CA1073379A - Method of obtaining ethanol by the utilization of unicellular green algae - Google Patents
Method of obtaining ethanol by the utilization of unicellular green algaeInfo
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- CA1073379A CA1073379A CA243,024A CA243024A CA1073379A CA 1073379 A CA1073379 A CA 1073379A CA 243024 A CA243024 A CA 243024A CA 1073379 A CA1073379 A CA 1073379A
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Abstract
ABSTRACT OF THE DISCLOSURE
The invention provides a process for obtaining ethanol by the saccharification and alcohol fermentation of starch produced intra- and/or extra-cellularly by unicellu-lar green algae, such as, for example, Chloretta, Chlamy-domonus and Scenedesmus, all of which are aerobes adapted for heterotrophic culture. This ensures a high yield, economical ethanol production.
The invention provides a process for obtaining ethanol by the saccharification and alcohol fermentation of starch produced intra- and/or extra-cellularly by unicellu-lar green algae, such as, for example, Chloretta, Chlamy-domonus and Scenedesmus, all of which are aerobes adapted for heterotrophic culture. This ensures a high yield, economical ethanol production.
Description
` ~73379 ~
This invention relates to a process for producing ethanol from unicellular green algae. More particularly, the present invention relates to a process for producing ethanol by the utilization of starch produced either intra-cellularly or extra-cellularly, or both intracellularly and extracellularly, by unicellular green algae.
This invention is concerned with improvements in ethanol production, based upon the discovery of particular species of unicellular green algae which produce and store starch. More particularly these species will be referred to as Chlorella vulgaris Al-ly-3(11), Chlamydomonas sp.
Al-5 and Scenedosmus _asilensis IAM C-66, all of which are aerobes adapted for heterotrophic culture in a dark, liquid medium containing carbon, nitrogen and inorganic salts.
These strains are found in and are collected from slops and sewages near Tokyo, Japan, and isolated single cellularly.
It is known in the art that ethanol is obtained from the distillation of fermented starch provided by higher plants, such as, for example, corn, wheat, sweet potatoes, ; 20 etc. ~lowever 9 the growth of these plants is influenced by the weather, and their yields greatly depend upon the climatic condition. The current threat of food shortages makes it likely that it will become difficult to obtain ethanol from such agricultural products.
The present invention is thus directed to ethanol production without using such plants. An object of a main aspect of this invention is to provide a process for obtain-ing ethanol by utilizing unicellular green algae as suppliers of ethanol material, the species of algae being advantageously adapted for heterotrophic culture even in places of indus-trial waste.
~L~3733~9 By one broad aspect of the present invention, a process is provided for producing ethanol, the process comprising: aerobically culturing starch-producing unicellular green algae in a culture medium containing an organic com-pound providing a source of assimilable carbon, a sourceof assimilable nitrogen and inorganic salts and other factors necessary for the growth of the algae; rupturing the cellu-lar material to release intra-cellular starch; saccharify-ing the starch produced; fermenting the saccharified starch in the presence of a saccharified starch-utilizing, ethanol- :
producing microorganism; and recovering the ethanol pro-duced from the fermentation medium.
By a prepared aspect of this invention, the algae used is Chlorella vul~aris Al-ly-3(11), Chlamydomonas sp.
Al-5, or Scenedesmus basilensis lAMC-66, as will be described further hereinafter.
By one variant of the preferred aspect, the algae is Chlorella vulgaris Al-ly-3~11) and is cultured at a temperature in the range of 25-42C and at a pH in the range of 5 9, especially where the algae is cultured at a temperature of 37C at a pH in the range of 7-8.
By another variant of the preferred aspect, the algae used is Chlamydomonas sp. Al-5, and is cultured at a temperature in the range of 25-35C and at a pH in the range of 5-9, especially where the ~emperature is 28C
and the pH is from 7-8.
By yet another variant of the preferred aspect, the algae used is Scenedesmus basilensis lAM C-66, and is cultured at a temperature in the range of `25-37C and at a pH in the range of 5-9, especially where the temperature ~73379 is 30C and the pH is from 7-8.
By another aspect of this invention, the culture medium for the algae contains as the source of assimilable carbon, glucose, maltose or acetic acid.
By still another aspect of this invention, the culture medium for the algae is or comprises an industrial wasteO
By still other variants of this invention, the starch product is saccharified using dilute aqueous sulphuric acid or is saccharified by means of enzymes.
Three preferred species of algae are as follows:
(l) Chlorella vulgaris Al-ly-3(11):
The cell is spllerical and 3.3 to 5.0 X3.3 to 5.2 micron large in size, and normally multiplies while splitting into four auto spores. The colour of its colony is initially green, but as it grows, it fades and becomes yellowish.
This species grows at a temperature of 25 to 42~C, but the optimum condition is 37C with pH 5 to 9; preferably pH 7 or 8. A feature of this species is that it produces and accumulates starch both inside and outside the cell, the starch amounting to 70 to 80% of its dry weight, as much as the starch content of cereals. A further advantage is that it discharges two-thirds or less of the in~ernal starch out of the cell.
The strain of this species can be cultured either in the dark or in the light. In the dark, glucose, maltose, and acetic acid are preferably utilized as carbon sources.
Potassium nitrate, polypepton, and ammonium salts (such as, for example, urea, ammonium sulfate, ammonium acetate, and ammonium nitrate) are preferably utilized as nitrogen sources.
~;
~ 73~79
This invention relates to a process for producing ethanol from unicellular green algae. More particularly, the present invention relates to a process for producing ethanol by the utilization of starch produced either intra-cellularly or extra-cellularly, or both intracellularly and extracellularly, by unicellular green algae.
This invention is concerned with improvements in ethanol production, based upon the discovery of particular species of unicellular green algae which produce and store starch. More particularly these species will be referred to as Chlorella vulgaris Al-ly-3(11), Chlamydomonas sp.
Al-5 and Scenedosmus _asilensis IAM C-66, all of which are aerobes adapted for heterotrophic culture in a dark, liquid medium containing carbon, nitrogen and inorganic salts.
These strains are found in and are collected from slops and sewages near Tokyo, Japan, and isolated single cellularly.
It is known in the art that ethanol is obtained from the distillation of fermented starch provided by higher plants, such as, for example, corn, wheat, sweet potatoes, ; 20 etc. ~lowever 9 the growth of these plants is influenced by the weather, and their yields greatly depend upon the climatic condition. The current threat of food shortages makes it likely that it will become difficult to obtain ethanol from such agricultural products.
The present invention is thus directed to ethanol production without using such plants. An object of a main aspect of this invention is to provide a process for obtain-ing ethanol by utilizing unicellular green algae as suppliers of ethanol material, the species of algae being advantageously adapted for heterotrophic culture even in places of indus-trial waste.
~L~3733~9 By one broad aspect of the present invention, a process is provided for producing ethanol, the process comprising: aerobically culturing starch-producing unicellular green algae in a culture medium containing an organic com-pound providing a source of assimilable carbon, a sourceof assimilable nitrogen and inorganic salts and other factors necessary for the growth of the algae; rupturing the cellu-lar material to release intra-cellular starch; saccharify-ing the starch produced; fermenting the saccharified starch in the presence of a saccharified starch-utilizing, ethanol- :
producing microorganism; and recovering the ethanol pro-duced from the fermentation medium.
By a prepared aspect of this invention, the algae used is Chlorella vul~aris Al-ly-3(11), Chlamydomonas sp.
Al-5, or Scenedesmus basilensis lAMC-66, as will be described further hereinafter.
By one variant of the preferred aspect, the algae is Chlorella vulgaris Al-ly-3~11) and is cultured at a temperature in the range of 25-42C and at a pH in the range of 5 9, especially where the algae is cultured at a temperature of 37C at a pH in the range of 7-8.
By another variant of the preferred aspect, the algae used is Chlamydomonas sp. Al-5, and is cultured at a temperature in the range of 25-35C and at a pH in the range of 5-9, especially where the ~emperature is 28C
and the pH is from 7-8.
By yet another variant of the preferred aspect, the algae used is Scenedesmus basilensis lAM C-66, and is cultured at a temperature in the range of `25-37C and at a pH in the range of 5-9, especially where the temperature ~73379 is 30C and the pH is from 7-8.
By another aspect of this invention, the culture medium for the algae contains as the source of assimilable carbon, glucose, maltose or acetic acid.
By still another aspect of this invention, the culture medium for the algae is or comprises an industrial wasteO
By still other variants of this invention, the starch product is saccharified using dilute aqueous sulphuric acid or is saccharified by means of enzymes.
Three preferred species of algae are as follows:
(l) Chlorella vulgaris Al-ly-3(11):
The cell is spllerical and 3.3 to 5.0 X3.3 to 5.2 micron large in size, and normally multiplies while splitting into four auto spores. The colour of its colony is initially green, but as it grows, it fades and becomes yellowish.
This species grows at a temperature of 25 to 42~C, but the optimum condition is 37C with pH 5 to 9; preferably pH 7 or 8. A feature of this species is that it produces and accumulates starch both inside and outside the cell, the starch amounting to 70 to 80% of its dry weight, as much as the starch content of cereals. A further advantage is that it discharges two-thirds or less of the in~ernal starch out of the cell.
The strain of this species can be cultured either in the dark or in the light. In the dark, glucose, maltose, and acetic acid are preferably utilized as carbon sources.
Potassium nitrate, polypepton, and ammonium salts (such as, for example, urea, ammonium sulfate, ammonium acetate, and ammonium nitrate) are preferably utilized as nitrogen sources.
~;
~ 73~79
(2) Chlamydomonas sp. Al-5:
The cell is spherical or ellipsoidal, being 5.5 to 8.3 X 6.1 to 9.5 micron large in si7e ~ and normally reproduces as it developes four auto spores. The colour of its colony is dark green irrespective of whether it is in the dark or in the light. The species grows at a tempera-ture of 25 to 35C and at a pH of 5 to 9, but the optimum condition is that the temperature is 28C, and the pH is 7 or 8. Inside the body, a starch of 45 to 60% of its dry weight accululates, but no extra-cellular starch is produced.
The strain of this species can be cultured either in the dark or in the light. ~ the dark, glucose, galactose, and maltose are preferably utilized as carbon sources.
The cell is spherical or ellipsoidal, being 5.5 to 8.3 X 6.1 to 9.5 micron large in si7e ~ and normally reproduces as it developes four auto spores. The colour of its colony is dark green irrespective of whether it is in the dark or in the light. The species grows at a tempera-ture of 25 to 35C and at a pH of 5 to 9, but the optimum condition is that the temperature is 28C, and the pH is 7 or 8. Inside the body, a starch of 45 to 60% of its dry weight accululates, but no extra-cellular starch is produced.
The strain of this species can be cultured either in the dark or in the light. ~ the dark, glucose, galactose, and maltose are preferably utilized as carbon sources.
(3) Scenedesmus basilensis IAM C-66:
The cell is lemon-shaped and 4.2 to 8.4 X 7.7 to 11.2 micron large in si~e. This species grows at a tem-perature of 25 to 37C. and at a pH of 5 to 9, but the optimum conditions are a temperature of 30~C. and a pH of 7 or 8. The colour of its colony is green in the dark as well as in the light. Inside the cell, a starch of 40 to 60% of its dry weight is produced and stored. The strain can be cultured either in the dark or in the light, and in the dark, glucose, fructose, maltose, etc. are preferably utilized as carbon sources.
In common with the aforementioned species, it is preferred to utili~e a place of agriculture waste contain-ing sugar and/or industrial waste containing acetate as a cultural source. This serves an economical purpose.
Glucose, maltose, and acetic acid are prefPrably used as carbon sources. KN03~ NaN03, tNH4)2S0~, ammonium acetate, urea, etc. are preferably used as nitrogen sources. A
small portion of phosphate such asJ for example, K2HPO~, K~2PO4, Na2HPO4 can be added as inorganic salts and a small portion of metallic salts such as, for example, MgSo4.7H2O, CaC12.2H2O, FeS04.7H~O can be added to the culture bed. Preferably a strain is cultured at a temperature of 30 to 35C, and at a pH of 7 to 8. On a small scale, a shaking culture can be made, and on a large scale an aera-tion or stirring culture can be performed. Either a batch culture or a continuous culture is possible. When a batch culture is used the growing volume reaches its maximum in two or four days. Normally the carbohydrate contained in a unicellular alga amounts to 25% or less of its dry weight, but Chlorella vulgaris Al-ly-3(11) has an advantageous ability of storing starch of 70 to 80% of its dry weight both inside and outside the cell.
When a strain is cultured in a batch, the starch begins to accumulate after the logarithmic phase, which occurs in 24 to 48 hours after the culture is started, and the accumulation reaches its maximum during the station-ary phase.
The green algae mentioned above are adapted for culture in the dark, and on mass production they can be cultivated in a tank..
A feature of the present invention lies in the utilization of the starch stored in unicellular algae, so as to obtain ethanol through the saccharification and alcohol fermentation thereof. The alcohol production by fermen-tation is a conventional practice, but this procedure cannotapply to the ethanol production by the utilization of starch produced by green algae because of the characteristica of the cell walls of the green algae. Once the starch is taken out of the cells, saccharification and fermentation of such starch are not different from the conventional processes.
The following is a description of certain steps in the process of aspects of ~his invention.
(1~ Pretreatment:
A culture medium is sub~ected to centrifugal action at 3,000rpm for 10 minutes, and the packed precipitate is liqu~fied with the addition of a small portion of water, thereby obtaining a pasty state of medium. Subsequently, the pasty medium is subjected to a sonic treatment in a lOkHz oscillator for 10 to 20 minutes, 50 as substantially to destroy the alga cells. This treatment increases the starch yield, and facilitates the saccharification of starch.
(2) Saccharification and alcohol fermentation:
(a) Acid saccharification and alcohol fermentation:
A sulphuric acid solution havin~ two- to five- fold quantity of water is added to the pretreated medium~ wherein the concentration of sulphuric acid is adjusted to 25% at maximum and 5% at minimum with respect to the quantity of the starch to be treated. Then the medium is heated in boiling water for 30 minutes, and with the addition of two-to five-fold quantity of water, a saccharifying operation is performed at 1~0C under pressure (2kg/cm2) for 30 minutes.
The obtained saccharified liquor is neutrali~ed to pH value 5.0 with milky lime, and after filtration, appropriate amount-of MgS04.7H20, KH2P04 and urea are added. In addition, an inoculum is added by 10% to this neutralized liquor.
Then the liquor is left as it is, for 3 or 4 days to ensure fermentation. As a result the liquor contains alcohol of 33~79 approximately 6%. ~inally the obtained broth ls distilled ln a known manner to produce alcohol.
(b) Enzymatic saccharification and alcohol fermentation:
A diluted hydrochloric acid is added to the pretreated medium, which is heated at 120C under pressure (2kg/cm2) for 30 minutes. Subsequently the liquid is approximately neutralized with caustic soda, and left as it is, at 55C
for 1.5 hours. The liquid is saccharified with the addition of a suitable enæyme to obtain a saccharified liquor, which is fermented in the same manner as in the case of acid sac-charification.
The following are examples of processes of aspects of thls invention EXAMPLE ~ Culture by glucose Table (1) shows the components of an industrial waste liquid obtained from a glucose manufacturer engaged in the enæymatic saccharification of starch to produce an inverted glucose:
TABLE 1 (mg/ml Organic substances Glucose Inorganic substances 23.8 20.6 0.04 Two g of NaN03, 0.2g of MgS04.7H20, O.O5g of CaC12.
2H20~ 0-025g of FeS04.7H20, 0.8 of K2HP04 and 0.2g of KH2PO~
we~e dissolved in l,OOOml of the above described liquid, and the mixture was distributed by lOOml into 500ml shaking flasks. The liquid in each flask was steriliæed at 120C
under 1.5 atmospheric pressure for 15 minutes, and 10 to 20mg by dry weight of Chlorella vu~aris Al-ly-3(11) was givenp as an inoculum, in each flask, which was subJected to shaking at 130 strokes/min. After 3 days' culturing 1~733~ !
the growth of Chlorella reached its maxlmum condition, and from 1 litre of the culture medium algae as well as extra-cellular starch, totally amounting to 9.5g by dry weight, were obtained, of which the intra- and extra-cellular starch amounted to 7.lg.
In the same manner Chlamydocomas sp.Al-5 and Scendesmus_ basilensis IAM C-66 were cultured. After 3 days the growth thereof reached their maximum condltion, and from 1 litre of the respective liquid 10.3g by dry weight of Chlamydomonas 10 Al-5 and 9.8g by dry weight of Scenedesmus C-66 were obtained, respectively.
EXAMPLE (2) - Culture by acetic acid The medium was cultured with acetic acid, utilizing Chlorella vulgaris Al-ly-3(11), as the carbon source, wherein 15 the aqueous medium contained lg of yeast, 2g of NaN03, 0.2g of MgS04.7H20, 0.05g of CaC12.2~120, 0.025g of FeSO4.7H2O, 0.8g of K2HP04, 0.2g of KH2P04 and lOg of CH3CQONa.3H20.
Then the mixture was distributed by lOOml into 500ml shaking flasks. The initial p~ value o~ the mediu~ was 7.2. The 20 medium in each flask was sterili~ed at 120C under 1.5 atmospheric pressure for 15 minutes, and then 10 to 20ml by dry weight of C~llorella vulgaris Al-ly-3(11) was put in each flask, so as to ensure a shaking culture at 30C.
During the culture the pH value was adjusted to 7.2 with lN acetic acid. After 4 days the growth of Chlorella reached its maximum condition, and from 1 litre of the medium 12.3g by dry weight of algae and extra-cellular starch was obtained, of which the intra- and extra-cellular starch amounted to 8.7g. Throughout the period of 4 days 460ml/1 f lN acetic acid (glacial acetic acid~27.6g) was used to .~
7337~
adjust the pH value.
EXAMPLE (3) - Acid saccharification Chlorella vulgaris Al-ly-3(11), Chlamydomonas sp.Al-5 and Scenedesmus basilensis IAM C-66, each being in a sedi-mentary state under centrifugal action, were liquefledinto paste with the addition of a small portion of pure water, and subgected to a sonic treatment in a lOkHz oscillator for 10 to 30 minutes, thereby substantially destroying their alga cells. Ten g by dry weight of each medium was put into a 200ml conical flask, and each medium in the flask was heated in boiling water for 30 minutes in the presence of 2g of sulphuric acid and 25ml of H20.
Then, with the addition of 25ml of water, the medium was saccharified under pressure (2kg/cm ) for 30 minutes. As a result the rate of saccharification was 90 to 95%.
The saccharified liquid was put in a neutralization tank, where, after cooling, it was neutralized to pH value of 5.0 with milky lime. After the neutralized liquid w~s filtered, MgSO4.7H2O of 0.02%, K2HP04 of 0.2% and urea of 0.1% were added thereto so as to obtain a saccharified liquor. To 50ml of the saccharified liquor (sugar concen-tration~l4.8%), an alcohol fermentation yeast (cf. Brennereihefe Rasse II, XII) of 10% by weight of the liquor was added for innoculation.
EXAMPLE (4) - En~ymatic saccharification ~ach species was liquefied into paste and subjected to a sonic treatment in the same manner as in EXAMPLE (3), and lOg by dry weight of each medium was put in a 200ml triangular flask, where the medium was heated under pressure (2kg/cm2) for 30 minutes in the presence of O.Olg of HCl ~ 3379 and 25ml of H20. In addition, 25ml of H20 was added and an aqueous sodium hydroxide solution was added to neutra-lize the liquid, which was subsequently kept in a water bath having the constant temperature of 55C for 1.5 hours.
Finally saccharification enzyme (cf. that is known by the Trade Mark of Glucenzyme AF-3 produced by Amano Pharmaceutical Co. Ltd.) was added by 0.09% by weight. The saccharifying operation was continued at 55C for 20 hourfi. As a result the rate of saccharification was 95%. The saccharified liquid was filtered so~as to obtain a saccharified liquor.
EXAMPLE (5) - Alcohol fermentation After the saccharified liquor was innoculated with an alcohol fermentation yeast the liquor was sub~ected to fermentation at the temperature of 32C. Preferably, immedi-ately after ~he innoculation the liquor should be well shaked,but after that once a day is enough. Table 2 hereinbelow shows the results of analysis after 4 days' fermentation:
~7337~
~,,,.,~ ... ...
O ~ ~ 1-- n u, ~ ~ ~
o .,. ..
,,, .
o U ~ o~
o oo,~ U~
o ~ ', V o ,, ¢
~0 d :~ o ~ o a~ o o ~ ~o :
The cell is lemon-shaped and 4.2 to 8.4 X 7.7 to 11.2 micron large in si~e. This species grows at a tem-perature of 25 to 37C. and at a pH of 5 to 9, but the optimum conditions are a temperature of 30~C. and a pH of 7 or 8. The colour of its colony is green in the dark as well as in the light. Inside the cell, a starch of 40 to 60% of its dry weight is produced and stored. The strain can be cultured either in the dark or in the light, and in the dark, glucose, fructose, maltose, etc. are preferably utilized as carbon sources.
In common with the aforementioned species, it is preferred to utili~e a place of agriculture waste contain-ing sugar and/or industrial waste containing acetate as a cultural source. This serves an economical purpose.
Glucose, maltose, and acetic acid are prefPrably used as carbon sources. KN03~ NaN03, tNH4)2S0~, ammonium acetate, urea, etc. are preferably used as nitrogen sources. A
small portion of phosphate such asJ for example, K2HPO~, K~2PO4, Na2HPO4 can be added as inorganic salts and a small portion of metallic salts such as, for example, MgSo4.7H2O, CaC12.2H2O, FeS04.7H~O can be added to the culture bed. Preferably a strain is cultured at a temperature of 30 to 35C, and at a pH of 7 to 8. On a small scale, a shaking culture can be made, and on a large scale an aera-tion or stirring culture can be performed. Either a batch culture or a continuous culture is possible. When a batch culture is used the growing volume reaches its maximum in two or four days. Normally the carbohydrate contained in a unicellular alga amounts to 25% or less of its dry weight, but Chlorella vulgaris Al-ly-3(11) has an advantageous ability of storing starch of 70 to 80% of its dry weight both inside and outside the cell.
When a strain is cultured in a batch, the starch begins to accumulate after the logarithmic phase, which occurs in 24 to 48 hours after the culture is started, and the accumulation reaches its maximum during the station-ary phase.
The green algae mentioned above are adapted for culture in the dark, and on mass production they can be cultivated in a tank..
A feature of the present invention lies in the utilization of the starch stored in unicellular algae, so as to obtain ethanol through the saccharification and alcohol fermentation thereof. The alcohol production by fermen-tation is a conventional practice, but this procedure cannotapply to the ethanol production by the utilization of starch produced by green algae because of the characteristica of the cell walls of the green algae. Once the starch is taken out of the cells, saccharification and fermentation of such starch are not different from the conventional processes.
The following is a description of certain steps in the process of aspects of ~his invention.
(1~ Pretreatment:
A culture medium is sub~ected to centrifugal action at 3,000rpm for 10 minutes, and the packed precipitate is liqu~fied with the addition of a small portion of water, thereby obtaining a pasty state of medium. Subsequently, the pasty medium is subjected to a sonic treatment in a lOkHz oscillator for 10 to 20 minutes, 50 as substantially to destroy the alga cells. This treatment increases the starch yield, and facilitates the saccharification of starch.
(2) Saccharification and alcohol fermentation:
(a) Acid saccharification and alcohol fermentation:
A sulphuric acid solution havin~ two- to five- fold quantity of water is added to the pretreated medium~ wherein the concentration of sulphuric acid is adjusted to 25% at maximum and 5% at minimum with respect to the quantity of the starch to be treated. Then the medium is heated in boiling water for 30 minutes, and with the addition of two-to five-fold quantity of water, a saccharifying operation is performed at 1~0C under pressure (2kg/cm2) for 30 minutes.
The obtained saccharified liquor is neutrali~ed to pH value 5.0 with milky lime, and after filtration, appropriate amount-of MgS04.7H20, KH2P04 and urea are added. In addition, an inoculum is added by 10% to this neutralized liquor.
Then the liquor is left as it is, for 3 or 4 days to ensure fermentation. As a result the liquor contains alcohol of 33~79 approximately 6%. ~inally the obtained broth ls distilled ln a known manner to produce alcohol.
(b) Enzymatic saccharification and alcohol fermentation:
A diluted hydrochloric acid is added to the pretreated medium, which is heated at 120C under pressure (2kg/cm2) for 30 minutes. Subsequently the liquid is approximately neutralized with caustic soda, and left as it is, at 55C
for 1.5 hours. The liquid is saccharified with the addition of a suitable enæyme to obtain a saccharified liquor, which is fermented in the same manner as in the case of acid sac-charification.
The following are examples of processes of aspects of thls invention EXAMPLE ~ Culture by glucose Table (1) shows the components of an industrial waste liquid obtained from a glucose manufacturer engaged in the enæymatic saccharification of starch to produce an inverted glucose:
TABLE 1 (mg/ml Organic substances Glucose Inorganic substances 23.8 20.6 0.04 Two g of NaN03, 0.2g of MgS04.7H20, O.O5g of CaC12.
2H20~ 0-025g of FeS04.7H20, 0.8 of K2HP04 and 0.2g of KH2PO~
we~e dissolved in l,OOOml of the above described liquid, and the mixture was distributed by lOOml into 500ml shaking flasks. The liquid in each flask was steriliæed at 120C
under 1.5 atmospheric pressure for 15 minutes, and 10 to 20mg by dry weight of Chlorella vu~aris Al-ly-3(11) was givenp as an inoculum, in each flask, which was subJected to shaking at 130 strokes/min. After 3 days' culturing 1~733~ !
the growth of Chlorella reached its maxlmum condition, and from 1 litre of the culture medium algae as well as extra-cellular starch, totally amounting to 9.5g by dry weight, were obtained, of which the intra- and extra-cellular starch amounted to 7.lg.
In the same manner Chlamydocomas sp.Al-5 and Scendesmus_ basilensis IAM C-66 were cultured. After 3 days the growth thereof reached their maximum condltion, and from 1 litre of the respective liquid 10.3g by dry weight of Chlamydomonas 10 Al-5 and 9.8g by dry weight of Scenedesmus C-66 were obtained, respectively.
EXAMPLE (2) - Culture by acetic acid The medium was cultured with acetic acid, utilizing Chlorella vulgaris Al-ly-3(11), as the carbon source, wherein 15 the aqueous medium contained lg of yeast, 2g of NaN03, 0.2g of MgS04.7H20, 0.05g of CaC12.2~120, 0.025g of FeSO4.7H2O, 0.8g of K2HP04, 0.2g of KH2P04 and lOg of CH3CQONa.3H20.
Then the mixture was distributed by lOOml into 500ml shaking flasks. The initial p~ value o~ the mediu~ was 7.2. The 20 medium in each flask was sterili~ed at 120C under 1.5 atmospheric pressure for 15 minutes, and then 10 to 20ml by dry weight of C~llorella vulgaris Al-ly-3(11) was put in each flask, so as to ensure a shaking culture at 30C.
During the culture the pH value was adjusted to 7.2 with lN acetic acid. After 4 days the growth of Chlorella reached its maximum condition, and from 1 litre of the medium 12.3g by dry weight of algae and extra-cellular starch was obtained, of which the intra- and extra-cellular starch amounted to 8.7g. Throughout the period of 4 days 460ml/1 f lN acetic acid (glacial acetic acid~27.6g) was used to .~
7337~
adjust the pH value.
EXAMPLE (3) - Acid saccharification Chlorella vulgaris Al-ly-3(11), Chlamydomonas sp.Al-5 and Scenedesmus basilensis IAM C-66, each being in a sedi-mentary state under centrifugal action, were liquefledinto paste with the addition of a small portion of pure water, and subgected to a sonic treatment in a lOkHz oscillator for 10 to 30 minutes, thereby substantially destroying their alga cells. Ten g by dry weight of each medium was put into a 200ml conical flask, and each medium in the flask was heated in boiling water for 30 minutes in the presence of 2g of sulphuric acid and 25ml of H20.
Then, with the addition of 25ml of water, the medium was saccharified under pressure (2kg/cm ) for 30 minutes. As a result the rate of saccharification was 90 to 95%.
The saccharified liquid was put in a neutralization tank, where, after cooling, it was neutralized to pH value of 5.0 with milky lime. After the neutralized liquid w~s filtered, MgSO4.7H2O of 0.02%, K2HP04 of 0.2% and urea of 0.1% were added thereto so as to obtain a saccharified liquor. To 50ml of the saccharified liquor (sugar concen-tration~l4.8%), an alcohol fermentation yeast (cf. Brennereihefe Rasse II, XII) of 10% by weight of the liquor was added for innoculation.
EXAMPLE (4) - En~ymatic saccharification ~ach species was liquefied into paste and subjected to a sonic treatment in the same manner as in EXAMPLE (3), and lOg by dry weight of each medium was put in a 200ml triangular flask, where the medium was heated under pressure (2kg/cm2) for 30 minutes in the presence of O.Olg of HCl ~ 3379 and 25ml of H20. In addition, 25ml of H20 was added and an aqueous sodium hydroxide solution was added to neutra-lize the liquid, which was subsequently kept in a water bath having the constant temperature of 55C for 1.5 hours.
Finally saccharification enzyme (cf. that is known by the Trade Mark of Glucenzyme AF-3 produced by Amano Pharmaceutical Co. Ltd.) was added by 0.09% by weight. The saccharifying operation was continued at 55C for 20 hourfi. As a result the rate of saccharification was 95%. The saccharified liquid was filtered so~as to obtain a saccharified liquor.
EXAMPLE (5) - Alcohol fermentation After the saccharified liquor was innoculated with an alcohol fermentation yeast the liquor was sub~ected to fermentation at the temperature of 32C. Preferably, immedi-ately after ~he innoculation the liquor should be well shaked,but after that once a day is enough. Table 2 hereinbelow shows the results of analysis after 4 days' fermentation:
~7337~
~,,,.,~ ... ...
O ~ ~ 1-- n u, ~ ~ ~
o .,. ..
,,, .
o U ~ o~
o oo,~ U~
o ~ ', V o ,, ¢
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4~ p, ~ . . . .
o ~
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~ U
c~ h rd W bO
~ l ¢ ~0 1~ ~ ~ ~Cl~
E~ ~ ~_ . . . . . .
O O O O O O
~q a~
~ .
~d .~
t;OL'~ 11'1 a~ ~D 11') E~ O
O Y
C~ . i r~
~, I ~a ¢ ~ ¢ ¢
Ti N
U~ ¢ ~'~
.
o ~
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~ U
c~ h rd W bO
~ l ¢ ~0 1~ ~ ~ ~Cl~
E~ ~ ~_ . . . . . .
O O O O O O
~q a~
~ .
~d .~
t;OL'~ 11'1 a~ ~D 11') E~ O
O Y
C~ . i r~
~, I ~a ¢ ~ ¢ ¢
Ti N
U~ ¢ ~'~
.
Claims (24)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for producing ethanol, which comprises:
aerobically culturing starch-producing unicellular green algae in a culture medium containing an organic compound providing a source of assimilable carbon, a source of as-similable nitrogen and inorganic salts and other factors necessary for the growth of the algae; rupturing the cel-lular material to release intra-cellular starch; saccharify-ing the starch produced; fermenting the saccharified starch in the presence of a saccharified starch-utilizing, ethanol-producing microorganism; and recovering the ethanol pro-duced from the fermentation medium,
aerobically culturing starch-producing unicellular green algae in a culture medium containing an organic compound providing a source of assimilable carbon, a source of as-similable nitrogen and inorganic salts and other factors necessary for the growth of the algae; rupturing the cel-lular material to release intra-cellular starch; saccharify-ing the starch produced; fermenting the saccharified starch in the presence of a saccharified starch-utilizing, ethanol-producing microorganism; and recovering the ethanol pro-duced from the fermentation medium,
2. A process according to claim 1, wherein the algae used is Chlorella vulgaris A1-ly-3-(11) Chlamydomonas sp. A1-5, or Scenedesmus basilensis lAMC-66 ,
3. A process according to claim 2, wherein the algae used is Chlorella vulgaris A1-ly-3(11) and is cultured at a temperature in the range of 25-42°C and at a pH in the range of 5-9.
4. A process according to claim 3, wherein the algae is cultured at a temperature of 37°C at a pH in the range of 7 8.
5. A process according to claim 2, wherein the algae is Chlamydomonas sp. Al-5 and is cultured at a temperature in the range of 25-35°C and at a pH in the range of 5-9.
6. A process according to claim 5, wherein said temperature is 28°C and said pH is from 7-8.
7. A process according to claim 2, wherein the algae used is Scenedesmus basilensis lAM C-66, and is cul-tured at a temperature in the range of 25-37°C and at a pH in the range of 5-9.
8. A process according to claim 7, wherein said temperature is 30°C and said pH is from 7-8.
9. A process according to claims 1 or 2, wherein the culture medium for the algae contains as tha source of assimilable carbon, glucose, maltose or acetic acid.
10. A process according to claims 3 or 4, wherein the culture medium for the algae contains as the source of assimila-ble carbbn, glucose, maltose or acetic acid.
11. A process according to claims 5 or 6, wherein the culture medium for the algae contains as the source of assimilable carbon, glucose, maltose or acetic acid.
12. A process according to claims 7 or 8 wherein the culture medium for the algae contains as the source of assimilable carbon, glucose, maltose or acetic acid.
13. A process according to claims 1 or 2, wherein the culture medium for the algae is or comprises an indus-trial waste.
14. A process according to claims 3 or 4, wherein the culture medium for the algae is or comprises an in-dustrial waste.
15. A process according to claims 5 or 6, wherein the culture medium for the algae is or comprises an in-dustrial waste.
16. A process according to claims 7 or 8, wherein the culture medium for the algae is or comprises an in-dustrial waste.
17. A process according to claims 1 or 2, wherein the starch product is saccharified using dilute aqueous sulphuric acid.
18. A process according to claims 3 or 4, wherein the starch product is saccharified using dilute aqueous sulphuric acid.
19. A process according to claims 5 or 6, wherein the starch product is saccharified using dilute aqueous sulphuric acid.
20. A process according to claims 7 or 8, wherein the starch product is saccharified using dilute aqueous sulphuric acid.
21. A process according to claims 1 or 2, wherein the starch product is saccharified using an enzyme.
22. A process according to claims 3 or 4, wherein the starch product is saccharified using an enzyme.
23. A process according to claims 5 or 6, wherein the starch product is saccharified using an enzyme.
24. A process according to claims 7 or 8, wherein the starch product is saccharified using an enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA243,024A CA1073379A (en) | 1976-01-06 | 1976-01-06 | Method of obtaining ethanol by the utilization of unicellular green algae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA243,024A CA1073379A (en) | 1976-01-06 | 1976-01-06 | Method of obtaining ethanol by the utilization of unicellular green algae |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1073379A true CA1073379A (en) | 1980-03-11 |
Family
ID=4104908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA243,024A Expired CA1073379A (en) | 1976-01-06 | 1976-01-06 | Method of obtaining ethanol by the utilization of unicellular green algae |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1073379A (en) |
-
1976
- 1976-01-06 CA CA243,024A patent/CA1073379A/en not_active Expired
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