decks 2. A bed 6 of particulate solid cellulosic materials, such as wood chips or the like is provided on each of the decks 2. The depth of the bed is generally a function of the size of the particles. As extremely fine materials (of the order of -20 to +40 mesh) tend to compact, such materials are generally screened out and preferably particles retained on a 5 mesh (1/5") screen andlarger are employed. With 5 mesh particles bed depths up to about 3 inches are effective with larger particles, of the order of 3" x bed depths up to about 8 inches have been found effective.
Clearly, the size of the perforations in the decks 2 is related to the size of the material treated, and generally the perfora-tions are of the order of 1/8" in diameter so as to prevent pas-sage of the particles. If the perforations are too small, of the order of 1/16" diameter, clogging tends to be a problem. It is important to appreciate that no provision for stirring or otherwise agitating the beds 6 are provided as it has been found that agitation tends to shear or break up the filamentous mycelia of the organisms and hence reduce product yield. Provided that bed and reactor conditions are controlled as noted above so that a non-compacting, non-clogging system through which sufficient air to prevent anaerobic conditions and sufficient liquid nutri-ent medium to sustain the microorganisms may pass freely, agita-tion or stirring of the bed is unnecessary and indeed undesirable.
The rate of aeration may be simply controlled by carbon dioxide analysis of the off-gases. Carbon dioxide should always be pre-sent in the reactor and off-gases and for optimum results the rate of aeration should be sufficient to maintain the carbon dioxide level in the off-gases in the range of 2-3% by volume.
Excessive aeration tends to dry out the microorganisms, while insufficient aeration causes anaerobiosis. Sufficient nutrient solution to maintain the fermenting charge in at least a moist condition is introduced under controlled and monitored conditions and recirculated through the tower at least once per day. The externally circulated medium may be controlled and monitored by means of any conventional control and monitoring devices, illustrated in schematic block form 7 in Figure 1.
Anycellulolytic or white rot fungi may be employed, although Polyporus anceps (DAOM 21401 Agriculture Canada), Pleurotus ostreatus (P 11, Glasshouse Crops Research Inst., f-~
Littlehampton England) or Phanerochaete chrysosporium (A387 Canadian Forestry Services) have been found particularly effec-tive. Fermentation causes a fairly rapid rise in temperature so that no externally applied heat is necessary.
Silver maple (Acer saccharinum) wood shavings (20 x 10 x 0.3 - 0.5 mm) were obtained from 25 cm - 50 cm logs and pretreated by autoclaving (a 2.5% slurry of the wood with alkali) for 2 hr at 121C. A sodium hydroxide to wood ratio of 1.2:1 was used. The wood was removed by filtration, back-washed with tap water until the effluent was neutral, filtered and air-dried. The sodium hydroxide pretreatment resulted in a 26% loss in dry weight. 20 g, 5.5% moisture samples of the shavings were introduced into respective jar fermenters and mixed with 100 ml of a salts media comprising 1.0 g/lNH4Cl, and
Heretofo.re, most large scale aerobic fermentation processes for the produc-tion of S.C.P. and the like have been in a substantially liquid medium or at best in a slurry medium which contains up to about 10% solids. Such liquid mediums generally preclude or, at least, limit the use of such abundant waste cellulosic materials as wood, baqasse, straw and the like all of which have been shown to be eminently suitable source materials for the production of S.C.P. While solid state fer-mentation, i.e. Eermentation in mediums containing greater than about 10% by volume solids, has several advantages compared to submerged cultivation including smaller working volumes, less technology and less waste water, it is not without its diffi-culties and disadvanta~es. Firstly, i.t is relatively difficult to control fermenter operating conditions, secondly deep tray fermenters give rise to the possibility of anae:robiosis, and thirdly use of a tumble fermenter frequently inhibits growth as a result of shearing of the filamentous mycelia of the organisms employed.
It is therefore an object of the present invention to provide a process for solid state fermentation, as de.Eined hereinabove, of lignocellulosic materials to produce relatively high yields of single cell proteins and other economically valuable conversion products.
Another o~ject of the invention is to provide a novel tower-type bioreactor in which to carry out solid state fermentation processes of the present invention.
Thus by one aspect of this invention there is provided an aerobic fermentation process for conversion of solid organic materials in a tower bioreactor containing a plurality of horizontal perforated trays stacked in vertical spaced relationship therein comprising: (a) providing each of said trays with an air pervious layer of particulate organic material; (b) inoculating at least one said layer with a selected microorganism for a desired conversion; and (c) recirculating a liquid nutrient medium for said microorganism through said re-actor in an amount sufficient tOmoi5ten said particulate material and maintain said microorganism in viable form and passing air through said reactor in an amount sufficient to maintain aerobic fermentation and ensure the presence of carbon dioxide in the exhaust gas for a sufficient time to effect said desired conver-sion.
By another aspect of this invention there is provided an apparatus for solid state aerobic fermentation of particulate organic materials comprising: (a) a reactor shell; (b) a plurality of horizontally disposed and vertically spaced per-forated trays contained within said shell and each adapted to receive a layer of said particulate material thereon; (c) means to circulate air through said reactor shell and said layers of particulate material; (d) means to inoculate at least one of said layers with a microorganism; and (e) means to recirculate a liquid nutrient medium through said layers in said reactor shell.
The invention will be described in more detail hereinafter with reference to the accompanying drawings in which:-Figure 1 is a vertical section through a tower reactoraccording to one embodiment of the present invention;
Figure 2 is a graph illustrating protein production versus time in a jar fermenter;
Figure 3 is a ~raph illustrating lignin content versus time in a jar fermenter; and Figure 4 is a graph illustrating cellulase production versus time in a recirculating tower reactor of the present invention.
Referring to Figure 1 there is shown an oriented, vertically cylindrical tower 1 having a plurality of perforated horizontal.ly disposed decks or trays 2 contained therein to support the charged mass of cellulosic particles. An air inlet port 3 is provided adjacent the bottom of the tower and a gas outlet port 4 is provided at the top. Nutrient medium intro-duction and culture inoculation ports 5 are provided above each of the perforated decks 2. A bed 6 of particulate solid cellulosic materials, such as wood chips or the like is provided on each of the decks 2. The depth of the bed is generally a function of the size of the particles. As extremely fine materials (of the order of -20 to +40 mesh) tend to compact, such materials are generally screened out and preferably particles retained on a 5 mesh (1/5") screen andlarger are employed. With 5 mesh particles bed depths up to about 3 inches are effective with larger particles, of the order of 3" x bed depths up to about 8 inches have been found effective.
Clearly, the size of the perforations in the decks 2 is related to the size of the material treated, and generally the perfora-tions are of the order of 1/8" in diameter so as to prevent pas-sage of the particles. If the perforations are too small, of the order of 1/16" diameter, clogging tends to be a problem. It is important to appreciate that no provision for stirring or otherwise agitating the beds 6 are provided as it has been found that agitation tends to shear or break up the filamentous mycelia of the organisms and hence reduce product yield. Provided that bed and reactor conditions are controlled as noted above so that a non-compacting, non-clogging system through which sufficient air to prevent anaerobic conditions and sufficient liquid nutri-ent medium to sustain the microorganisms may pass freely, agita-tion or stirring of the bed is unnecessary and indeed undesirable.
The rate of aeration may be simply controlled by carbon dioxide analysis of the off-gases. Carbon dioxide should always be pre-sent in the reactor and off-gases and for optimum results the rate of aeration should be sufficient to maintain the carbon dioxide level in the off-gases in the range of 2-3% by volume.
Excessive aeration tends to dry out the microorganisms, while insufficient aeration causes anaerobiosis. Sufficient nutrient solution to maintain the fermenting charge in at least a moist condition is introduced under controlled and monitored conditions and recirculated through the tower at least once per day. The externally circulated medium may be controlled and monitored by means of any conventional control and monitoring devices, illustrated in schematic block form 7 in Figure 1.
Anycellulolytic or white rot fungi may be employed, although Polyporus anceps (DAOM 21401 Agriculture Canada), Pleurotus ostreatus (P 11, Glasshouse Crops Research Inst., f-~
Littlehampton England) or Phanerochaete chrysosporium (A387 Canadian Forestry Services) have been found particularly effec-tive. Fermentation causes a fairly rapid rise in temperature so that no externally applied heat is necessary.
Silver maple (Acer saccharinum) wood shavings (20 x 10 x 0.3 - 0.5 mm) were obtained from 25 cm - 50 cm logs and pretreated by autoclaving (a 2.5% slurry of the wood with alkali) for 2 hr at 121C. A sodium hydroxide to wood ratio of 1.2:1 was used. The wood was removed by filtration, back-washed with tap water until the effluent was neutral, filtered and air-dried. The sodium hydroxide pretreatment resulted in a 26% loss in dry weight. 20 g, 5.5% moisture samples of the shavings were introduced into respective jar fermenters and mixed with 100 ml of a salts media comprising 1.0 g/lNH4Cl, and
2.0 g/l yeast extract. 15 g of the shavings and 75 ml of the salts media were also added to each stage of a recirculating tower reactor (RTB) as described herein. Additionally 400 ml of salts media were added to the RTB holding tank.
The jar fermenters consisted of polymethypentene (PMP) wide-mouth jars (11 cm x 7 cm) with polypropylene screw closures fitted with a 2.5 cm porous plug. Deep glass jars (5.7 cm x 20 cm) were fitted with a rubber stopper and two glass tubes (5 mm, OD), one extending to the bottom of the jar and the other to 2 cm below the stopper. The recirculating tower bioreactor (RTB) (10 cm x 25 cm) was constructed of polypropylene and consisted of two stages. The closed unit had two outlets at the top, one for venting gases and the other an inoculation port which was used during the fermentations as an entrance port for the recirculating salts medium. The bottom of the unit was fitted with an inlet aeration port and a liquid exit which was connected to a 2 1 holding tank. A peristaltic pump was employed to recirculate the salts medium.
The jar fermenters were inoculated with 25 ml and the RTB with 80 ml of a fragmented mycelial suspension of Polyporus anceps.
The fermentations were carried out at 30C. Additional salts media was added to the PMP jars as follows: 40 ml on day 4 and 8, 20 ml on day 12 and 10 ml on day 16. One PMP jar, inoculated with 75 ml of mycelial suspension received all additional salts except those added at day 4. After day 4, each jar unit was gently rotated once a day to ensure the fermenting shavings were wetted. No tumbling or mixing of solids occurred. Aeration oE the glass unit and the RTB was for 1 hr at 12 hour intervals at a flow rate of 100 ml.min 1 The salts media in the RTB holding tank were recirculated through the tower for 2 hr once a day at a rate of 60 ml. min 1 The wet contents of the vessels were pressed and the liquid analyzed for pH, carboxymethylcellulase activity, and reducing sugars. Pressed solids were dried at 105C for 48 hr.
A portion of the dried solids was extracted with lN NaOH (50 ml g solids 1) for 20 hr at room temperature. Extracted solids were washed and redried. Extracted and non-extracted solids were ground in a Wiley mill to a 40 mesh size. The solids were analyzed for Klason lignin and nitrogen using the Kjeldahl-nesslerization method. Crude protein was calculated as N
content x 6.25. To determine the weight of the biomass pre-sent, calculations were made on the basis of 6.9% N content of fungi. The difference in this weight and the non-extracted L'7~
dried solids gave the mass of unused substrate. The results were plotted as shown in Figures 2 and 3.
One week after inoculation, the mycelium had penetrated the mass of wood shavings in the PMP jar fermenters and the observed chemical changes were tabulated as is shown in Figures 1 and 2. There was a progressive loss in dry weight and a linear increase of the lignin fraction. The alkali sol-uble fraction varied as growth proceeded. This fraction repre-sents solubilization of the cell contents and possible extraction of partially degraded lignin. Associated with these chemical changes, the wood shavings lightened in colour and compac-tness.
Throughout the major portion of the growth, pEI was 3.0 and did not afEect the growth, as indicated by lignin content and the loss of substrate. In the case of the RTB pH changes (Figure 4) were similar to those in the jar fermenters and the cellulase activity increased dramatically only in the final stages of the fermentation. Table 1 compares the cellulase activity and analysis of the three types of fermenters at day 40.
Comparison of Various Fermentations After 40 Da~s % Loss of Cellulase Unit pH Substrate %Protein mg glucose.ml~l.hr~
A 4.1 54.2 9.6 122 B 3.8 57.0 9.36 166 C 3.8 47.1 6.4 158 D 3.5 44.1 7.9 79 E 4.1 69.6 16.0 288 F 4.1 68.8 16.8 288 A - Standard PMP fermenter B - PMP fermenter with thoroughly mixed inoculum C - PMP fermenter with 3X inoculum and thorough mixing D - Deep glass jar fermenter E - Top stage of RTB
F - Second stage of RTB
Surface inoculation chosen as the standard because of ease of handling was used for those units used in the kinetic study.
Initial thorough mixing of the substrate and the inoculum resulted in increased cellulase activity as well as a slight increase in substrate loss. A three fold increase in the inoculum size (Unit C) on the other hand resulted in less substrate utiliza-tion and also lower protein content. Gentle contacting of the liquid and solids showed itself to be bene-ficial to the growth process whereas tumbler mixing was found to considerably inhibit the growth of Polyporus anceps. The deep jar fermenter (Unit D) was not as effective. This could be due to the submersion of one-third of the wood shavings in the aerated salts media.
Of the three static fermentation systems used, the RTB proved most successful in degrading the wood (70~ loss) and producing high levels of protein (16.0~ and 16.8%) with con-comitant high cellulase activity (Unit E and F). Intermittent aeration and recirculation of liquid is more effective than con-tinuous air and liquid contact (values similar to A obtained).
Over the 12 hour non-aeration phase, carbon dioxide levels increased to approximately 4.0%. Nitrogen balance for the RT~
showed that 315 mg N added as ammonium chloride, yeast extract and inoculum resulted in 230 mg N being recovered as protein after 40 days. In these fermenters the mycelium grew throughout the mass of wood shavings and formed a thick mat at the solid/
air in-terface.
Heretofore growth of fungi on wood has generally resulted in low yields of protein, due to the nature of the organisms which are concerned with mycelial penetration and L'71 degradation of substrate. This has been overcome by the RTB
which provides a medium of liquid contact between the fungus and the wood. As this liquid contains degradative enzymes, nutrients and possible mycelial fragments, a distribution of fluid throughout the wood mass accelerates the conversion of wood to biomass. Extent of wetting is important as partial immersion of the solids in the excess salts media in deep jars inhibits further rapid degradation. Carbon dioxide is an important gas for the growth of basidiomycetes as shown by different tolerances and levels of CO2 requirements for Pleurotus species and Agaricus bisporus. The concentration of C2 may be of importance for the growth of Polyporus anceps in an RTB, as intermittent aeration has proven to be the most effective method of supplying oxygen and flushing c~ases and volatiles.
It will be appreciated that many modifications can be effected within the scope of the present invention. For example, the tower reactor has been described herein with particular reference to a batch type reactor but the invention may equally well be carried out in a continuous mode. As stirring or other movement of the fermenting mass on the trays or decks 2 tends to break up the filaments ofthe microorganism with attenden-t lowering of yield, the usual rabbles employed to move material from one level to another should not be employed.
Instead, the trays themselves with the fermenting mass thereon are moved upwardly, counter current to the flow of nutrient medium, by a simple chain elevating device. A tray inlet port arrangement may be provided at the bottom of the tower, and a tray outlet port arrangement will likewise be provided at the 7~
~op. Assuming a 40 day cycle, it is convenient to provide for 40 layers or tiers of trays within the reactor, so that one new tray can be inserted and one fully treated tray can be removed each day.
The jar fermenters consisted of polymethypentene (PMP) wide-mouth jars (11 cm x 7 cm) with polypropylene screw closures fitted with a 2.5 cm porous plug. Deep glass jars (5.7 cm x 20 cm) were fitted with a rubber stopper and two glass tubes (5 mm, OD), one extending to the bottom of the jar and the other to 2 cm below the stopper. The recirculating tower bioreactor (RTB) (10 cm x 25 cm) was constructed of polypropylene and consisted of two stages. The closed unit had two outlets at the top, one for venting gases and the other an inoculation port which was used during the fermentations as an entrance port for the recirculating salts medium. The bottom of the unit was fitted with an inlet aeration port and a liquid exit which was connected to a 2 1 holding tank. A peristaltic pump was employed to recirculate the salts medium.
The jar fermenters were inoculated with 25 ml and the RTB with 80 ml of a fragmented mycelial suspension of Polyporus anceps.
The fermentations were carried out at 30C. Additional salts media was added to the PMP jars as follows: 40 ml on day 4 and 8, 20 ml on day 12 and 10 ml on day 16. One PMP jar, inoculated with 75 ml of mycelial suspension received all additional salts except those added at day 4. After day 4, each jar unit was gently rotated once a day to ensure the fermenting shavings were wetted. No tumbling or mixing of solids occurred. Aeration oE the glass unit and the RTB was for 1 hr at 12 hour intervals at a flow rate of 100 ml.min 1 The salts media in the RTB holding tank were recirculated through the tower for 2 hr once a day at a rate of 60 ml. min 1 The wet contents of the vessels were pressed and the liquid analyzed for pH, carboxymethylcellulase activity, and reducing sugars. Pressed solids were dried at 105C for 48 hr.
A portion of the dried solids was extracted with lN NaOH (50 ml g solids 1) for 20 hr at room temperature. Extracted solids were washed and redried. Extracted and non-extracted solids were ground in a Wiley mill to a 40 mesh size. The solids were analyzed for Klason lignin and nitrogen using the Kjeldahl-nesslerization method. Crude protein was calculated as N
content x 6.25. To determine the weight of the biomass pre-sent, calculations were made on the basis of 6.9% N content of fungi. The difference in this weight and the non-extracted L'7~
dried solids gave the mass of unused substrate. The results were plotted as shown in Figures 2 and 3.
One week after inoculation, the mycelium had penetrated the mass of wood shavings in the PMP jar fermenters and the observed chemical changes were tabulated as is shown in Figures 1 and 2. There was a progressive loss in dry weight and a linear increase of the lignin fraction. The alkali sol-uble fraction varied as growth proceeded. This fraction repre-sents solubilization of the cell contents and possible extraction of partially degraded lignin. Associated with these chemical changes, the wood shavings lightened in colour and compac-tness.
Throughout the major portion of the growth, pEI was 3.0 and did not afEect the growth, as indicated by lignin content and the loss of substrate. In the case of the RTB pH changes (Figure 4) were similar to those in the jar fermenters and the cellulase activity increased dramatically only in the final stages of the fermentation. Table 1 compares the cellulase activity and analysis of the three types of fermenters at day 40.
Comparison of Various Fermentations After 40 Da~s % Loss of Cellulase Unit pH Substrate %Protein mg glucose.ml~l.hr~
A 4.1 54.2 9.6 122 B 3.8 57.0 9.36 166 C 3.8 47.1 6.4 158 D 3.5 44.1 7.9 79 E 4.1 69.6 16.0 288 F 4.1 68.8 16.8 288 A - Standard PMP fermenter B - PMP fermenter with thoroughly mixed inoculum C - PMP fermenter with 3X inoculum and thorough mixing D - Deep glass jar fermenter E - Top stage of RTB
F - Second stage of RTB
Surface inoculation chosen as the standard because of ease of handling was used for those units used in the kinetic study.
Initial thorough mixing of the substrate and the inoculum resulted in increased cellulase activity as well as a slight increase in substrate loss. A three fold increase in the inoculum size (Unit C) on the other hand resulted in less substrate utiliza-tion and also lower protein content. Gentle contacting of the liquid and solids showed itself to be bene-ficial to the growth process whereas tumbler mixing was found to considerably inhibit the growth of Polyporus anceps. The deep jar fermenter (Unit D) was not as effective. This could be due to the submersion of one-third of the wood shavings in the aerated salts media.
Of the three static fermentation systems used, the RTB proved most successful in degrading the wood (70~ loss) and producing high levels of protein (16.0~ and 16.8%) with con-comitant high cellulase activity (Unit E and F). Intermittent aeration and recirculation of liquid is more effective than con-tinuous air and liquid contact (values similar to A obtained).
Over the 12 hour non-aeration phase, carbon dioxide levels increased to approximately 4.0%. Nitrogen balance for the RT~
showed that 315 mg N added as ammonium chloride, yeast extract and inoculum resulted in 230 mg N being recovered as protein after 40 days. In these fermenters the mycelium grew throughout the mass of wood shavings and formed a thick mat at the solid/
air in-terface.
Heretofore growth of fungi on wood has generally resulted in low yields of protein, due to the nature of the organisms which are concerned with mycelial penetration and L'71 degradation of substrate. This has been overcome by the RTB
which provides a medium of liquid contact between the fungus and the wood. As this liquid contains degradative enzymes, nutrients and possible mycelial fragments, a distribution of fluid throughout the wood mass accelerates the conversion of wood to biomass. Extent of wetting is important as partial immersion of the solids in the excess salts media in deep jars inhibits further rapid degradation. Carbon dioxide is an important gas for the growth of basidiomycetes as shown by different tolerances and levels of CO2 requirements for Pleurotus species and Agaricus bisporus. The concentration of C2 may be of importance for the growth of Polyporus anceps in an RTB, as intermittent aeration has proven to be the most effective method of supplying oxygen and flushing c~ases and volatiles.
It will be appreciated that many modifications can be effected within the scope of the present invention. For example, the tower reactor has been described herein with particular reference to a batch type reactor but the invention may equally well be carried out in a continuous mode. As stirring or other movement of the fermenting mass on the trays or decks 2 tends to break up the filaments ofthe microorganism with attenden-t lowering of yield, the usual rabbles employed to move material from one level to another should not be employed.
Instead, the trays themselves with the fermenting mass thereon are moved upwardly, counter current to the flow of nutrient medium, by a simple chain elevating device. A tray inlet port arrangement may be provided at the bottom of the tower, and a tray outlet port arrangement will likewise be provided at the 7~
~op. Assuming a 40 day cycle, it is convenient to provide for 40 layers or tiers of trays within the reactor, so that one new tray can be inserted and one fully treated tray can be removed each day.
Claims (16)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An aerobic fermentation process for conversion of solid organic materials in a tower bioreactor containing a plurality of horizontal perforated trays stacked in vertical spaced relationship therein comprising:
(a) providing each of said trays with an air pervious layer of particulate organic material;
(b) inoculating at least one said layer with a selected microorganism for a desired conversion; and (c) recirculating a liquid nutrient medium for said micro-organism through said reactor in an amount sufficient to moisten said particulate material and maintain said microorganism in viable form and passing air through said reactor in an amount sufficient to maintain aerobic fermentation and ensure the presence of carbon dioxide in the exhaust gas, for a sufficient time to effect said desired conversion.
(a) providing each of said trays with an air pervious layer of particulate organic material;
(b) inoculating at least one said layer with a selected microorganism for a desired conversion; and (c) recirculating a liquid nutrient medium for said micro-organism through said reactor in an amount sufficient to moisten said particulate material and maintain said microorganism in viable form and passing air through said reactor in an amount sufficient to maintain aerobic fermentation and ensure the presence of carbon dioxide in the exhaust gas, for a sufficient time to effect said desired conversion.
2. A process as claimed in claim 1 for the production of single cell proteins, in which said solid organic material is a lignocellulosic material.
3. A process as claimed in claim 2 wherein said micro-organism is a white rot fungi.
4. A process as claimed in claim 3 wherein said micro-organism is selected from Polyporus anceps, Pleurotus ostreatus and Phanerochaete chrysosporium.
5. A process as claimed in claim 1, 2 or 3 wherein said nutrient medium is recirculated countercurrent to the direction of passage of air.
6. A process as claimed in claim 1, 2 or 3 wherein said nutrient medium is recirculated countercurrent to the direction of passage of air and wherein said trays are moved upwardly through said reactor during said fermentation and countercurrent to said nutrient medium.
7. A process as claimed in claim 1, 2 or 3 wherein air is passed through said reactor at a rate sufficient to obtain about 1-3% by volume carbon dioxide in said exhaust gas.
8. A process as claimed in claim 2 or 3 wherein said lignocellulosic material comprises wood chips having a minimum diameter of about 1/5".
9. A process as claimed in claim 2 or 3 wherein said lignocellulosic material comprises wood chips having a size range between about 1/5" and about 3".
10. A process as claimed in claim 1, 2 or 3 wherein each said layer is up to about 8" thick.
11. An apparatus for solid state aerobic fermentation of particulate organic materials comprising:
(a) a reactor shell;
(b) a plurality of horizontally disposed and vertically spaced perforated trays contained within said shell and each adapted to receive a layer of said particulate material thereon;
(c) means to circulate air through said reactor shell and said layers of particulate material;
(d) means to inoculate at least one of said layers with a microorganism; and (e) means to recirculate a liquid nutrient medium through said layers in said reactor shell.
(a) a reactor shell;
(b) a plurality of horizontally disposed and vertically spaced perforated trays contained within said shell and each adapted to receive a layer of said particulate material thereon;
(c) means to circulate air through said reactor shell and said layers of particulate material;
(d) means to inoculate at least one of said layers with a microorganism; and (e) means to recirculate a liquid nutrient medium through said layers in said reactor shell.
12. An apparatus as claimed in claim 11 wherein said air circulates countercurrent to said nutrient medium.
13. An apparatus as claimed in claim 11 or 12 including means to introduce said air upwardly through said reactor.
14. An apparatus as claimed in claim 11, 12 or 13 including means to move said horizontally disposed trays upwardly through said shell.
15. An apparatus as claimed in claim 11, 12 or 13 including: means to move said horizontally disposed trays upwardly through said shell; means adjacent a lower end of said shell to introduce said trays sequentially thereinto and means adjacent an upper end of said shell to withdraw said trays sequentially therefrom.
16. An apparatus as claimed in claim 11, 12 or 13 wherein said means to recirculate said liquid nutrient medium includes peristaltic pump means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000378900A CA1160171A (en) | 1981-06-02 | 1981-06-02 | Recirculation tower bioreactor for solid-state fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000378900A CA1160171A (en) | 1981-06-02 | 1981-06-02 | Recirculation tower bioreactor for solid-state fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1160171A true CA1160171A (en) | 1984-01-10 |
Family
ID=4120128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000378900A Expired CA1160171A (en) | 1981-06-02 | 1981-06-02 | Recirculation tower bioreactor for solid-state fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1160171A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2572091A1 (en) * | 1984-10-24 | 1986-04-25 | Inst Biokhim Fiziol | Process for culturing mycelium-containing fungi on a solid substrate and the fungi obtained by the said process |
| EP0225479A2 (en) * | 1985-11-11 | 1987-06-16 | Keller & Bohacek GmbH & Co KG | Process and apparatus for the production of an enzymatic biomass from sugar beet pulp |
| EP0676466A2 (en) * | 1994-03-01 | 1995-10-11 | Dietmar Dr. Vollbrecht | Bioreactor for solid medium |
| CN106723219A (en) * | 2016-10-31 | 2017-05-31 | 黑龙江省科学院微生物研究所 | Suitable for the fermentation tank of dregs of beans solid aerobic fermentation |
| CN107002003A (en) * | 2015-08-14 | 2017-08-01 | 国家开发投资公司 | A kind of photo-biological culture multifunctional assisting device |
| CN107674826A (en) * | 2017-09-04 | 2018-02-09 | 陕西众兴高科生物科技有限公司 | A kind of liquid bacterial culture device |
-
1981
- 1981-06-02 CA CA000378900A patent/CA1160171A/en not_active Expired
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2572091A1 (en) * | 1984-10-24 | 1986-04-25 | Inst Biokhim Fiziol | Process for culturing mycelium-containing fungi on a solid substrate and the fungi obtained by the said process |
| EP0225479A2 (en) * | 1985-11-11 | 1987-06-16 | Keller & Bohacek GmbH & Co KG | Process and apparatus for the production of an enzymatic biomass from sugar beet pulp |
| EP0225479A3 (en) * | 1985-11-11 | 1988-07-20 | Keller & Bohacek GmbH & Co KG | Process and apparatus for the production of an enzymatic biomass from sugar beet pulp |
| EP0676466A2 (en) * | 1994-03-01 | 1995-10-11 | Dietmar Dr. Vollbrecht | Bioreactor for solid medium |
| EP0676466A3 (en) * | 1994-03-01 | 1997-08-20 | Dietmar Dr Vollbrecht | Bioreactor for solid medium. |
| CN107002003A (en) * | 2015-08-14 | 2017-08-01 | 国家开发投资公司 | A kind of photo-biological culture multifunctional assisting device |
| CN106723219A (en) * | 2016-10-31 | 2017-05-31 | 黑龙江省科学院微生物研究所 | Suitable for the fermentation tank of dregs of beans solid aerobic fermentation |
| CN107674826A (en) * | 2017-09-04 | 2018-02-09 | 陕西众兴高科生物科技有限公司 | A kind of liquid bacterial culture device |
| CN107674826B (en) * | 2017-09-04 | 2020-10-27 | 陕西众兴高科生物科技有限公司 | Liquid strain incubator |
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