DESCRIPTION Technical field

The invention provides a consortium of microorganisms capable of hydrolyzing cellulose, preferably lignocellulosic biomass, a preparation comprising it, use of the consortium of microorganisms and/or the preparation for catalyzing the hydrolysis of cellulose, preferably lignocellulosic biomass and/or increasing the efficiency of biogas production in the methane fermentation process and/or reviving and/or propagating methanogenic consortia, and/or methanogenic microorganisms themselves, comprising a use of the consortium of microorganisms capable of hydrolyzing cellulose, preferably lignocellulosic biomass, and/or the preparation comprising it. The invention also provides a a supplement preparation for reviving and propagating groups of methanogenic microorganisms, produced using the consortium of microorganisms capable of hydrolyzing cellulose, preferably lignocellulosic biomass, , and/or the preparation comprising it. The invention also provides a combination preparation, comprising the consortium of microorganisms capable of hydrolyzing cellulose and/or the preparation comprising the consortium, as well as a supplement preparation.

Background art

Degradation of lignocellulosic biomass is a process used in many branches of industry, mainly in the production of biofuels. Various kinds of energy crops are used to produce high- quality biofuels, such as biogas (biomethane) or bioethanol. The key step, limiting the production of the aforementioned biofuels, is the process of hydrolysis of lignocellulosic material to simpler carbohydrates. In the production of bioethanol, hydrolysis of the plant matter leads to the formation of simple sugars, which undergo alcoholic fermentation. On the other hand, in the production of biogas, plant substrates undergo hydrolysis and then the processes of acidogenesis, acetogenesis and finally methanogenesis. Too slow rate of hydrolysis of lignocellulose leads to stalling of the entire process of degradation of plant biomass and, consequently to the reduction of the efficiency of the fermentation process. On the other hand, excessive hydrolysis of plant substrates can lead to too great an accumulation of intermediate products, which may contribute to system overload and deceleration (or complete inhibition) of the activity of microorganisms carrying out the final stages of biofuel production. The efficiency of hydrolysis and plant biomass degradation processes is dependent on the presence and activity of cellulolytic microorganisms. In agricultural biogas plants and in wastewater treatment plant digesters, the hydrolysis process is dependent on the presence of cellulolytic microorganisms in the input material (i.a. slurry, manure or sewage sludge). If the input material used is poor in cellulolytic microflora, the hydrolysis process will be slow and unstable. Stabilization of an appropriate microflora can take up to several months, which, obviously has a direct impact on the efficiency of the process and on economic benefits.

Currently, the biogas market is trying to respond to the problems caused by the unstable activity of microbial consortia. A great need for a biologically active preparation, comprising a stable and well-controlled consortium of microorganisms for the hydrolysis and degradation of lignocellulosic biomass, is observed. Due to the unstable activity and the lack of the ability to control plant biomass degradation, nearly 20% of biogas plants close their operation within the first two years on the market. Preparations that would enable efficient hydrolysis and operation of the process in a controlled manner are sought. Preparations for increasing the viability and efficiency of the microorganisms involved in the production of biogas in the methane fermentation process as well as their revival are also sought.

Microbiological preparations (i.a. produced by German and Danish companies), are available on the biogas market, typically being used for faster ensilage of corn, grass or other batch plants. They are derived from preparations that has been used for preparation of animal feeds for years. Silasil Energy. C, Jbs progas, or AntaSil BG comprise a mixture of bacteria from the genus Lactobacillus, whose presence in the silage improves the content of lactic acid, which is a substrate for the microorganisms responsible for methane fermentation. According to the manufacturers' advertising, through such activity, the efficiency of obtaining methane increases by approx. 5%.

In the group of preparations dispensed directly into digesters, mainly mineral supplements of methane fermentation are found. Products such as IPUSmeth-Max are mixtures of micronutrients and substances buffering the resulting ammonia, too high concentrations of which is a methanogenesis inhibitor. According to the manufacturer, its use can bring up to 10% increase of the efficiency of methane production, but it requires constant expenses to purchase the preparation, the dosage of which should be almost daily. In this group, one of the very few products containing microorganisms is AntaFerm BG, consisting of both cultures of microorganisms and trace elements. In the case of using the above preparations, investments of 25-42 euros per 30 tons of input are required. These are expensive solutions, which do not always give a radical improvement in the functioning of the biogas plant.

Preparations intended directly for plant biomass hydrolysis are also known. These are enzymatic preparations which are stable, but they are usually specific for a particular pool of substrates, because they are derived from one strain (typically from fungi). Moreover, such preparations are expensive, because they must be systematically and regularly added to the working biogas plant. Such preparations include, among others, Celuferm (Eurozyme) and BG Max (Novozymes).

Currently preparations for the hydrolysis of lignocellulosic biomass, which: (i) after a single addition, would ensure long-term and stable operation of the biogas plant, (ii) would be relatively inexpensive, (iii) would have a broad spectrum of activity, would be universal and resistant to the changing environmental conditions, are sought. In summary, preparations employing a bacterial biomass, capable of propagation using the utilized lignocellulosic material are sought. Thus, preparations, which will increase the viability of methanogenic microorganisms and eventually support the efficiency of gas production by methanogenic microorganisms using lignocellulosic material are also sought.

Disclosure of the invention

The invention provides a consortium of microorganisms capable of hydrolyzing cellulose, preferably lignocellulosic biomass, comprising the following mixtures of bacterial strains, which have been deposited on the 14 ^th May 2014 in the Polish Collection of Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, in Wroclaw, Poland: Bacillus sp. KP7, KP20 and Ochrobactrum sp. KP8 (Digest- Prep A - a mixture deposited under the no. B/00064), Providencia sp. KP14; Bacillus sp. KP6 and KP16 (Digest- Prep B - a mixture deposited under the no. B/00065), Bacillus sp. KP4, KP5, KP17 and KP22 (Digest- Prep C - a mixture deposited under the no. B/00066), Providencia sp. KP10; Bacillus sp. KP1 and KP19 (Digest- Prep D - a mixture deposited under the no. B/00067), Ochrobactrum sp. KP13; Bacillus sp. KP9 and KP12 (Digest- Prep E - a mixture deposited under the no. B/00068). The consortium according to the invention was named the Digest-Prep consortium. In the preferred embodiments of the present invention, particular strains in each mixture are mixed in equal proportions. Preferably all the prepared mixtures are combined at an equal quantitative ratio.

The invention also provides a preparation named Digest-Prep for catalyzing the hydrolysis of cellulose, preferably lignocellulosic biomass and/or increasing the efficiency of biogas production in the methane fermentation process and/or reviving and/or propagating methanogenic consortia, and/or methanogenic microorganisms themselves, comprising the consortium of microorganisms according to the invention. Preferably, such preparation also comprises supplementary and/or auxiliary substances.

The present invention is based on a mixture of strains developed by the inventors and the preparation comprising it, named Digest-Prep comprising Digest-Prep A - the mixture deposited under the no. B/00064, Digest-Prep B - the mixture deposited under the no. B/00065, Digest- Prep C - the mixture deposited under the no. B/00066, Digest-Prep D - the mixture deposited under the no. B/00067, Digest-Prep E - the mixture deposited under the no. B/00068) for the hydrolysis of cellulose, preferably lignocellulosic biomass. The Digest-Prep preparation comprises a consortium including 16 strains of bacteria specialized in lignocellulosic biomass degradation, isolated from different environments (agricultural biogas plant hydrolyzer from Miedzyrzec Podlaski, raw sludge from the wastewater treatment plant Czajka in Warsaw, slurry and cattle manure from farm households in Niemoglowy and Trzebieszow Pierwszy). The consortium consists of strains selected from strains representing the bacteria of the genera Bacillus, Ochrobactrum, and Providencia. The invention is based upon an unexpected finding that the bacterial strains isolated from the aforementioned environments, which are part of the consortium according to the invention and the Digest-Prep preparation according to the invention are characterized by a high activity of cellulose degradation (determined on the basis of tests with carboxymethylcellulose) and the ability to function in a broad range of stress factors (under aerobic and anaerobic conditions, at pH 4-10, temperature, salinity). The Digest-Prep consortium and the Digest-Prep preparation accelerate the degradation of lignocellulosic biomass, releasing many organic compounds and allowing supplementation of the fermentation and increase of the biogas production by 10 to 40%, depending on the substrate used.

The present invention also relates to a supplement preparation named Supp-Digest-Prep for supplementation of methane fermentation, which comprises organic and inorganic substances derived from the degradation of biomass produced using a consortium of microorganisms, and which is produced using the consortium of microorganisms according to the invention and/or the preparation comprising the consortium according to the invention. Such preparation supports the viability and efficiency of methanogenic microorganisms. The consortium of microorganisms and/or the preparation according to the invention can provide effective hydrolysis of cellulose, preferably lignocellulosic biomass and/or increase in the efficiency of biogas production, even after a single administration, also having influence on the supporting of viability and efficiency of methanogenic microorganisms.

In another aspect, the invention comprises the use of the consortium of microorganisms according to the invention and/or the preparation according to the invention for catalyzing the hydrolysis of cellulose, preferably lignocellulosic biomass. Preferably, the consortium of microorganisms and/or the preparation are used directly in digesters. In the preferred embodiment, the use according to the invention leads to increase in the efficiency of biogas production in the methane fermentation process. In another aspect of the invention, the Digest- Prep consortium and preparation according to the invention enable the production of methane fermentation supplements, which increase the efficiency of biogas production by up to 40%, and decrease the retention time in the digester.

In one of the embodiments the Digest-Prep consortium and/or preparation according to the invention are used for manufacturing a supplement preparation for supplementation of methane fermentation, named the Supp-Digest-Prep preparation. In the preferred embodiment, the supplement preparation for supplementing methane fermentation according to the invention comprises the supernatant obtained after centrifugation of cultures of bacterial strains included in the consortium of microorganisms according to the invention and/or the preparation comprising the consortium according to the invention.

Preferably, the supplement preparation for supplementation of the methane fermentation according to the invention is added to the medium for methanogenic consortia, and/or methanogenic microorganisms themselves at a ratio of 1 :20 of the medium. In another aspect of the invention, the Digest-Prep consortium according to the invention and/or the Digest-Prep preparation comprising the consortium according to the invention and/or the Supp-Digest-Prep supplement preparation for supplementation of the methane fermentation according to the invention, are used for reviving and/or propagating methanogenic consortia and/or methanogenic microorganisms themselves, including methanogenic archaea. Thus, the invention also relates to the use of the consortium of microorganisms according to the invention and/or the preparation according to the invention and/or the supplement preparation according to the invention and/or the Supp-Digest-Prep-Plus combination preparation according to the invention for reviving and/or propagating methanogenic consortia, and/or methanogenic microorganisms themselves.

The invention also relates to the Supp-Digest-Prep-Plus combination preparation for catalyzing the hydrolysis of cellulose, preferably lignocellulosic biomass and/or increasing the efficiency of biogas production in the methane fermentation process and/or reviving and/or propagating methanogenic consortia, and/or methanogenic microorganisms themselves. The Supp-Digest-Prep-Plus combination preparation comprising the consortium and/or the Digest- Prep preparation in combination with the supplement preparation, Supp-Digest-Prep, and is used for supporting the viability of methanogenic microorganisms and productivity of methanogenic microorganisms, supplementing methane fermentation and ensuring effective hydrolysis of cellulose, preferably lignocellulosic biomass and/or increasing the efficiency of biogas production, preferably after a single administration.

The Supp-Digest-Prep-Plus combination preparation used for enhancing viability and supplementing methane fermentation yields a synergistic effect and mediates an increase in the production of biogas by 25 to 48% and methane concentration in the obtained biogas by even 75%, and a decrease of the retention time in the digester (what directly affects the speed of the conducted process).

The invention also relates to a method of catalyzing the hydrolysis of cellulose, preferably lignocellulosic biomass, comprising the use of the Digest-Prep consortium of microorganisms according to the invention and/or the Digest-Prep preparation according to the invention and/or the Supp-Digest-Prep-Plus combination preparation according to the invention. In the preferred embodiment of the method, the use of the consortium and/or the preparation according to the invention and/or the combination preparation according to the invention leads to the supporting of viability and productivity of methanogenic microorganisms and increase in the efficiency of biogas production in the methane fermentation process.

Preferably, in the method according to the invention, the consortium of microorganisms and/or the preparation according to the invention, are used directly in the digesters.

In a preferred embodiment, the hydrolysis is carried out under anaerobic conditions at 30°C. In a preferred embodiment, the hydrolysis is carried out at pH=7.

In a preferred embodiment, the consortium of microorganisms and/or the preparation according to the invention are used together with the methanogenic consortium.

The invention also relates to a method of reviving and/or propagating methanogenic consortia, and/or methanogenic microorganisms themselves, comprising the use of the consortium of microorganisms according to the invention (the Digest-Prep consortium) and/or the preparation according to the invention (the Digest-Prep preparation) and/or the preparation for supplementation of the methane fermentation according to the invention (the Supp-Digest-Prep preparation) and/or the combination preparation according to the invention (the Supp-Digest- Prep-Plus preparation). To prepare the Digest-Prep consortium and/or preparation according to the invention, five smaller mixtures of the Digest-Prep strains A, B, C, D and/or E must first be prepared, each of which is composed of several different microbial cultures. The mixtures comprise the following cultures:

• Digest-Prep A: Bacillus sp. KP7, KP20; Ochrobactrum sp. KP8 (the mixture deposited in PCM under the no. B/00064)

• Digest-Prep B: Providencia sp. KP14; Bacillus sp. KP6, KP16 (the mixture deposited in PCM under the no. B/00065)

• Digest-Prep C: Bacillus sp. KP4, KP5, KP17, KP22 (the mixture deposited in PCM under the no. B/00066)

• Digest-Prep D: Providencia sp. KP10; Bacillus sp. KP1, KP19 (the mixture deposited in PCM under the no. B/00067)

• Digest-Prep E: Ochrobactrum sp. KP13; Bacillus sp. KP9, KP12 (the mixture deposited in PCM under the no. B/00068)

In all the cases, the procedure for preparing components of the mixtures is identical. This procedure is based on the obtaining of pure cultures of bacteria in a liquid medium with cellulose or derivative thereof, as the sole source of carbon. On the basis of the growth period of bacteria in the medium, the number of cells can be determined by known techniques (for example by fluorescence staining with DAPI dye) and the particular strains can be mixed, preferably in equal proportions. The density of cells in each of the Digest-Prep mixtures (A, B, C, D, E) is preferably about 10 ⁷ -10 ⁸ . The so prepared mixture/s of microorganisms can be stored using the methods known in the art, for example, after lyophilization.

In order to obtain the Digest-Prep cellulolytic consortium, all the prepared mixtures should be mixed together, preferably in an equal quantitative ratio (so as the number of cells per ml of each strain in each mixture was the same). The Digest-Prep preparation can be further supplemented with additional supplementary, auxiliary, stabilizing substances, etc. The added substances may also affect e.g. the obtaining of a high density of biomass, maintaining an increased biochemical activity, increase in the access to nutrients or additionally increase in the survival rate.

In order to obtain the preparation for the supplementation of methane fermentation, named the Supp-Digest-Prep preparation, culture of microorganisms included in the Digest-Prep consortium and/or the preparation according to the invention must be carried out, and subsequently, the supernatant from this culture should be obtained by methods known in the art, for example, by centrifugation. The obtained supernatant should be sterilized. The so-obtained preparation for the supplementation of methane fermentation can be used to supplement the medium for methanogenic consortia, as well as methanogenic microorganisms themselves, including methanogenic archaea.

The term "consortium" or "consortium of microorganisms", as used herein is intended to mean a group of bacterial strains capable of growing together, and interacting in lignocellulose degradation.

The term "consortium", also called the Digest-Prep consortium, as used herein is intended to mean a group of 16 strains (forming the Digest-Prep mixtures: A - the mixture deposited in PCM under the no. B/00064, B - the mixture deposited in PCM under the no. B/00065, C - the mixture deposited in PCM under the no. B/00066, D - the mixture deposited in PCM under the no. B/00067, E - the mixture deposited in PCM under the no. B/00068), capable of growing together, and interacting in lignocellulose degradation.

The term "preparation", also called the Digest-Prep preparation, as used herein is intended to mean a mixture comprising the Digest-Prep consortium and supplementary and/or auxiliary substances. The supplementary and/or auxiliary substances can be for example any media components, carriers, stabilizers, supplements for culturing bacteria, and mixtures thereof known in the art.

The term "supplement preparation", also called the Supp-Digest-Prep preparation or Supp-Digest-Prep, as used herein is intended to mean a sterile solution containing organic (i.a. volatile fatty acids, sugars, vitamins) and inorganic substances (i.a. ammonia, phosphates) deriving from the degradation of biomass, preferably plant biomass, most preferably from the degradation of corn silage, using the Digest-Prep consortium or preparation according to the invention.

The term "combined preparation", also called Supp-Digest-Prep or the Supp-Digest-Prep preparation, as used herein is intended to mean a mixture of the Digest-Prep consortium of microorganisms and/or the Digest-Prep preparation according to the invention with the Supp- Digest-Prep preparation. Such a combined preparation, named Supp-Digest-Prep-Plus will be used for catalyzing the hydrolysis of cellulose, preferably lignocellulosic biomass and/or increasing the efficiency of biogas production in the methane fermentation process and/or reviving and/or propagating methanogenic consortia, and/or the methanogenic microorganisms themselves. Figures

Publications cited in the description, and the references given therein, are in their entirety incorporated herein as references. For a better understanding of the invention, it has been illustrated by the embodiments and in the accompanying figures, wherein:

Fig. 1 shows the results of DGGE electrophoretic separation for: pure cultures being the components of the mixtures Digest-Prep A, Digest-Prep B, Digest-Prep C, Digest-Prep D, Digest-Prep E; the Digest-Prep consortium; the Digest-Prep consortium grown at 3% corn silage, and indigenous microflora present in corn silage, which is used as a substrate in the experiments.

Fig. 2 is a graph showing the effect of hydrolysis of lignocellulosic biomass under the influence of the Digest-Prep preparation (control sample - 3% silage without the addition of the Digest- Prep consortium and/or the Digest-Prep preparation; 3% silage+Digest-Prep - culture enriched with the Digest-Prep consortium; 3% silage+GB+Digest-Prep - methanogenic consortium enriched with the Digest-Prep consortium).

Fig. 3 shows the influence of the activity of the Digest-Prep preparation on biogas production by methanogenic consortium with the use of corn silage as a substrate (GB - methanogenic consortium from the culture collection of the Laboratory of Environmental Pollution Analysis).

Fig. 4 is a graph showing the cumulative efficiency of biogas production (dm ³ CiL kg ^"1 d.w. of corn silage) in the culture of the GB methanogenic consortium before revival, operating in a stable mode (GB-WT) and during revival on minimal medium with the addition of the Supp- Digest-Prep preparation (GB-Supp-Digest-Prep) and without the addition of Supp-Digest-Prep (GB).

Fig. 5 is a graph showing the cumulative efficiency of biogas production (dm ³ CH ₄ kg ^_1 d.w. of corn silage) in the culture of the GB methanogenic consortium working without the addition of the Digest-Prep preparation, Supp-Digest-Prep, with the addition of the Digest-Prep preparation only, and with the addition of the Supp-Digest-Prep-Plus combined preparation (the Digest-Prep preparation in combination with Supp-Digest-Prep).

Fig. 6 is a graph showing the effect of the activity of the Digest-Prep preparation and the synergistic effect of the Supp-Digest-Prep-Plus combined preparation (the Digest-Prep preparation in combination with Supp-Digest-Prep) on the percentage content of methane in the obtained biogas in the culture with methanogenic consortium (GB - from the culture collection of the Laboratory of Environmental Pollution Analysis). The following examples are presented merely to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims.

In the following examples, unless it was otherwise indicated, standard materials and methods described in Sambrook and Russell. 2001. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York, were used, or the manufacturers' instructions for specific materials and methods were followed.

Examples

Example 1. Selection and identification of bacterial strains with an increased hydrolytic activity of lignocellulosic biomass.

Bacteria capable of degrading cellulose were isolated from inoculum samples from: (i) agricultural biogas plant hydrolyser from Miedzyrzec Podlaski, (ii) raw sludge from the wastewater treatment plant Czajka in Warsaw, (iii) slurry and cattle manure from farm households in Niemglowy and Trzebieszow Pierwszy. Selection was performed using the modified minimal medium of the following composition: peptone 5 g/1; yeast extract 5 g/1; NaCl

5 g/1; KH ₂ P0 ₄ 1 g/1; MgS0 ₄ 0.2 g/1; carboxymethylcellulose (CMC) 10 g/1; pH 7.0 (Bushnell and

Haas, 1941). Cultures were carried out in 200 ml of the medium with the addition of 0.5% of dry weight of the initial inoculum, at 30 C, 120 rpm for 72 hours. Subsequently, the culture was passaged onto minimal medium with CMC. After each of the 6 passages made, bacteria were plated on minimal medium with CMC solidified with agar, and incubated for 72 hours at 30 C.

Pure cultures, characterized by varying morphology were selected by replica method onto a fresh medium. From all the conducted cultures, a pool of -100 pure cultures was obtained. Strains which were characterized by an increased ability to degrade carboxymethylcellulose were selected for further analysis. Preliminary studies on the CMC degradation activity were carried out on a modified medium with carboxymethylcellulose and Congo Red of the following composition: KH ₂ P0 ₄ 0.5 g/1; MgS0 ₄ 0.25 g/1; carboxymethylcellulose 2 g/1; gelatin 2 g/1; Red

Congo 0.2 g/1. The pH of the medium was adjusted to 6.8 - 7.2 and solidified with agar 15 g/1

(Hendricks et al., 1995). The use of Congo Red as indicator and an inductor of cellulose degradation on solid medium constitutes the basis for a rapid and sensitive assay for cellulolytic bacteria. Discolorations around a colony indicate a positive result regarding the degradation.

After the initial selection a pool of -50 strains capable of degrading CMC and characterized by an increased cellulolytic activity (i.e. clear zones on CMC+Congo Red medium were > 15mm) was obtained (Tab. 2). The next step was the elimination of the same strains. For this purpose, Amplified Ribosomal DNA Restriction Analysis (ARDRA) was used. Each of the investigated strains was subjected to the so-called rapid cell lysis in lysis buffer (0.05M NaOH, 0.25% SDS). Subsequently, the resulting lysate was used as template DNA in the amplification of the 16S rRNA gene using the primers 27F and 1492R (Lane, 1991). The obtained PCR products were digested with Haelll enzyme and electrophoretic analysis was performed. Based on this, unique strains, representing each of the selected environments (hydrolyzer, slurry, manure) were chosen (Tab. 1)

In order to identify the selected strains, 16S rRNA genes of each strain were amplified, cloned in the pGEM-T-Easy vector, and then sequenced. Sequencing was performed using the primers 27F and 1492R. Computer analyses included the processing of the obtained sequences using programs: FinchTV ver. 1.4; Clone Manager Professional Suite ver. 8.0; Blast (http://blast.ncbi.nlm.nih.gov/). The sequences of the 16S rRNA genes of the identified strains were deposited in the GenBank database, NCBI. The results of the identification of pure cultures are presented in Table 1.

Table 1. Identification of bacteria included the "DigestPrep" consortium, degrading lignocellulosic biomass

Classification based on the analysis of 16S rDNA

Strain no. Origin 16S

(accession (site of isolation) rDNA number in Organism Identity

sequence GenBank) (accession number in GenBank) (%) (SEQ ID.

NO)

KP1 Biogas plant hydrolyzer Bacillus licheniformis strain Pb- 99 1

(KJ777134) Miedzyrzec Podlaski HK09002 (HM006898.1)

KP16 Biogas plant hydrolyzer Bacillus aerius strain RGS230 99 12

(KJ777149) Miedzyrzec Podlaski (KC469617.1)

KP22 Biogas plant hydrolyzer Bacillus licheniformis strain AnBa7 99 16

(KJ777154) Miedzyrzec Podlaski (AY887129.1)

KP4 Cattle manure Bacillus pumilus strain SAFR-032 98 2

(KJ777137) Niemglowy (NR_074977.1)

KP5 Cattle manure Bacillus pumilus strain Jo2 99 3

(KJ777138) Niemglowy (KF734912.1)

KP6 Cattle manure Bacillus pumilus strain 43 (KF923453.1) 99 4

(KJ777139) Niemglowy

KP7 Cattle manure Bacillus altitudinis strain EH36 96 5

(KJ777140) Niemglowy (GU339265.1)

KP8 Cattle manure Uncultured Ochrobactrum sp. clone 98 6

(KJ777141) Niemglowy DM11-150 (KC172366.1)

KP9 Cattle manure Bacillus sp. B2066 (JX266376.1) 99 7

(KJ777142) Niemglowy

KP10 Cattle manure Providencia vermicola strain FFA6 99 8

(KJ777143) Niemglowy (JN092794.1)

KP12 Cattle manure Uncultured Bacillus sp. clone Filt.123 99 9 (KJ777145) Niemglowy (HM152710.1)

KP17 Cattle manure Bacillus subtilis isolate SCS-3 99 13

(KJ777150) Niemglowy (EU257431.1 )

KP13 Cattle manure Uncultured Ochrobactrum sp. clone 98 10

(KJ777146) Trzebieszow Pierwszy DM11-150 (KC172366.1)

KP14 Cattle manure Providencia vermicola strain FFA6 95 11

(KJ777147) Trzebieszow Pierwszy (JN092794.1)

KP19 Cattle manure Bacillus pumilus strain 38 (KF923448.1) 99 14

(KJ777151) Trzebieszow Pierwszy

KP20 Cattle manure Uncultured Bacillus sp. clone Filt.123 99 15

(KJ777152) Trzebieszow Pierwszy (HM152710.1)

Example 2. Determination of the activity of extracellular cellulolytic enzymes.

In order to show that the selected strains are characterized by a high cellulolytic activity, a detailed, quantitative assay of the activity of cellulolytic enzymes was carried out using the modified method developed by Ghose T.K. (1987). Cellulolytic activity was determined indirectly based on the quantity of reducing sugars (glucose) in the reaction mixture, resulting from the hydrolysis of carboxymethylcellulose by enzymes secreted from the selected microorganisms cultured on minimal medium with CMC. To 0.5 ml of double-diluted culture supernatant an equal volume of 2% solution of carboxymethylcellulose was added, and incubated for 30 minutes at 50°C. After the incubation, 3 ml of 3,5-dinitrosalicylic acid (DNS) were added to the reaction mixture and it was incubated again for 5 minutes in boiling water. Subsequently, the samples were cooled and 20 ml of distilled water were added, and vigorously mixed. Absorbance of the reaction mixture was measured at a wavelength of 540 nm. Glucose standards in the range of 0.5 - 2 mg/ml and a blank, to which, instead of the culture, 0.1 M of sodium acetate of pH 5,5 was added, were prepared analogously to the method above. The amount of reducing sugars was determined from the standard curve constructed as the dependency of absorbance on the amount of glucose. The enzymatic activity of endoglucanase (CMCase) is defined in international units (IU). One unit of enzymatic activity is defined as the amount of enzyme which releases 1 μπιοΐ of reducing sugars (measured as glucose) per ml within 1 minute. Cellulolytic activity was determined for the pure cultures as well as for the mixture of strains, included in the consortium labelled as Digest-Prep.

Among the 16 isolated bacterial strains there are microorganisms characterized by increased endoglucanase cellulolytic activity (Tab. 2). The range of activity for all the strains was from 0.127 IU/ml for the KP11 strain, to 0.547 IU/ml for the KP16 strain. The KP16, KP22 strains, isolated from the agricultural biogas plant hydrolyzer and the KP19 strain, from cattle manure, are characterized by the highest activity of extracellular enzymes. All these bacteria belong to the Bacillus genus. For comparison, exemplary literature data report that the CMCase activity for the reference cellulolytic strain Cellulomonas sp. ASN2 may be about 0.400 IU/ml, depending on the composition and pH of the medium, as well as the temperature at which the cultures were carried out.

The results of cellulolytic activity, obtained by carrying out the cultures of strains on the medium with carboxymethylcellulose as the sole source of carbon, pH 7, at 30°C, are shown in Tab. 2. In the case of the results of cellulolytic activity obtained on the basis of the test carried out on the medium with Congo Red as the inducer for degradation of cellulose, the results obtaineddiffered from those for CMCase. The basis of the test was the investigation of the size of clear zones around the colonies of bacteria. All the results were higher than the minimal value found in the literature, i.e. 15 mm. The highest results were obtained for the strains KP1, KP20, KP22, KP16. Lack of correlation between the results of the two variants of the cellulolytic activity studies ,may be explained by the technique of the performed tests and the main principle of action. In the case of CMCase, the amount of the end product formed as a result of action of cellulolytic enzymes was investigated, whereas in the case of the test with Congo Red in the medium, the result may depend on the diffusion of the enzymes in the medium, the amount of the enzymes, or the size of the colony.

Table 2. The results of cellulolytic activity and the optimal conditions for the growth of the selected bacteria.

Cellulolytic activity Optimal conditions for growth

KP19 0.497 30

K P20

Example 3. Determination of the optimal conditions for the growth of pure cultures and the Digest-Prep consortium.

In order to determine the optimal conditions for the growth for individual strains of bacteria and the consortium, microorganisms were cultured on medium with CMC in different growth conditions (carboxymethylcellulose substrate concentration, temperature, pH).

To determine the optimum pH, the tested strains of bacteria were passaged onto minimal medium with carboxymethylcellulose, having pH from 4 to 10, respectively. The density of the cultures at the beginning of the experiment was set at approx. 10 ⁶ cfu/ml. The cultures were incubated for 96 hours at 37°C. The OD ₆ oonm value measurements were performed every 24 h.

In order to determine the optimal growth temperature, the tested strains of bacteria were passaged onto minimal medium with carboxymethylcellulose, having pH of 7. The cultures were incubated for 96 h at: 23°C, 30°C, 37°C, 45°C. The OD ₆ oonm value measurements were performed every 24 h.

In order to determine the optimal concentration of the substrate - carboxymethylcellulise, the tested strains of bacteria were passaged onto minimal medium with 0.5%, 1%, 1.5%, 2% concentration of carboxymethylcellulose (CMC) having pH of 7.0. The cultures were incubated for 96 hours at 37°C. The OD ₆ oonm value measurements were performed every 24 h.

In all the tested bacterial strains growth was observed in the above-described ranges of growth conditions, which in fact may somewhat constitute stress factors for these organisms. However, for each strain, different optimum growth conditions were obtained. For most strains, the optimum temperature for growth was about 30°C and about 37°C. This temperature was higher only for the KP8 strain and it was 45°C. In case of pH of the medium on which the cultures were carried out, the range of the best conditions for growth was from pH 4, for the KP9 strain to pH 10 for the strains: KP4, KP6, KP7, KP8, KP20. The most optimal pH of the medium for most of the strains was 7. As for the concentration of carboxymethylcellulose as the substrate, the range was also quite broad for the isolated bacteria and it ranged between 0.5 and 2% of the CMC concentration. Most of the strains cope very well with the hydrolysis of CMC at high concentrations and prefer such conditions for optimal growth.

The above results may indicate a quite flexible range of optimal conditions, which allow bacterial growth. The obtained results allow to determine the best conditions for growth (temp. 30-37°C; pH 7-10 and 1-2% CMC concentration) for the artificially constructed mixtures of strains as well as the Digest-Prep consortium and preparation. Moreover, a broad range of tolerance for external factors allows a mixture of microorganisms to survive even in these extreme and adverse external conditions. The results are shown in Table 2.

Example 4: Construction of the Digest-Prep consortium and preparation, and its component Digest-Prep A, B, C, D, E mixtures.

To prepare the Digest-Prep consortium and preparation, smaller mixtures of the Digest-Prep strains A, B, C, D, E, obtained in Example 1., consisting of several different cultures of bacteria were prepared first. The mixtures comprise the following cultures of bacteria:

• Digest-Prep A: Bacillus sp. KP7, KP20; Ochrobactrum sp. KP8 (the mixture deposited under the no. B/00064)

• Digest-Prep B: Providencia sp. KP14; Bacillus sp. KP6, KP16 (the mixture under the no.

B/00065)

• Digest-Prep C: Bacillus sp. KP4, KP5, KP17, KP22 (the mixture deposited under the no.

B/00066)

• Digest-Prep D: Providencia sp. KP10; Bacillus sp. KP1, KP19 (the mixture deposited under the no. B/00067)

• Digest-Prep E: Ochrobactrum sp. KP13; Bacillus sp. KP9, KP12 (the mixture deposited under the no. B/00068)

In all the cases, the procedure for preparing components of the mixtures was identical. This procedure is based on obtaining pure cultures of bacteria on a liquid medium with CMC. For this purpose, night culture was cultivated at 30°C with shaking 120 rpm. After a period of bacterial growth on the medium, the number of cells per ml was determined (fluorescence staining using DAPI dye) and the individual strains were mixed in equal proportions. The density of cells per ml in each of the Digest-Prep mixtures (A, B, C, D, E) was set at 10 ⁷ -10 ⁸ . The so prepared mixtures of microorganisms were lyophilized for storage purposes and deposited in the Polish Collection of Microorganisms at Lud.wik Hirszfeld Institute of Immunology and Experimental Therapy in Wroclaw.

In order to obtain the cellulolytic Digest-Prep consortium itself all prepared mixtures were mixed together in equal quantitative ratio (so that cell number/ml of each strain of each mixture was equal).

Example 5. Testing for the presence of individual strains in the Digest-Prep preparation. In order to verify the presence of bacteria in the mixtures of strains constructed in Example 4, isolation of the total DNA of each of the pure cultures according to the method developed by Chen and Kuo in 1993, and isolation of the total DNA in the mixture of strains cultured in LB medium and mixed at a ratio of 1 : 1 (v/v) was performed. In order to verify the stability of the strains in the consortium growing on a plant substrate, DNA was isolated (using a method developed by Zhou et al., 1996) from cultures on corn silage as the cellulose substrate (concentration of the substrate was 3% of dry weight). On the template of the total isolated DNA, fragments of the 16S rRNA gene were amplified (using the primers specific for the bacteria and according to the method described in Example 1) and then, using the obtained fragment of the 16S rRNA as template, the V3 16S rRNA variable region was amplified. The reaction mixture was prepared according to the instructions of the manufacturer of the Taq DNA Polymerase kit, in a volume of 25 μΐ. Amplification of DNA of the V3 16S rRNA region was performer using primers 357F and 519R (Muyzer et al., 1993). The PCR products were subjected to qualitative analysis by denaturing gradient gel electrophoresis (DGGE). Separation was carried out in the DCode Universal Mutation Detection (BioRad) system according to the modified method described by Nakatsu et al. (2000). The denaturing factor gradient was in the range from 30 to 55%, whereas a mixture of 7 M urea and 40% formamide constituted 100% solution of the denaturing factor. 6% polyacrylamide gel was used in the electrophoretic separation. Electrophoresis was carried out in lx TAE buffer at 60°C, for 30 minutes at a voltage of 30V, and then at a voltage of 200V for 3.5 hours. After electrophoresis, the gel was stained in SYBR gold solution (1 : 10 000) and the results of the separation were visualized using the Image Quant software (GE Healthcare). DGGE electrophoresis results indicate bands corresponding to various bacterial genes, characteristic for the individual pure cultures. The results for the individual strains resemble the electrophoretic DNA profile of the constructed consortium, which includes all the strains. DGGE electrophoresis results allowed to roughly estimate and compare the diversity of microorganisms in the consortium. Moreover, the obtained results prove the stability of the individual strains in the mixture. The results are shown in Fig. 1.

Example 6. Degradation of lignocellulosic biomass - corn silage.

To verify what is the real cellulolytic activity of the Digest-Prep preparation constructed in Example 4, tests were carried out using corn silage - a substrate which is commonly used in biogas plants world wide. Corn silage is a substrate, the basic component of which is cellulose, which after hydrolysis to simple sugars (glucose) constitutes an assimilable carbon source for most microorganisms. The tests were carried out under conditions reflecting the operation of the industrial hydrolysers from the two-component biogas plants. The cultures were carried out in a medium containing low-mineralized water with the addition of 3% d.w. of corn silage, under anaerobic conditions at 30°C for 72 hours. The inoculum (the Digest-Prep preparation, obtained in Example 4), constituted 10% (v/v) of the entire volume of the conducted culture (giving a density of the culture at the level of ~10 ⁷ cells/ml). Due to the acidic pH of the corn silage, the initial pH of the culture was increased to 7 using a sodium hydrogencarbonate solution. During the cultivation, samples were taken to determine the pH of the culture, cellulolytic activity of the mixture of strains in the consortium, as well as to determine the changes in the number of bacteria in the consortium mixture and the species composition of the consortium. In order to determine the stability of the strains in the mixture in the culture, DNA isolation and DGGE electrophoresis was performed according to the description in Example 5. The stability of all the 16 strains in the Digest-Prep consortium was confirmed. The results of the cellulolytic activity of the mixture of strains is shown in Fig. 2.

The results of the experiment based on the estimation of the amount of reducing sugars, according to the test with DNS, show that the highest cellulolytic activity of the Digest-Prep preparation occurs after 72 hours of conducting the culture. It is after this incubation period that the highest concentration of glucose in the culture enriched with the cellulolytic consortium was observed. The concentration of glucose in this culture reaches up to 3.25 mg/ml and is 5-times higher than the concentration in the control sample devoid of supplementation with the consortium. With time, the concentration of glucose in the culture decreases. After 144 hours a 4-fold decrease of concentration of glucose is observed, which is probably due to the use of the obtained sugars in the metabolic processes of the strains included in Digest-Prep. The product of cellulose degradation - glucose is also observed in the control sample, probably because bacteria naturally present in corn silage have developed. The concentration level in the control is, however, definitely lower than in the tested sample, and the concentration of sugar decreases in the course of the experiment. Another result presented on the graph is the concentration of glucose during degradation of corn silage in the experiment carried out with methanogenic consortium and the Digest-Prep preparation. The result after 72 hours also illustrates a high concentration of the resulting glucose, which proves the cooperation of the microorganisms included in the methanogenic consortium and the strains of bacteria from the Digest-Prep preparation. The obtained result indicates the possibility of using the Digest-Prep preparation for the degradation of lignocellulosic substrate - corn silage to glucose, in order to provide a source of nutrients for methanogenic microorganisms.

Example 7. The effect on the efficiency of biogas production - supporting methanogens. The main purpose of the Digest-Prep consortium and preparation is to enable the pre- treatment and degradation of lignocellulosic biomass. The products resulting from the hydrolysis of corn silage can be used by other groups of microorganisms in the entire methane fermentation process, having an influence on the level and quality of the obtained biogas. In order to verify the effect of the activity of the consortium on the quality and efficiency of biogas production, an experiment was set with the mixtures: the strains included in the Digest-Prep preparation with the GB methanogenic consortium from the culture collection of the Laboratory of Environmental Pollution Analysis. The following variants of the experiment with the mixtures of the Digest- Prep preparation with the GB methanogenic consortia were performed:

(1) 20% (v/v) of the GB methanogenic consortium with a density of the culture at the level of ~10 ⁹ cells/ml (determined using DAPI fluorescence staining and determination by fluorescence microscopy) was mixed with 10% (v/v) mixture of the Digest-Prep strains with a density of the culture at the level of ~10 ⁸ cells/ml.

(2) 20%) (v/v) of the GB methanogenic consortium (which corresponds to the density of the culture at the level of ~10 ⁹ cells/ml) was mixed with 5% (v/v) mixture of the Digest-Prep strains (which corresponds to the density of the culture at the level of ~10 ⁷ cells/ml).

(3) 20%) (v/v) of the GB methanogenic consortium (which corresponds to the density of the culture at the level of ~10 ⁹ cells/ml) without the addition of Digest-Prep, as the control culture.

The cultures were carried out in glass bioreactors with the active volume of 400 ml using gas bags for measuring the quality of gases. The substrate in the experiment was corn silage at a final concentration of 1%> by dry weight. After 72 hours of incubation at 30°C, the temperature of the culture was raised to 37°C. The methane fermentation process was carried out for 21 days, monitoring the volume and quality of the resulting biogas every 7 days. In all the three variants production of biogas was at a similar level (maximally 15 dm ³ of biogas/day/kg of d.w. of corn silage) was observed. The observed differences were related to the quality of the produced biogas (Fig. 3.). GC-MS chromatographic analyses showed that the addition of the Digest-Prep preparation influences the improvement of the quality of the produced biogas. The maximum concentration of methane, observed in the control culture was 28.45%, and in the variants with the addition of 10% and 5% (v/v) of the Digest-Prep preparation 56.19% and 60.66% CH _4, respectively. These results prove the effectiveness of the Digest-Prep preparation as the "enhancer" of the production of a high-quality biogas. The addition of the Digest-Prep preparation supports the viability and efficiency of methanogenic microorganisms. Example 8. The use of the Digest-Prep preparation for producing supplements necessary in reviving and functioning of methanogens.

During the degradation of the combined lignocellulosic biomass by cellulolytic bacteria many organic compounds, i.a. volatile fatty acids, simple carbohydrates and many intermediate metabolites are released. These compounds are used by successive groups of microorganisms in the alcoholic or methane fermentation process. In the presented example it was shown that the constructed Digest-Prep consortium/preparation can be used for preparation of supplements of the methane fermentation process. Such supplements can be used in both well-working biogas plants in order to increase the efficiency of the process and increase the rate of the biogas production process by shortening the retention time in the digester, as well as in order to revive the fixed (e.g. frozen of lyophilized) methanogenic microorganisms.

To demonstrate that the Digest-Prep consortium can be used in the production of supplements for methanogenic microorganisms a procedure for preparing and using such formulations was developed. Preparation of the preparations for revival comprised several steps. The first was a partial degradation of lignocellulosic biomass, namely running the culture of the Digest-Prep preparation for 72 hours at 30°C on minimal medium enriched with 3% of d.w. of corn silage at pH 7. After 72 hours of incubation, the entire culture was centrifuged (10 000 rpm, 30 min), culture supernatant was poured into a new vessel and subjected to extraction in an autoclave at 121°C for 25 minutes, in order to eliminate the remains of microorganisms of the initial culture. The obtained extract (called the Supp-Digest-Prep preparation) was kept at 4°C until the use in reviving of the methanogenic consortia.

The next step was the enrichment with the Supp-Digest-Prep preparation in a ratio 1 :20 of the medium (e.g. 50 ml Supp-Digest-Prep+950ml of the medium) used for culturing microorganisms, which were to be revived or propagated.

In order to verify the effectiveness of the Supp-Digest-Prep preparation, revival of a consortium of methanogenic microorganisms was performed in two variants: (i) control - without the enrichment of the culture with the Supp-Digest-Prep preparation and (ii) basic - with the addition of the Supp-Digest-Prep preparation. For revival, consortia of methanogenic microorganisms labeled as GB in the culture collection of the Laboratory of Environmental Pollution Analysis were used. The methanogenic consortia were kept at -70°C in the mixture of 10% glycerol and 10% DMSO. In order to revive the consortia, they were gradually thawed at 4°C, and then cultures were started in laboratory reactors with a final volume of 900 ml. As the substrate 1% of d.w. of shredded corn silage was used, and 0, 1% yeast extract was added as an additional source of carbon. The cultures were supplemented with a mixture of vitamins 10 ml/1 and Touvinen's salts 2 ml/1. In the basic variant, the cultures were supplemented with 50 ml/1 of the Supp-Digest-Prep preparation, and in the control variant - without the enrichment with the Supp-Digest-Prep preparation. The cultures were started under anaerobic conditions in an anaerobic chamber and were carried out for 14/21 days. During culture, the increase in the volume of the produced biogas was monitored. In the control variant, without the addition of the Supp-Digest-Prep preparation, trace biogas production (at the level of 0.8 dm /day/kg of d.w. of corn silage) was observed, while in the basic variant revival of the consortium of methanogenic microorganisms and biogas production was reported (Fig. 4). The efficiency of biogas production in the revived methanogenic consortium was maximally -11 dm ³ biogas/day/lkg of d.w. of corn silage, and in the stabilized methanogenic consortium working before freezing, the efficiency was ~8 dm ³ biogas/day/lkg of d.w. of corn silage. The obtained results indicate that the use of the Supp-Digest-Prep preparation affects the revival and promotes viability and efficiency of the consortium of methanogenic microorganisms.

Example 9. The influence of the Supp-Digest-Prep-Plus combination preparation on the quality and efficiency of biogas produced in methane fermentation.

Due to the constructed Digest -Prep consortium and preparation and the Supp-Digest-Prep preparation it is possible to affect various stages of the methane fermentation process. Both at the hydrolysis step, with the use of the Digest-Prep consortium and/or preparation, and during methanogenesis, by providing the produced supplements, contained in the Supp-Digest-Prep preparation, to methanogenic microorganisms.

In order to investigate the combined effect of the Digest-Prep consortium/preparation combined with the Supp-Digest-Prep preparation, methane fermentation process was carried out using methanogenic consortia (GB - from the culture collection of the Laboratory of Environmental Pollution Analysis) in the following variants: (i) control (GB-WT) - without the addition of preparations to the culture, (ii) GB+Digest-Prep - with the addition of the Digest-Prep preparation, (iii) GB+ Supp-Digest-Prep-Plus - culture enriched with both the Digest-Prep preparation and the supplements of Supp-Digest-Prep. The cultures were carried out in laboratory reactors with a final volume of 900 ml.

As the substrate, 1% d.w. of shredded corn silage was used. The cultures were supplemented with a mixture of vitamins 10 ml/1 and Touvinen's salts 2 m/1. In all the variants, 75% (v/v) GB methanogenic consortium was used. In the variant with the Digest-Prep preparation, the cultures were enriched with 10% and 5% (v/v) Digest-Prep preparation, respectively. In the variant with the combined Supp-Digest-Prep-Plus preparation, 10%> (v/v) Digest-Prep preparation and 10%> Supp-Digest-Prep were used. The cultures were started under anaerobic conditions in an anaerobic chamber and were carried out for 14 days. During culture, the volume of the produced biogas and methane content in the biogas were monitored by GC-MS chromatographic analyses. In all the variants differences in the amount of the produced biogas were observed (Fig. 5). In the control variant, biogas production at the level of 15-18 dm /day/kg of d.w. of corn silage was observed. In the variant with the addition of the Digest-Prep preparation and the addition of the Supp-Digest-Prep-Plus combination preparation, biogas production at the level of 30-35 dm /day/kg of d.w. of corn silage was reported. The GC-MC chromatographic analyses supplemented the results concerning the quality of the produced biogas (Fig. 6). The maximum concentration of methane in the control culture was 37%, in the culture with the Digest-Prep preparation - 40-45%, and in the culture with the Supp-Digest-Prep-Plus combination preparation, it reached up to 58%. The obtained results indicate an unexpected synergistic effect of the preparation on the methane fermentation process. The results indicate the enhanced effectiveness of the combined effect of these preparations. As a result, the use of such a combination preparation enables and facilitates the production of a high quality biogas.

Literature:

Bushnell D. L. i Haas H. F., 1941. The utilization of certain hydrocarbon by microorganisms; Kansas Agricultural Experiment Station, 199: 653 - 673,

Chen W. P. i Kuok T. T. 1993. A simple Rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res. 21: 2260.

Gathogo E. W., Waugh A. C., Peril N., Red path M. B., Long P. F., 2003 Colony PCR amplification of actinomycetes DNA. J. Antibiot. 56: 423-424.

Ghose T. K. 1987. Measurement of cellulase activities. Pure Appl Chem 59: 257 - 268.

Hendricks C. W., Doyle J. D., Hugley B, 1995. A new solid medium for enumerating cellulose- utilizing bacteria in soil; Applied and Environmental Microbiology , 61: 2016 - 2019,

Lane D. J., 16S/23S rRNA sequencing. 1991. (Stackebrandt E., Goodfellow M., red.) Nucleic Acid Techniques in Bacterial Systematics. John Wiley and Sons: New York

Muyzer G., Waal de E. C. Uitterlinden A. G., 1993. Profiling of combined microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction- amplified genes coding for 16S rRNA. Appl. Environ, Microbiol. 59: 695 - 700 SEQUENCE LISTING

<110> University of Warsaw

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<213> Bacillus pumilus

<400> 4

gattaccagt ttgatcctgg ctcaggacga acgctggcgg cgtgcctaat acatgcaagt 60 cgagcggaca gaagggagct tgctcccgga tgttagcggc ggacgggtga gtaacacgtg 120 ggtaacctgc ctgtaagact gggataactc cgggaaaccg gagctaatac cggatagttc 180 cttgaaccgc atggttcaag gatgaaagac ggtttcggct gtcacttaca gatggacccg 240 cggcgcatta gctagttggt ggggtaatgg ctcaccaagg cgacgatgcg tagccgacct 300 gagagggtga tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca 360 gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg agtgatgaag 420 gttttcggat cgtaaagctc tgttgttagg gaagaacaag tgcgagagta actactcgca 480 ccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 540 gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa 600 gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat tggaaactgg gaaacttgag 660 ttgcagaaga ggagagtgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720 acaccagtgg cgaaggcgac tctctggtct gtaaactgac gctgaggagc gaaagcgtgg 780

2 ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 840 agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc tggggagtac 900 ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 960 gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaaccctag 1020 agatagggct ttcccttcgg ggacagagtg acaggtggtg catggttgtc gtcagctcgt 1080 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccagca 1140 tttagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1200 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac agaacaaagg 1260 gctgcgagac cgcaaggttt agccaatccc ataaatctgt tctcagttcg gatcgcagtc 1320 tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1380 atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgc aacacccgaa 1440 gtcggtgagg taacctttat ggagccagcc gccgaaggtg gggcagatga ttggggtgaa 1500 gtcgtaacaa ggtaaccaat cactagt 1527

<210> 5

<211> 1408

<212> DNA

<213> Bacillus altitudinis

<400> 5

aaggtaacca agtagagttt gatcctggct cattaagtcg taacaaggta accaagtaga 60 gtttgatcct ggcgcaaacc gtcgcaacaa gggacccgtt ccttgaaccg catggttcaa 120 ggatgaaaga cggtttcggc tgtcacttac agatggaccc gcggcgcatt agctagttgg 180 tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca 240 ctgggactga gacacggccc acactcctac gggaggcagc agtagggaat cttccgcaat 300 ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct 360 ctgttgttag ggaagaacaa gtgcaagagt aactgcttgc accttgacgg tacctaacca 420 gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 480 cggaattatt gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc 540 ggctcaaccg gggagggtca ttggaaactg ggaaacttga gtgcagaaga ggagagtgga 600 attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac 660 tctctggtct gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac 720 cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt 780 gctgcagcta acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa 840 aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 900 agaaccttac caggtcttga catcctctga caaccctaga gatagggctt tcccttcggg 960 gacagagtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 1020 tcccgcaacg agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt 1080 gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1140 acctgggcta cacacgtgct acaatggaca gaacaaaggg ctgcgagacc gcaaggttta 1200 gccaatccca caaatctgtt ctcagtgatc atggctgtgt aagtcgtaac aaggtaacca 1260 agtagagttt gatcctggct cagtaagttg taacaaggta accaagtaga gtttgatcct 1320 ggctcagtaa gtcgtaacaa ggtaaccaag tagagtttga tcctggctca gtaagtcgga 1380 acaaaggaac caacttgggt tgtctgcg 1408

<210> 6

<211> 1459

<212> DNA

<213> Ochrobactrum sp

<400> 6

gattagagtt tgatcatggc tcagaacgaa cgctggcggc aggcttaaca catgcaagtc 60 gaacggtctc ttcggaggca gtggcagacg ggtgagtaat gcatgggaat ctaccattct 120 ctacggaata actcagggaa acttgtgcta ataccgtata cgcccttttg gggaaagatt 180 tatcggagag tgatgagccc atgttggatt agctagttgg tggggtaaag gcctaccaag 240 gcgacgatcc atagctggtc tgagaggatg atcagccaca ctgggactga gacacggccc 300 agactcctac gggaggcagc agtggggaat attggacaat gggcgcaagc ctgatccagc 360 catgccgcgt gagtgatgaa ggtcttagga ttgtaaagct ctttcaccgg tgaagataat 420 gacggtaacc ggagaagaag ccccggctaa cttcgtgcca gcagccgcgg taatacgaag 480 ggggctagcg ttgttcggat ttactgggcg taaagcgcac gtaggcggac ttttaagtca 540 ggggtgaaat cccagagctc aactctggaa ctgcctttga tactggaagt cttgagtatg 600 gaagaggtga gtggaattcc gagtgtagag gtgaaattcg tagatattcg gaggaacacc 660 agtggcgaag gcggctcact ggtccattac tgacgctgag gtgcgaaagc gtggggagca 720 aacaggatta gataccctgg tagtccacgc cgtaaacgat gaatgttagc cgtcggggtg 780 tttacacttc ggtggcgcag ctaacgcatt aaacattccg cctggggagt acggtcgcaa 840

3 gattaaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900 cgaagcaacg cgcagaacct taccagccct tgacataccg gtcgcggaca cagagatgtg 960 tctttcagtt cggctggacc ggatacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020 agatgttggg ttaagtcccg caacgagcgc aaccctcgcc tttagttgcc atcatttggt 1080 tgggcactct aaagggactg ccagtgataa gctggaggaa ggtggggatg acgtcaagtc 1140 ctcatggccc ttacgggctg ggctacacac gtgctacaat ggtggtgaca gtgggcagca 1200 agcgtgcgag cgcaagctaa tctccaaaag ccatctcagt tcggattgca ctctgcaact 1260 cgagtgcatg aagttggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1320 cccgggcctt gtacacaccg cccgtcacac catgggagtt ggttctgccc gaaggcactg 1380 tgctaaccgt aaggaggcag gtgaccacgg tagggtcagc gactggggtg aagtcgtaac 1440 aaggtaacca atcactagt 1459

<210> 7

<211> 1526

<212> DNA

<213> Bacillus sp.

<400> 7

gattagagtt tgatcctggc tcaggacgaa cgctggcggc gtgcctaata catgcaagtc 60 gagcggacag aagggagctt gctcccggat gttagcggcg gacgggtgag taacacgtgg 120 gtaacctgcc tgtaagactg ggataactcc gggaaaccgg agctaatacc ggatagttcc 180 ttgaaccgca tggttcaagg atgaaagacg gtttcggctg tcacttacag atggacccgc 240 ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt agccgacctg 300 agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360 tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg 420 ttttcggatc gtaaagctct gttgttaggg aagaacaagt gcaagagtaa ctgcttgcac 480 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg aaacttgagt 660 gcagaagagg gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa 720 caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg 780 agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag 840 ggggtttccc gccccttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900 gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg aagcatgtgg 960 tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caaccctaga 1020 gatagggctt tcccttcggg gacagagtga caggtggtgc atggttgtcg tcagctcgtg 1080 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatcttag ttgccagcat 1140 tcagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggaca gaacaaaggg 1260 ttgcgagacc gcaaggttta gccaatccca caaatctgtt ctcagttcgg atcgcagtct 1320 gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgca acacccgaag 1440 tcggtgaggt aacctttatg gagccagccg ccgaaggtgg ggcagatgat tggggtgaag 1500 tcgtaacaag gtaaccaatc actagt 1526

<210> 8

<211> 1518

<212> DNA

<213> Providencia vermicola

<400> 8

gattagagtt tgatcatggc tcagattgaa cgctggcggc aggcctaaca catgcaagtc 60 gagcggtaac aggggaagct tgcttcccgc tgacgagcgg cggacgggtg agtaatgtat 120 ggggatctgc ccgatagagg gggataacca ctggaaacgg tggctaatac cgcataatct 180 ctcaggagca aagcagggga acttcggtcc ttgcgctatc ggatgaaccc atatgggatt 240 agctagtagg tgaggtaatg gctcacctgg gcgacgatcc ctagctggtc tgagaggatg 300 atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 360 attgcacaat gggcgcaagc ctgatgcagc catgccgcgt gtatgaagaa ggccctaggg 420 ttgtaaagta ctttcagtcg ggaggaaggc gttgatgcta atatcatcaa cgattgacgt 480 tatcgacaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc 540 aagcgttaat cggaattact gggcgtaaag cgcacgcagg cggttgatta agttagatgt 600 gaaatccccg ggcttaacct gggaatggca tctaagactg gtcagctaga gtcttgtaga 660 ggggggtaga attccatgtg tagcggtgaa atgcgtagag atgtggagga ttacccggtg 720 ggcgaaggcg gccccctgga caaagactga cgctcaggtg cgaaagcgtg gggagcaaac 780 aggattagat accctggtag tccacgctgt aaacgatgtc gatttgaagg ttgttccctt 840

4 gaggagtggc ttttcggagc taacgcgtta aatcgaccgc ctggggagta cggccgcaag 900 gttaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gatgcaacgc gaagaacctt acctactctt gacatccaga gaacttagca gagatgcttt 1020 ggtgccttcg ggaactctga gacaggtgct gcatggctgc cgtcagctcg tgttgtgaaa 1080 tgttgggtta agtcccgcaa cgagcgcaac ccttatcctt tgttgccagc gattcggtcg 1140 ggaactcaaa ggagactgcc ggtgataaac cggaggaagg tggggatgac gtcaagtcat 1200 catggccctt acgagtaggg ctacacacgt gctacaatgg cgtatacaaa gagaagcgac 1260 ctcgcgaggg caagcggaac tcataaagta cgtcgtagtc cggattggag tctgcaactc 1320 gactccatga agtcggaatc gctagtaatc gtagatcaga atgctacggt gaatacgttc 1380 ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgcaaaag aagtaggtag 1440 cttaacctgc gggagggcgc ttaccacttt gtgattcatg actggggtga agtcgtaaca 1500 aggtaaccaa tcactagt 1518

<210> 9

<211> 1526

<212> DNA

<213> Bacillus sp.

<400> 9

gattagagtt tgatcatggc tcaggacgaa cgctggcggc gtgcctaata catgcaagtc 60 gagcggacag aagggagctt gctcccggat gttagcggcg gacgggtgag taacacgtgg 120 gtaacctgcc tgtaagactg ggataactcc gggaaaccgg agctaatacc ggaaagttcc 180 ttgaaccgca tggttcaagg atgaaagacg gtttcggctg tcacttacag atggacccgc 240 ggcgcattag ctagttggtg gggtaatggc tcaccaaggc gacgatgcgt agccgacctg 300 agagggtggt cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360 tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg 420 ttttcggatc gtaaagctct gttgttaggg aagaacaagt gcgagagtaa ctactcgcac 480 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg aaacttgagt 660 gcagaagagg agagtgggat tccacgtgta gcggtgaaat gcgtagagat gtggagggaa 720 caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg 780 agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag 840 ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg 900 tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960 ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcctctgac aaccctagag 1020 atagggcttt cccttcgggg acagagtgac aggtggtgca tggttgtcgt cagctcgtgt 1080 cgtgagatgt tgggttaagt cccgcaacga gcgcaacccc tgatcttagt tgccagcatt 1140 tagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200 aatcatcatg ccccttatga cctgggctac acacgtgcta cagtggacag aacaaagggc 1260 tgcgagaccg caaggtttag ccaatcccat gaatctgttc tcagttcgga tcgcagtctg 1320 caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1380 acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgcaa cacccgaagt 1440 cggtgaggta acctttatgg agccagccgc cgaaggtggg gcagatgatt ggggtgaagt 1500 cgtaacaagg taaccaaatc actagt 1526

<210> 10

<211> 1488

<212> DNA

<213> Ochrobactrum sp

<400> 10

catatggtgc tgctggagcg ctgaagcgac tagtgattgg ttaccttgtt acgacttcac 60 cccagtcgct gaccctaccg tggtcacctg cctccttacg gttagcacag tgccttcggg 120 cagaaccaac tcccatggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg 180 cggcatgctg atccgcgatt actagcgatt ccaacttcat gcactcgagt tgcagagtgc 240 aatccgaact gagatggctt ttggagatta gcttgcgctc gcacgcttgc tgcccactgt 300 caccaccatt gtagcacgtg tgtagcccag cccgtaaggg ccatgaggac ttgacgtcat 360 ccccaccctc ctccagctta tcactggcag tccctttaga gtgcccaacc gaatgatggc 420 aactaaaggc gagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 480 gacgacagcc atgcagcacc tgtatccggt ccagccgaac tgaaagacac atctctgtgt 540 ccgcgaccgg tatgtcaagg gctggtaagg ttctgcgcgt tgcttcgaat taaaccacat 600 gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt tttaatctcg cgaccgtact 660 ccccaggcgg aatgtttaat gcgttagctg cgccaccgaa gtgtaaacac cccgacggct 720 aacattcatc gtttacggcg tggactacca gggtatctaa tcctgtttgc tccccacgct 780

5 ttcgcacctc agcgtcagta atggaccagt gagccgcctt cgccactggt gttcctccga 840 atatctacga atttcacctc tacactcgga attccactca cctcttccat actcaagact 900 ttccagtatc aaaggcagtt ccagagttga gctctgggat ttcacccctg acttaaaagt 960 ccgcctacgt gcgctttacg cccagtaaat ccgaacaacg ctagccccct tcgtattacc 1020 gcggctgctg gcacgaagtt agccggggct tcttctccgg ttaccgtcat tatcttcacc 1080 ggtgaaagag ctttacaatc ctaagacctt catcactcac gcggcatggc tggatcaggc 1140 ttgcgcccat tgtccaatat tccccactgc tgcctcccgt aggagtctgg gccgtgtctc 1200 agtcccagtg tggctgatca tcctctcaga ccagctatgg atcgtcgcct tggtaggcct 1260 ttaccccacc aactagctaa tccaacatgg gctcatcact ctccgataaa tctttcccca 1320 aaagggcgta tacggtatta gcacaagttt ccctgagtta ttccgtagag aatggtagat 1380 tcccatgcat tactcacccg tctgccactg cctccgaaga gaccgttcga cttgcatgtg 1440 ttaagcctgc cgccagcgtt cgttctgagc caggatcaaa ctctaatc 1488

<210> 11

<211> 1499

<212> DNA

<213> Providencia vermicola

<400> 11

agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60 ggtaacaggg gaagcttgct tcccgctgac gagcggcgga cgggtgagta atgtatgggg 120 atctgcccga tagaggggga taaccactgg aaacggtggc taataccgca taatctctca 180 ggagcaaagc aggggaactt cggtccttgc gctatcggat gaacccatat gggattagct 240 agtaggtgag gtaatggctc acctaggcga cgatccctag ctggtctgag aggatgatca 300 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg 360 cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaaggcc ctagggttgt 420 aaagtacttt cagtcgggag gaaggcgttg atgctaatat catcaacgat tgacgttacc 480 gacagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc 540 gttaatcgga attactgggc gtaaagcgca cgcaggcggt tgattaagtt agatgtgaaa 600 tccccgggct taacctggga atggcatcta agactggtca gctagagtct tgtagagggg 660 ggtagaattc catgtgtagc ggtgaaatgc gtagagatgt ggaggaatac cggtggcgaa 720 ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt 780 agataccctg gtagtccacg ctgtaaacga tgtcgatttg aaggttgttc ccttgaggag 840 tggctttcgg agctaacgcg ttaaatcgac cgcctgggga gtacggccgc aaggttaaaa 900 ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa 960 cgcgaagaac ctacctactc ttgacatcca gagaacttag cagagatgct ttggtgcctt 1020 cgggaactct gagacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttgtca ttagttgcca gcattcagtt gggcactcta 1140 atgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtcc tcatggccct 1200 tatgggtagg gcttcacacg tcatacaatg gtcgggacag agggttgcca aaccgcgagg 1260 tggagccaat ctcagaaacc cgatcgtagt ccggattgca ggctgcaact cgcctgcatg 1320 aagtcggaat cgctagtaat cgcggatcag catgtcgcgg tgaatacgtt cccgggtctt 1380 gtacacaccg cccgtcacac catgggagtg ggttttacca gaagtagtta gcctaaccgc 1440 aaggggggcg attaccacgg taggattcat gactggggtg aagtcgtaac aaggtaacc 1499

<210> 12

<211> 1545

<212> DNA

<213> Bacillus aerius

<400> 12

gattgtttga tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60 cggacagatg ggagcttgct ccctgatgtc agcggcggac gggtgagtaa cacgtgggta 120 acctgcctgt aagactggga taactccggg aaaccggggc taataccaga tgcttgattg 180 aaccgcatgg ttcaattata aaaggtggct tttagctacc acttacagat ggacccgcgg 240 cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag 300 agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt 420 ttcggatcgt aaaactctgt tgttagggaa gaacaagtac cgttcgaata gggcggtacc 480 ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 540 aggtggcaag cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg tttcttaagt 600 ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg 660 cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tgggaggaac 720 accagtggcg aaggcgactc tctggtctgt aactgacgct gaggcggcga aagcgtgggg 780 agcgaacagg attagatacc ctggtagtcc cccccgtaaa cgatgagtgc taagtgttag 840

6 agggtttccc ccctttagtg ctgcagcaaa cgcattaagc actccgcctg gggagtacgg 900 gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaaccggtg gagcatgtgg 960 tttaattcga agcaacgcga agaaccttac caggtcttga catcctctgc caacccctag 1020 agatagggct tccccttcgg gggcagagtg acaggtggtg catggttgtc cgtcagctcg 1080 tgtcgtgaga tgttgggtta agtcccgcac cgagcgcaac ccttgatctt agttgccagc 1140 attcagttgg gcactctaag gtgcctcccg gtgacaaacc ggaggaaggt ggggatgacg 1200 tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctccaatggg cagaacaaag 1260 ggcagcgaag ccgcgaggct aagccaatcc cacaaatctg ttctcagttc ggatcgcagt 1320 ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggcg 1380 aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440 agtcggtgag gtaacctttt ggagccagcc gccgaaggtg ggacagatga ttggggtgaa 1500 gtcgtaacaa ggtaaccaag tagagtttga tcctgaatca ctagt 1545

<210> 13

<211> 1529

<212> DNA

<213> Bacillus subtilis

<400> 13

gattagagtt tgatcatggc tcaggacgaa cgctggcggc gtgcctaata catgcaagtc 60 gagcggacag atgggagctt gctccctgat gtcagcggcg gacgggtgag taacacgtgg 120 gtaacctgcc tgtaagactg ggataactcc gggaaaccgg ggctaatacc aggtgcttga 180 ttgaaccgca tggttcaatt ataaaaggtg gcttttagct accacttaca gatggacccg 240 cggcgcatta gctagttggt gaggtaacgg ctcaccaagg caacgatgcg tagccgacct 300 gagagggtga tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca 360 gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg agtgatgaag 420 gttttcggat cgtaaaactc tgttgttagg gaagaacaag taccgttcga atagggcggt 480 accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540 cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600 agtctgatgt gaaagccccc ggctcaaccg gggagggtca ttggaaactg gggaacttga 660 gtgcagaaga ggagagtggg aattccacgt gtagcggttg aaatgcgtag agatgtggag 720 gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggcg cgaaagcgtg 780 gggagcgaac aggattagat acccctggta gtccacgccg taaacgatga gtgctaagtg 840 ttagagggtt tcccgccctt tagtgctgca gcaaacgcat taagcactcc gcctggggag 900 tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 960 gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaaccc 1020 tagagatagg gcttcccctt cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct 1080 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1140 gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1200 cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg ggcagaacaa 1260 agggcagcga agccgcgagg ctaagccaat cccacaaatc tgttctcagt tcggatcgca 1320 gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1380 tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1440 gaagtcggtg aggtaacctt ttggagccag ccgccgaagg tgggacagat gattggggtg 1500 aagtcgtaac aaggtaacca atcactagt 1529

<210> 14

<211> 1526

<212> DNA

<213> Bacillus pumilus

<400> 14

gattagagtt tgatcctggc tcaggacgaa cgctggcggc gtgcctaata catgcaagtc 60 gagcggacag aagggagctt gctcccggat gttagcggcg gacgggtgag taacacgtgg 120 gtaacctgcc tgtaagactg ggataactcc gggaaaccgg agctaatacc ggatagttcc 180 ttgaaccgca tggttcaagg atgaaagacg gtttcggctg tcacttacag atggacccgc 240 ggcgcattag ctagttggtg gggtaatggc tcaccaaggc gacgatgcgt agccgacctg 300 agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360 tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg 420 ttttcggatc gtaaagctct gttgttaggg aagaacaagt gcgagagtaa ctgctcgcac 480 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg aaacttgagt 660 gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720 accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga 780

7 gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg 840 gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt 900 cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960 taattcgaag caacgcgaag aaccttacca ggtcttgaca tcgtctgata accctagaga 1020 tagggctttc ccttcgggga cagagtgaca ggtggtgcat ggtcagtcgt cagctcgtgt 1080 cgtgagatgt tgggttaagt cccgcaacgg gcgcaaccct tgatcttagt tgccagcatt 1140 tagttgggca ctcttaaggt gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggaca gaacaaaggg 1260 ctgcgagacc gcaaggttta gccaatccca taaatctgtt ctcagttcgg atcgcagtct 1320 gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggccttgtac acaccgcccg tcacaccacg ggagtttgca acacccgaag 1440 tcggtgaggt aacctttatg gagccagccg ccgaaggtgg ggcagatgat tggggtgaag 1500 tcgtaacaag gtaaccaatc actagt 1526

<210> 15

<211> 1526

<212> DNA

<213> Bacillus sp

<400> 15

gattagagtt tgatcatggc tcaggacgaa cgctggcggc gtgcctaata catgcaagtc 60 gagcggacag aagggagctt gctcccggat gttagcggcg gacgggtgag taacacgtgg 120 gtaacctgcc tgtgagactg ggataactcc gggaaaccgg agctaatacc ggatagttcc 180 ttgaaccgca tggttcaagg atgaaagacg gtttcggctg tcacttacag atggacccgc 240 ggcgcattag ctagttggtg gggtaatggc tcaccaaggc gacgatgcgt agccgacctg 300 agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360 tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg 420 ttttcggatc gtaaagctct gttgttgggg aagaacaagt gcgagagtaa ctgctcgcac 480 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg aaacttgagt 660 gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720 accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga 780 gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg 840 gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt 900 cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960 taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgata accctagaga 1020 tagggctttc ccttcgggga cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080 gtgagatgtt gggttaagtc ccgcaacgag cgcagggggg gggggcttag tagccagcat 1140 ttagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcca cacacgtgct acaatggaca gaacaaaggg 1260 ctgcgagacc gcaaggttta gccaatccca taaatctgtt ctcagttcgg atcgcagtct 1320 gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggccttgtgc acaccgcccg tcacaccacg agagtttgca acacccgaag 1440 tcggtgaggt aaccattatg gagccagccg ccgaaggtgg ggcagatgat tggggtgaag 1500 tcgtaacaag gtaaccaatc actagt 1526

<210> 16

<211> 1524

<212> DNA

<213> Bacillus licheniformis

<400> 16

actagtgatt agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg 60 caagtcgagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac 120 acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat 180 gcttgattga accgcatggt tcaattataa aaggtggctt cggctaccac ttacagatgg 240 acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcaacg atgcgtagcc 300 gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 360 cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 420 tgaaggtttt cggatcgtaa aactctgttg ttagggaaga acaagtaccg ttcgaatagg 480 gcggtacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 540 taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagcgcgc gcaggcggtt 600 tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa 660 cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg 720

8 gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gcgcgaaggc 780 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 840 tgttagaggg tttccgccct ttagtgctgc agcaaacgca ttaagcactc cgcctgggga 900 gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 960 tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaacc 1020 ctagagatag ggcttcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc 1080 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1140 agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1200 acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat gggcagaaca 1260 aagggcagcg aagccgcgag gctaagccaa tcccacaaat ctgttctcag ttcggatcgc 1320 agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1380 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1440 cgaagtcggt gaggtaacct tttggagcca gccgccgaag gtgggacaga tgattggggt 1500 gaagtcgtaa caaggtaacc aatc 1524

9