WO2012071443A1 - Milieux de fermentation et procédés de conversion du glycérol en éthanol - Google Patents

Milieux de fermentation et procédés de conversion du glycérol en éthanol Download PDF

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Publication number
WO2012071443A1
WO2012071443A1 PCT/US2011/061904 US2011061904W WO2012071443A1 WO 2012071443 A1 WO2012071443 A1 WO 2012071443A1 US 2011061904 W US2011061904 W US 2011061904W WO 2012071443 A1 WO2012071443 A1 WO 2012071443A1
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nitrogen source
unhydrolyzed
culture medium
glycerol
ethanol
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PCT/US2011/061904
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English (en)
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Matthew S. Wong
Reza Khankal
Paul Campbell
Daniel J. Monticello
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Glycos Biotechnologies, Inc.
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Publication of WO2012071443A1 publication Critical patent/WO2012071443A1/fr

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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
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present disclosure generally relates to fermentation media and fermentation conditions that result in the efficient conversion of glycerol to ethanol by microorganisms.
  • One alternative strategy is to perform a first fermentation to convert glycerol to ethanol, and a second fermentation to convert glucose or another appropriate sugar source to ethanol, followed by mixing the two separate fermentations to achieve a final ethanol titer that is high enough to distill economically.
  • this approach requires running separate fermentation lines.
  • a more practical approach would be to combine the benefits of these two separate fermentations into a single process using the same equipment.
  • the present invention provides methods of converting a carbon source into ethanol efficiently and at reduced costs compared to prior art processes.
  • the method comprises culturing an ethanologenic microorganism that produces an
  • extracellular enzyme such as an exogenous (i.e. extracellular) protease capable of
  • the method comprises converting a carbon source into ethanol using ethanologenic microorganisms wherein the method does not require the addition of enzymes to the cell culture or cell culture medium that are produced outside the cell culture or cell culture medium such as the addition of semi-purified or purified enzyme preparations.
  • all of the enzymes needed for the fermentation process of converting the carbon source to ethanol are inherent to the ethanologenic microorganism.
  • the invention provides a method for the production of ethanol from glycerol comprising culturing an ethanologenic microorganism that produces at least one extracellular enzyme capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for growth, in a suitable culture medium comprising a mixture of glycerol and an unhydrolyzed nitrogen source, thereby converting the ethanol to glycerol, preferably in the absence of adding additional enzyme to the cell culture medium.
  • the invention provides a method for the production of ethanol from sugars and complex carbohydrates comprising culturing an ethanologenic
  • the microorganism that produces at least one extracellular enzyme capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for cell growth, in a suitable culture medium comprising a mixture of an unhydrolyzed nitrogen source, and one or more of sugars and complex carbohydrates, preferably in the absence of the addition of enzymes produced outside the cell culture medium including purified or semi-purified hydrolases for the conversion of complex carbohydrates to mono-, di-, tri- and/or tetrasaccharides.
  • the unhydrolyzed nitrogen source is a plant-based unhydrolyzed nitrogen source including but not limited to, soybean meal, soybean flour, cottonseed meal, cotton seed flour, or a combination thereof.
  • the unhydrolyzed nitrogen source includes sources other than plants such as fish meal and certain meat extracts or a combination thereof.
  • the ethanologenic microorganism is a member of the genus Bacillus, Clostridium, Lactobacillus, Lactococcus, or Paenibacillus.
  • the amount of unhydrolyzed nitrogen source in the culture medium is about 1 to about 50 grams per liter and preferably about 5 to about 40 grams per liter.
  • glycerol is the carbon source and the glycerol in the culture medium is about 1 to about 200 grams per liter, preferably about 5 to about 100 grams per liter, preferably about 10 to about 100 grams per liter and more preferably about 20 to about 50 grams per liter.
  • the present invention provides a method of producing ethanol by culturing an ethanologenic microorganism that produces an extracellular protease that is capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for cell growth, in a suitable culture medium comprising a mixture of glycerol, yeast extract, and one or more of soybean meal, soybean flour, cottonseed meal, and cottonseed flour.
  • the present invention provides a culture medium for culturing an ethanologenic microorganism to convert glycerol into ethanol, the culture medium comprising an ethanologenic microorganism that produces at least one extracellular enzyme capable of converting an unhydrolyzed nitrogen source into a hydrolyzed nitrogen source suitable for use by the microorganism as a nutrient for growth, yeast extract and one or more of soybean meal, soybean flour, cottonseed meal and cottonseed flour, preferably with the proviso that the cell culture medium does not comprise additional enzymes produced outside the cell culture.
  • Figure 1 shows ethanol production from glycerol in various fermentation media at 24 and 48 hour (h) time points during the fermentation.
  • the various media include MM1 , MM14, MM15, simplified MM15 ("Simpl. MM 15") and MM17, as described below.
  • Figure 2 shows ethanol production from glycerol in MM 17 fermentation medium with alterations as follows: MM 17 - SBM refers to MM 17 with the soybean meal omitted; MM 17 - YE refers to MM 17 with the yeast extract omitted; MM 17 - NG refers to MM 17 with the NutriGo 1500 omitted; MM 17 + CM refers to MM 17 in which the soybean meal has been replaced with an equal weight of cottonseed flour.
  • Figure 3 shows the ethanol production from glycerol of a non-sporulating mutated isolate of Paenibacillus macerans Strain LMG 13285 on MM23 fermentation medium which uses soybean flour as the unhydrolyzed nitrogen source.
  • ethanologenic microorganism refers to a microorganism with the ability to convert at least a portion of a carbon source including, but not limited to, glycerol, to ethanol.
  • the ethanologenic microorganisms are ethanologenic by virtue of their ability to express one or more enzymes that individually or together convert at least a portion of a carbon source such as glycerol to ethanol.
  • Examples of ethanologenic organisms include yeast, bacteria or fungi.
  • suitable microorganisms include, but are not limited to, a member of the genus Bacillus, Clostridium, Lactobacillus, Lactococcus, or Paenibacillus.
  • the ethanologenic microorganism is Paenibacillus macerans.
  • One preferred strain of Paenibacillus macerans is Strain LMG13285 of the Belgian Coordinated Collections of Micro-Organisms.
  • enzymes suitable produced by ethanologenic microorganisms include but are not limited to: glycerol dehydrogenase and dihydroxyacetone kinase (conversion of glycerol to dihydroxyacetone phosphate, a common intermediate with the glucose pathway); a CoA-linked aldehyde dehydrogenase and an alcohol dehydrogenase (Gupta, et al. (2009) Appl. Env. Microbiol. 75: 5871-5883).
  • ethanologenic microorganisms in accordance with the invention also express extracellular enzymes that are particularly suitable for utilizing unhydrolyzed nitrogen sources.
  • extracellular enzymes include proteases capable of converting unhydrolyzed proteins to hydrolyzed nitrogen sources such as amino acids suitable for use as a nitrogen source by the microorganisms.
  • Suitable proteases expressed by preferred microorganisms of the invention include, but are not limited to serine proteases which are typically present in the Bacillus/Paenibacillus species and glutamic acid proteases also known to be present in certain Bacillus species
  • the microorganism used in the culture system express sufficient enzyme to convert adequate amounts of unhydrolyzed nitrogen sources to a nitrogen source to support growth of the organisms in the absence of the addition of proteases and other enzymes, purified enzymes or purified enzyme components from sources outside the cell culture system.
  • Ethanologenic microorganisms may also be genetically engineered to express the desired enzymes suitable for use in the present invention.
  • genetically engineered organisms include those expressing and secreting one or more enzymes particularly suitable for converting a carbon source such as glycerol to ethanol and/or organisms expressing enzymes particularly suitable for utilizing unhydrolyzed, nitrogen sources.
  • the microorganism may be genetically engineered to overexpress a suitable enzyme native to the organism, or to express/overexpress an enzyme that is heterologous.
  • exogenous protease is a protease produced or expressed by an ethanologenic microorganism that is secreted by the microorganism into the extracellular space.
  • exogenous protease is used interchangeably herein with the terms “extracellular protease” and “secreted protease”.
  • the phrase "culturing an ethanologenic microorganism that produces an exogenous protease in a suitable culture medium” refers to growing a population of microorganisms under suitable conditions in a liquid or solid medium in a fermentation process that involves the enzymatic and anaerobic breakdown of carbon based substances by microorganisms to produce simpler end products such as ethanol. While fermentation occurs under anaerobic conditions, it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation may also occur in the presence of oxygen.
  • microorganisms disclosed herein are known to the skilled worker trained in microbiological and recombinant DNA techniques.
  • Methods and techniques for growing microorganisms e.g., bacterial cells
  • transporting isolated DNA molecules into the host cell and isolating, cloning and sequencing isolated nucleic acid molecules, knocking out expression of specific genes, etc.
  • These methods are described in many items of the standard literature, which are incorporated herein in their entirety: "Basic Methods In Molecular Biology” (Davis, et al, eds.
  • a knocked-out gene is a gene whose encoded product, e.g., a protein, does not or substantially does not perform its usual function or any function.
  • a knocked-out gene can be created through deletion, disruption, insertion, or mutation.
  • microorganisms that lack one or more indicated knocked-out genes are also considered to have knock outs of the indicated gene(s).
  • the microorganisms themselves may also be referred to as knock outs of the indicated gene(s).
  • Such knock outs can also be conditional or inducible, using techniques that are well-known to those of skill in the art.
  • knock ins in which a gene, or one or more segments of a gene, are introduced into the microorganism in place of, or in addition to, the endogenous copy of the gene.
  • the present invention offers several advantages over currently employed methods of producing ethanol in a fermentation process.
  • One advantage is the economy of scale offered by the use of less expensive nitrogen sources employed in the fermentation medium.
  • the preferred methods according to the invention do not require the addition of any commercially generated enzymes such as those enzymes necessary for converting
  • the enzymes employed are preferably produced in sufficient amounts by the ethanologenic microorganisms used in the cell culture system. As commercially generated enzymes are expensive, the invention is more economical than those methods which do require additional commercial enzymes.
  • Yet another advantage of the methods of the invention is that the entire process can be conducted in a single fermentation process which yields almost entirely ethanol.
  • the methods of the invention also advantageously employ by-products of other processes (such as those generating glycerol) and avoid the need for hydrolysis of nitrogen sources which have their own waste disposal issues.
  • Proflo is a cottonseed protein product obtained from Archer Daniels Midland (Decatur, IL). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
  • MM1 was made by the following procedure.
  • An MM1 concentrate was prepared by mixing 80 ml of 1.0 M Tricine (adjusted to pH 7.4 by NaOH), 20 ml of 10 mM FeS0 4 , 100 ml of 1.9 M NH 4 C1, 20 ml of 276 mM K 2 S0 4 , 20 ml of 0.5 mM CaCl 2 , 20 ml of 528 mM MgCl 2 , 200 ml of 5 M NaCl, 2 ml of a micronutrient solution, and purified water up to 2 L.
  • the micronutrient solution contained 30 ⁇ ( ⁇ 4 ) ⁇ 7 ⁇ 2 4, 4 mM H 3 B0 3 , 300 ⁇ CoCl 2 , 100 ⁇ CuS0 4 , 800 ⁇ MnCl 2 , 100 ⁇ ZnS0 4 .
  • MM14 contained 3 g Na 2 HP0 4 , 12 g NaH 2 P0 4 , 0.5 g (NH 4 ) 2 S0 4 , 0.5 g K 2 S0 4 , 30 g tryptone, 25 g soy peptone enzymatic digest, and 3 g yeast extract. The medium was sterilized by autoclave.
  • Soybean meal was obtained from Hieden Feed and Supply (Houston, TX), yeast extract was obtained from Ohly Americas (Hutchison, MN), and all other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
  • MM 15 contained 0.25 g Na 2 HP0 4 , 1 g NaH 2 P0 4 , 1 g K 2 S0 4 , 6 g Ohly KAT, and 36.8 g soybean meal. The medium was sterilized by autoclave, and 20 ml of a Minerals /Vitamins mix was added.
  • the Minerals /Vitamins mix was a 50/50 v/v mixture of a Minerals solution and a Vitamins solution.
  • the Minerals solution was 1.22 g MgS0 4 and 108 mg FeCl 3 6H 2 0 in 25 ml water.
  • the Vitamins solution was 100 mg thiamine HCl, 2 mg biotin, and 1 ml Trace mix in 1 L of water.
  • the Trace mix was 40 mg calcium pantothenate, 20 mg pyridoxine HCl, and 8 mg cyanocobalamin in 1 L of water.
  • the Minerals /Vitamins mix was filter sterilized.
  • Soybean meal was obtained from Hieden Feed and Supply (Houston, TX)
  • yeast extract was obtained from Ohly Americas (Hutchison, MN)
  • NutriGo 1500 was obtained from Nutrients Incorporated (Manitowoc, WI).
  • Per liter, Simplified MM 15 contained 36.8 g soybean meal, 6 g yeast extract, and 1.8 g NutriGo 1500. The medium was sterilized by autoclave.
  • Soybean meal was obtained from Hieden Feed and Supply (Houston, TX), yeast extract was obtained from Ohly Americas (Hutchison, MN), and NutriGo 1500 was obtained from Nutrients Incorporated (Manitowoc, WI).
  • MM 17 contained 36.8 g soybean meal, 6 g yeast extract, and 0.3 g NutriGo 1500. Soybean meal and yeast were sterilized by autoclave. NutriGo 1500 was pasteurized for 1 min at 105°C.
  • P. macerans, strain LMG 13285 was sourced from the Belgian Coordinated Collections of Microorganisms and is capable of converting glycerol to ethanol in high yields. This strain was maintained on plates containing Luria-Bertani (LB) medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar per liter).
  • LB Luria-Bertani
  • Ethanol, glycerol, and organic acid concentrations were determined using a Shimadzu LC-20AD HPLC equipped with a UV-monitor (210 nm) and refractive index detector (RID). Products were separated using an Aminex HPX-87H column (Bio-Rad Laboratories) with 2.5 mM H 2 SO 4 as the mobile phase (0.6 ml min "1 , 55° C).
  • the fermentation seed culture was prepared as follows: the cultures (stored as glycerol stocks at -80°C) were streaked onto LB plates and incubated overnight at 42°C. Single colonies growing on these plates were used to inoculate 40 ml (NBYE medium as described above) seed cultures in 250 ml baffled flasks grown aerobically at 42°C and 175 rpm overnight. These cultures were used to provide inocula in the 0.5-L fermentor.
  • Fermentations were carried out for 48 hours. Samples were collected at 24 and 48 hours and analyzed for nutrients and metabolites, as described above.
  • Soy peptone present in MM 14 is an enzymatically hydrolyzed soy protein product.
  • the hydrolysis is necessary for many organisms that do not produce sufficient levels of protease to degrade the soy proteins and liberate free amino acids and short oligopeptides to use as nutrients.
  • the hydrolysis step whether by acid hydrolysis or protease hydrolysis, and subsequent purification steps add additional cost to the soy protein.
  • Soybean meal and a low-cost yeast extract were the most effective combination of nutrients, resulting in medium MM 15, a combination of soybean meal, yeast extract and trace minerals and vitamins. This medium cost $0.09/L; the productivity of the LMG13285 culture achieved 27.6 g/L ethanol produced from glycerol in 48 h. Although the media cost was reduced by two orders of magnitude, the amount of ethanol produced was less than desired.
  • MM 15 when used in large-scale fermentations, attempts were made to reduce the number of components in the medium.
  • the complex trace minerals and vitamins were replaced with 1.8 g/L NutriGo 1500.
  • Pasteurization of the NutriGo instead of autoclaving, reduced the amount used to 0.3 g/L.
  • MM 17 the productivity of the LMG 13285 culture reached 35.3 g/L ethanol produced in 48 h for a price of $0.09/L; thus, a simple medium consisting of an unhydrolysed nitrogen source (soybean meal), yeast extract, and NutriGo 1500 achieved good performance with a low cost.
  • each of the components affects the efficient conversion of glycerol to ethanol by LMG 13285.
  • the omission of soybean meal (MM 17 - SBM, Figure 2) reduced productivity by over 50%.
  • the omission of yeast extract (MM17 - YE, Figure 2) reduced productivity even more; performance fell from 35.3 g/L in 48 h to 6.9 g/L of ethanol; the absence of NutriGo 1500 had only a marginal impact on performance, reducing the ethanol titer to 29 g/L at 48 h.
  • Soybean meal may be replaced by other unhydrolyzed nitrogen sources, such as cotton flour (Proflo). At an equal weight (36.8 g/L), cotton flour can substitute soybean meal with comparable productivity: 31 g/L ethanol at 48 h.
  • cotton flour can substitute soybean meal with comparable productivity: 31 g/L ethanol at 48 h.
  • MM23 was prepared without the use of casein or NutriGo 1500 supplement to be more economical.
  • the soy flour was obtained from Honeyville Food Products (Rancho Cucamonga, CA) and the Ohly KAT was obtained from Ohly Americas (Hutchison, MN).
  • MM23 contained 36.8 g soy flour and 6 g Ohly KAT. The soy flour and Ohly KAT were sterilized by autoclave.
  • Ohly KAT is an expensive component of MM23, accounting for $0.07/L out of the $0.09/L total cost.
  • the soy flour component of the medium accounts for the other $0.02/L.
  • yeast products in an effort to replace Ohly KAT and cut medium costs. They included many samples of yeast extract or yeast autolysate (the extract is refined autolysate) and one sample of dried whole yeast. The table below lists all of our costs for media made with each yeast product. The ethanol production is listed, as well as the cost per ton ethanol for the medium.
  • the Hy-Yest 413 provides yeast extract provides the best compromise between cost and ethanol titer. Yeast Product Cost of $/L for $/L for Total EtOH 72 h $/te

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Abstract

La présente invention concerne des procédés permettant de convertir une source de carbone en éthanol de manière efficace et à moindre coût en comparaison avec les procédés de l'état de la technique. Dans un mode de réalisation, le procédé comprend la culture d'un micro-organisme éthanologène qui produit une enzyme extracellulaire, telle qu'une protéase, capable d'hydrolyser des sources d'azote non hydrolysées dans un milieu de culture approprié, le milieu de culture comprenant un mélange contenant une source de carbone, telle que du glycérol, des sucres et des glucides complexes, et une source d'azote non hydrolysée, telle qu'une farine brute, une farine de soja, une farine de graines de coton ou d'autres substances riches en azote, en tant que nutriments, et la conversion de la source de carbone en éthanol par le micro-organisme. Dans un mode de réalisation, le procédé comprend la conversion d'une source de carbone en éthanol, le procédé ne nécessitant pas l'ajout d'enzymes à la culture cellulaire ou au milieu de culture cellulaire, produites hors du système de culture cellulaire, comme l'ajout de préparations enzymatiques semi-purifiées ou purifiées.
PCT/US2011/061904 2010-11-26 2011-11-22 Milieux de fermentation et procédés de conversion du glycérol en éthanol WO2012071443A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009043012A1 (fr) * 2007-09-27 2009-04-02 Mascoma Corporation Fermentation progressive de biomasse de lignocellulose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009043012A1 (fr) * 2007-09-27 2009-04-02 Mascoma Corporation Fermentation progressive de biomasse de lignocellulose

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EUROPEAN COMMISSION: "Opinion of the Scientific Committee on Food on beta-cyclodextrin produced using cycloglycosyltransferase obtained from Paenibacillus macerans.", DIRECTORATE C - SCIENTIFIC OPINIONS C3 - MANAGEMENT OF SCIENTIFIC COMMITTEES II; SCIENTIFIC CO-OPERATION AND NETWORKS., 11 July 2000 (2000-07-11), RUE DE LA LOI 200, B-1049 BRUSSELS. *
GUPTA A: "Fermentative utilization of glycerol and lignocellulosic sugars and production of ethanol by Paenibacillus macerans.", THESIS, DOCTOR OF PHILOSOPHY., January 2010 (2010-01-01), HOUSTON TEXAS. *
OLAJUYIGBE ET AL.: "Production dynamics of extracellular protease from Bacillus species.", AFRICAN JOURNAL OF BIOTECHNOLOGY, vol. 4, no. 8, August 2005 (2005-08-01), pages 776 - 779 *

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