USOO642O147B1 (12) United States Patent (10) Patent No.: US 6,420,147 B1 Jones et al. (45) Date of Patent: Jul. 16, 2002

(54) HALOALKALIPHILIC MICROORGANISMS Zvyagintseva, I.S. and Tarasor, A.L. (1988) “Extreme Halo philic Bacteria From Saline Solis” Microbiologiya, (75) Inventors: Brian Edward Jones, Va 57:664-669. Leidschendam (NL); William Duncan Morth, S. and Tindall, B.J. (1985) “Variation of Polar Lipid Grant, Leicester (GB) Composition within Haloalkaliphilic Archaebacteria” Sys tem. Appl. Microbiol., 6:247-250. (73) Assignee: Genencor International, Inc., Upasani, V. and Desai, S. (1990) “Chemical composition of Rochester, NY (US) the brines and Studies on haloalkaliphilic archaeacteria' Arch. Microbiol., 154:589–593 (Sambhar Salt Lake). (*) Notice: Subject to any disclaimer, the term of this Grant, W.D., Mwatha, W.E. and Jones, B.E. (1990) “Alka patent is extended or adjusted under 35 liphiles: ecology, diversity and applications” FEMS Micro U.S.C. 154(b) by 0 days. biology Reviews, 75:255-270. Grant, W.D. and Tindall, B.J. (1986) “The Alkaline Saline (21) Appl. No.: 08/186,220 Environment” in Microbes in Extreme Environments (eds. R.A. Herbert and G.A. Codd), Academic Press, London, pp. (22) Filed: Jan. 25, 1994 22-54. Tindall, B.J. (1988) “Prokaryotic Life In the Alkaline, Related U.S. Application Data Saline, Athalassic Environment” in Halophilic Bacteria, (63) Continuation of application No. 08/077.942, filed on Jun. 1:31-70 (ed. F. Rodriguez-Valera), CRC Press Inc., Boca 15, 1993, now abandoned, which is a continuation of appli Raton, Florida. cation No. 07/789,186, filed on Nov. 7, 1991, now aban Horikoshi, K. and Akiba, T. (1982) “A New Microbial doned. World” in Alkalophilic Microorganisms (Springer-Verlag, Berlin/Heidelberg/New York). (51) Int. Cl." ...... C12N 9/14 Shiba, H. (1991) “New Strictly Anaerobic Halophiles and (52) U.S. Cl...... 435/183; 435/252.1; 435/170; Halopiles and Haloalkaliphiles form Surface Sediments of 435/195; 435/198; 435/219 Hypersaline Enviroments” in Superbugs (eds. K. Horikoshi (58) Field of Search ...... 435/252.1, 183, and W.D. Grant), Japan Scientific Socienties Press, Tokyo, 435/195, 170, 198, 219 and Springer-Verlag, Berlin, Heidelberg, New York, pp. 191-211. (56) References Cited Nakatsugawa, N., (1991) “Novel Methanogenic Archaebac teria Which Grow in Extreme Environments” in Superbugs PUBLICATIONS (eds. K. Horikoshi and W.D. Grant), Japan Scientific Soci eties PreSS, Tokyo, and Springer-Verlag, Berlin, Heidelberg, Ventura Et Al. Arch Microb. 149(4). 1988. pp. 273-279 New York, pp. 212-220. (ABS).* Biosis Previews Database, Philadelphia, Biosis No. Imhoff Et Al. Arch Microb 114(2). 1977. pp 115-122. 88.100773, Ivanova II; et al., & Mikrobiologiya 58 (2), 1989, (ABS).* 251-255, abstract. Imhoff Et Al. Arch Microb 130(3). 1981. p 238-242. Biosis Previews Database, Philadelphia, Biosis No. (ABS).* 89030525, Meyer B: Imhoff J.F. & J Gen Microbiol 135 (11) Imhoff Et Al. Arch Hyrobiology. 84(3) 1978. p 381-388 1989, 2829–2836, abstract. (ABS).* Biosis Previews Database, Philadelphia, Biosis No. Danson Et Al. Biochem Journal. 218. p 811-818.* 78046408, Danson M.j. et al., & Biochem J 218 (3), 1984, Woese, C.R., et al., “Toward a natural System of organisms: 811-818, abstract. Proposal for the domains Archaea, Bacteria, and Eucarya' Biosis Previews Database, Philadelphia, Biosis No. (1990) Proc. Natl. Acad. Sci. U.S.A., 87:4576-4579. 90003116, Davis J.E.; Jones L.P.; Zajic J.E. & ACTA Soliman, G.S.H. & Triper, H.G., “Halobacterium pharaonis Biotechnol 10 (1) 1990 99-104 abstract. sp. nov., a New, Extremely Haloalkaliphilic Archaebacte rium with Low Magnesium Requirement” (1982) Zbl Bakt. * cited by examiner Hyg., I. Abt. Orig. C3:318-329. Primary Examiner Leon B. Lankford, Jr. Tindall, B.J. & Tripre, H.G., “Ecophysiology of the Aerobic (74) Attorney, Agent, or Firm Morrison & Foerster LLP Halopilic Archaebacteria” (1986) System. Appl. Microbiol., 7:2O2-212. (57) ABSTRACT Rodriguez-Valera, F. (1988) “Characteristics and Microb Haloalkaliphilic bacteria have been isolated from samples of hial Ecology of Hypersaline Environments” in Halophilic soil, water, sediment, trona (NaHCO.NaCO2HO) and a Bacteria, 1, Rodriguez-Valera, F., ed., CRC Press, Inc., number of other Sources obtained from in and around Boca Raton, Florida, pp. 3-30. hypersaline Soda lakes. These bacteria have been analyzed Javor, B. (1989) “Solar Salterns” in Hypersaline Environ according to the principles of numerical taxonomy with ments, Springer-Verlag, Berlin/Heidelberg. respect to each other, as well as to other known haloalka Javor, B. (1989) “Hypersaline, Alkaline Lakes” in Hyper liphilic bacteria. In addition, these bacteria are further cir Saline Environments, Springer-Verlag, Berlin/Heidelberg. cumscribed by chemotaxonomic analysis. The bacteria pro Wang, D. and Tang, Q., “Natronobacterium from Soda duce various alkali- and Salt-tolerant enzymes which may be Lakes of China” in Recent Advances in Microbial Ecology used in various industrial processes requiring Such enzy (Proceeding of the 5th International Symposium on Micro matic activity in a high pH, Saline environment. bial Ecology, eds. T. Hattori et al., Japan Scientific Societies Press, Tokyo (1989), pp. 68–72. 6 Claims, 2 Drawing Sheets U.S. Patent Jul. 16, 2002 Sheet 1 of 2 US 6,420,147 B1

i g

0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 O. 95 1.00 Percentage Similarity (SS) FIG. 1 U.S. Patent Jul. 16, 2002 Sheet 2 of 2 US 6,420,147 B1

0.00 0 10 020 030 0.40 0.50 060 0.70 0.80 0.90 100 CluSter

1

q S. 4 SS 83 S& 93d X 3 SS 84 SS 90 S, s 3. SS is Y4 SS 102d SS 101 SS 103 S. 82 NSS 100 SSC 3: SS Sy2 SS RS2 RS3 96 102L 104 31

0.00 0.10 0.20 030 0.40 0.50 060 0.70 0.80 0.90 100 Percentage Similarity (S) F.G. 2 US 6,420,147 B1 1 2 HALOALKALIPHILIC MICROORGANISMS In situations where the concentrations of Ca" and Mg" exceed that of carbonate, or where they are equimolar, a Salt This application is a continuation of application Ser. No. lake is generated with pH 6-8, and whose ion composition 08/077.942, filed 15 June 1993, now abandoned, which is a is dependent on the local geology. The Dead Sea in Israel is continuation of Ser. No. 07/789,186 filed 7 November 1991 5 a typical example of a slightly acidic (pH 6-7) saline lake now abandoned. enriched with divalent cations, particularly Mg". On the The present invention is in the field of microbiology and other hand, the Great Salt Lake in Utah, U.S.A. is an more particularly in the field of halophilic, alkaliphilic example of a Mg"-depleted brine and is slightly alkaline microorganisms. (pH 7–8). The commercial production of common Salt from Sea BACKGROUND OF THE INVENTION water in Solar evaporation ponds (Salterns) generates man Alkaliphilic microorganisms are defined as organisms made hyperSaline environments. Salterns provide excellent which exhibit optimum growth in an alkaline pH model Systems over a range of Salinities (from Sea water to environment, particularly in exceSS of pH 8, and generally in Super-Saturation), and their chemistry and microbiology the range between pH 9 and 10. Alkaliphiles may also be 15 have been intensely studied (Javor, B., in Hypersaline found living in environments having a pH as high as 12. Environments, Springer-Verlag, Berlin/Heidelberg, 1989). Obligate alkaliphiles are incapable of growth at neutral pH. The African Rift Valley is probably unusual in having Alkaliphiles may be found in Such everyday environments lakes with Significant, largely permanent bodies of brine. as garden Soil, where transient alkaline conditions may arise The Kenyan-Tanzanian section of the Rift Valley contains a due to biological activity Such as ammonification, Sulphate number of alkaline Soda lakes with a range of total Salinities reduction or photosynthesis. A much richer Source of a from around 5% (w/v) in the more dilute lakes (e.g. greater variety of alkaliphilic organisms may be found in Elmenteita, Bogoria, Nakuru, etc.), to Saturation (30% or naturally occurring, Stable alkaline environments Such as greater) in parts of lakes Magadi, Little Magadi (Nasikie Soda lakes. Engida) and Natron. These lakes are devoid of significant Halophilic bacteria are defined as microorganisms that 25 amounts of Ca" and Mg" (in most cases below the level of grow optimally in the presence of Salt (Sodium chloride). detection) and have pH values in the range from 9 to above Since microorganisms are often capable of growth over a 11.5 in the most concentrated lakes. wide range of Salt concentrations, the term halophile is Despite this apparently harsh environment, Soda lakes are usually reserved for microorganisms having a minimum nevertheless home to a large population of prokaryotes, a requirement in excess of the concentration found in Sea few types of which may dominate as permanent or Seasonal water (ca. 0.5 M or 3%). blooms. The organisms range from alkaliphilic cyanobacte Extremely halophilic bacteria are defined as bacteria that ria to haloalkaliphilic archaeobacteria. At the higher Salini grow optimally at above 20% NaCl (3-4 molar). Extreme ties (characterized by higher conductivities) haloalkaliphilic halophiles inhabit hypersaline environments. The most archaeobacteria predominate. Moreover, it is not unusual to intensely Studied extremely halophilic bacteria belong to the 35 find common types of alkaliphilic organisms inhabiting Soda order Halobacteriales. With the exception of the genera lakes in various widely dispersed locations throughout the Natronobacterium and Natronococcus, all known Halobac world such as in the East African Rift Valley, in the western teria are obligate halophiles which require at least 12-15% U.S., Tibet, China and Hungary. For example, natronobac Salt for growth and a pH around neutrality. These bacteria teria have been isolated and identified from Soda lakes and belong to the Kingdom Euryarchaeota of the Domain 40 soils located in China (Wang, D. and Tang, Q., “Natrono Archaea (the archaeobacteria) (Woese, C. R., et al., Proc. bacterium from Soda Lakes of China' in Recent Advances in Natl. Acad. Sci. U.S.A., 87, (1990), 4576-4579). Microbial Ecology (Proceedings of the 5th International The term “haloalkaliphile” was first used by Soliman and Symposium on Microbial Ecology, eds. T. Hattori et al., Truper to describe bacteria that are both halophilic and 45 Japan Scientific Societies Press, Tokyo, (1989), pp. 68-72), alkaliphilic. (Soliman, G. S. H. & Truper, H. G., (1982), Zbl. the Soviet Union (Zvyagintseva, I. S. and Tarasor, A. L. Bakt. Hyg., I. Abt. Orig. C3, pp. 318-329). Until now the (1988) Microbiologiya, 57, 664-669) and in the western only known examples of Such bacteria belong to the King U.S. (Morth, S. and Tindall, B. J. (1985) System. Appl. dom Euryarchaeota (Tindall, B. J. & Truper, H. G., (1986), Microbiol., 6, 247-250). Natronobacteria have also been System. Appl. Microbiol., 7, 202-212). 50 found in Soda lakes located in Tibet (W. D. Grant, unpub The most extreme hypersaline environments are micro lished observations) and India (Upasani, V. and Desai, S. biologically the least diverse but nevertheless contain a (1990) Arch. Microbiol., 154, pp. 589-593). distinct, rich and complex flora of extreme halophilic bac A more detailed Study of Soda lakes and alkaliphilic teria. It has been Suggested that these environments are organisms in general is provided in Grant, W. D., Mwatha, dominated by the Euryarchaeota with few eubacteria present 55 W. E. and Jones, B. E. (1990) FEMS Microbiology Reviews, (Rodriguez-Valera, F., in Halophilic Bacteria, vol. 1, 75, 255-270, the text of which is hereby incorporated by (Rodriguez-Valera, F., ed.) CRC Press, Inc., Boca Raton, reference. Lists of alkaline Soda lakes may be found in the Fla., (1988), pp. 3–30). publications of Grant, W. D. and Tindall, B.J. in Microbes Soda lakes, an example of naturally-occurring alkaline in Extreme Environments, (eds. R. A. Herbert and G. A. environments which may also be hyperSaline, are found in 60 Codd); Academic Press, London, (1986), pp. 22-54); and various locations around the World. They are caused by a Tindall, B. J. in Halophilic Bacteria, Volume 1, (ed. F. combination of geological, geographical and climatic con Rodriguez-Valera); CRC Press Inc., Boca Raton, Fla., ditions. They are characterized by the presence or large (1988), pp. 31-70, both texts are also hereby incorporated by amounts of Sodium carbonate (or complexes of this salt) reference. A detailed Study of hypersaline environments is formed by evaporation concentration, as well as the corre 65 provided in Javor, B., in Hypersaline Environments, supra). sponding lack of Cat" and Mg" which would remove Alkaliphiles isolated from non-Saline environments are carbonate ions as insoluble Salts. also discussed by Horikoshi, K. and Akiba, T. in Alkalophilic US 6,420,147 B1 3 4 Microorganisms (Springer-Verlag, Berlin/Heidelberg/N.Y., (NaHCO.Na2CO2H2O) and a number of other sources in 1982). However, alkaliphilic organisms from saline envi and around alkaline, hyperSaline lakes. These Samples were ronments Such as Soda lakes are not discussed therein. obtained as part of an investigation over a period of three Strictly anaerobic bacteria from alkaline, hyperSaline envi years. The isolated bacteria are non-phototrophic eubacteria ronments have been recently described by Shiba, H., in and archaeobacteria. Up until now, only a few haloalka Superbugs (eds. K. Horikoshi and W. D. Grant); Japan Scientific Societies PreSS, Tokyo, and Springer-Verlag, liphilic archaeobacteria have been well characterized (see Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and by Table 4). Nakatsugawa, N., ibid, pp. 212-220. The Samples were collected in Sterile plastic bags. Sam Alkaliphiles have already made an impact in the applica pling was conducted at lakes Magadi, Little Magadi tion of biotechnology for the manufacture of consumer (Nasikie Engida) and Natron, all of which are located in products. Alkaliphilic enzymes produced by alkaliphilic Kenyan-Tanzanian Rift Valley of East Africa. Alkaline soda microorganisms have already found use in industrial pro lakes having Similar environments may also be found in ceSSes and have considerable economic potential. For Tibet, China, Egypt and the western U.S. At each Sampling example, these enzymes are currently used in detergent Site, physical parameterS Such as pH, conductivity and compositions and in leather tanning, and are foreseen to find 15 temperature were measured as well as the physical appear applications in the food, waste treatment and textile indus ance of the Site and the Sample. Some of the Samples were tries. Additionally, alkaliphiles and their enzymes are poten treated locally within 36 hours of collection of the sample tially useful for biotransformations, especially in the Syn but the majority were examined off-site, Several weeks after thesis of pure enantiomers. Also, many of the collection. microorganisms described herein are brightly pigmented Table 1 lists various strains which have been isolated. The and are potentially useful for the production of natural Strains are listed according to the location from which the colorants. Sample was taken and the physical appearance of the Sample SUMMARY OF THE INVENTION itself. Table 2 provides examples of chemical analyses of the The present invention provides pure cultures of novel lake waters at the Sampling locations at the time of extrac haloalkaliphilic bacteria. These bacteria have been isolated 25 tion of the Samples. These data are consistent with earlier from Samples of Soil, water, Sediment, trona analyses (Grant, W. D. and Tindall, B. J., in Microbes in (NaHCO.Na2CO2H2O) and a number of other sources, all Extreme Environments, (eds. R. A. Herbert and G. A. Codd); of which were obtained from in and around alkaline, hyper Academic Press, London, 1986). Saline lakes. These haloalkaliphiles have been analyzed according to the principles of numerical taxonomy with Table 3 provides a list of the isolated strains arranged respect to each other and also to other known haloalka according to the results of the numerical taxonomic analysis liphilic bacteria in order to confirm their novelty. In addition, (FIG. 1). Furthermore Table 3 provides physical properties these bacterial taxa are further circumscribed by chemot of the sample, in particular the temperature., conductivity axonomic analysis. and alkaline pH, as well as the numerous isolation media The present invention also provides data as to the com 35 employed for obtaining pure cultures of the new bacteria. position of the environments from which the Samples con These media are letter coded with reference to Appendix A. taining the microorganisms were obtained, as well as the Tables 1, 2 and 3 provide data from which the environ media required for their efficient isolation and culturing Such ment of the Sampling locations may be characterized. The that one of ordinary skill may easily locate Such an envi chemical and physical analysis of the Samples confirm the ronment and be able to, isolate the organisms of the present 40 presence of alkaline pH, as well as the presence of unusually invention by following the procedures described herein. high levels of Na2CO, coupled with low levels of Ca" and It is also an object of the present invention to provide Mg". It is known that the basic environments of soda lakes microorganisms which produce useful alkali- and Salt are stable with respect to their pH and ionic composition. tolerant enzymes, as well as methods for obtaining Substan Moreover, the microbial populations found at these Sites tially pure preparations of these enzymes. These enzymes 45 remain largely stable. Thus, it is to be expected that the are capable of performing their functions in high pH, Saline environment from which bacteria according to the present environments which makes them uniquely Suited for appli invention may be obtained may be determined from the data cations requiring Such extreme conditions. For example, presented in Tables 1-3. enzymes having alkali- and Salt-tolerance may be employed in detergent compositions, in leather tanning and in the food, 50 TABLE 1. waste treatment and textile industries, as well as for biotransformations Such as the production of pure enanti Alkaliphilic Strains Arranged According to Their Place of Origin OCS. ANAL BRIEF DESCRIPTION OF THE FIGURES STRAINS SAMPLE LOCATION SAMPLEAPPEARANCE YSIS 55 FIG. 1. Dendrogram showing clusters (phenons) obtained 30M.11, Lake Magadi (final Liquor and salts 1. 31M1 salt making pond) with the Ss coefficient and Unweighted Average Linkage 86M.4, procedure. 87M.4 FIG. 2. Dendrogram showing clusters (phenons) obtained 82M.4 Lake Magadi (south Black liquor 2 with the S. coefficient and Unweighted Average Linkage causeway pumping 60 83M.4 station) Surface trona crust procedure. 84M.4 Lake Magadi Red liquor and 3 DETAILED DESCRIPTION OF THE (pre-concentration salts salt pan Pl) INVENTION 85M.4 Lake Magadi (“fish Water and sediment 4 Sampling water canal) 65 88M.4 Lake Magadi “coarse salt Several hundreds of strains of bacteria have been isolated (salt factory) from Samples of Soil, water, Sediment, trona US 6,420,147 B1 S 6

TABLE 1-continued TABLE 3-continued Alkaliphilic Strains Arranged According to Their Place of Origin ORIGIN OF THE STRAINS ARRANGED BY CLUSTER ANAL STRAINS SAMPLE LOCATION SAMPLE APPEARANCE YSIS SAMPLE 89M.4 Lake Magadi (upper Water and sediment 5 NW arm) Con 90M.4 Lake Magadi (upper Soda crust and mud NW arm) 1O ductiv- ISOLA 93dLM.4, Little Lake Magadi Soda encrusted mud CLUS- LOCA- Temp. ity TION 931LM.4 (north west springs) g 95LM.4, Little Lake Magadi Water + sediment + 6 TER STRAN TON pH C. mS/cm MEDIUM 96LM.4 (south east lagoon) trona crust 97Nt.4, Lake Natron (East Spring water and 7 3 93dLM.4T Little NR NR NR E 98Nt.4 shore, lake-basin- surrounding sediment 15 margin soda spring Magadi 99Nt.4 Lake Natron (dried Black soda mud 3 84M.4 Magadi 12 44 87.5 B 1OONt.4 lake el. littoral Dried soda crust 4 90M.4 Magadi NR NR NR E ZOle 101Nt.4, Lake Natron Algal mat + water 4 95LM.4 Little 11 37 >100 F 102dNt.4, (soda seep) Soda crusts 2O Magadi 102INt.4, 103Nt.4 4 97Nt.4 Natron 10-10.5 35 35 C 104Nt.4 Stream flowing Orange surface scum 8 4 105Nt.4 Natron 11 44 60-90 B to lake and water 105Nt.4 Pool in stream Water and sediment 9 4 102dNt.4 Natron NR NR NR B 4 101Nt.4 Natron 11 40 26-36 C

TABLE 2 Chemical Analysis of Kenyan-Tanzanian Soda Lake Waters ANALYSIS Na' K' Ca?" Mg2+ SiO, PO, CI SO, CO2. TON* TA" 1. 9.22 238.9 &O.O1 O.OO8 28.30 O.13 3.49 53.62 4.92 3.86 4900 2 7.00 57.0 &O.O1 O.OO8 1488 1.82 3.15 17:49 3.90 1.50 4260 3 7.57 66.5 &O.O1 O.OO8 16.98 1.79 265 11.56 4.12 1.50 4490 4 1.34 11.89 O.O15 O.OO8 2.78 O.46 5.41 2.60 O.65 O.69 851 5 1.63 11.89 O.O97 O.O16 3.55 0.15 O.56 6.98 1.12 3.57 1110 6 4.63 61.13 O.O17 O.O29 F.54 O.31 186 1312 2.43 15.07 2710 7 0.52 4.6O O.O27 O.OO4 O.91 O-42 O.18 S.OO O.23 O.30 365 8 122 11.51 O.142 O.O25 O.77 1.18 O.39 14.78 O.63 1.43 832 9 4.52 43.73 O.042 O.O2S 3.OS 4.21 1.46 1.67 2.67 3.OO 3040 Note: Cations and anions given in millimoles (mM) except for Na", CI and CO’ which are in moles (M) *TON = Total Organic Nitrogen (mM) "TA = Total Alkalinity in milliequivalents/liter

45 TABLE 3 TABLE 3-continued

ORIGIN OF THE STRAINS ARRANGED BY CLUSTER ORIGIN OF THE STRAINS ARRANGED BY CLUSTER

SAMPLE SAMPLE 50 Con- Con ductiv- ISOLA- ductiv- ISOLA CLUS- LOCA- Temp. ity TION CLUS- LOCA- Temp. ity TION TER STRAN TON pH o C. mS/cm MEDIUM TER STRAN TON pH o C. mS/cm MEDIUM 1. 3OM.4 Magadi 12.5 55 60 A. 55 103Nt.4 Natron NR NR NR B 1. 87M.4 Magadi 12.3 56 >100 B 31 M.4 Magadi 12.5 55 60 A. 1. 85M.4 Magadi 10.5 33 80.4 B 931LM.4 Little NR NR NR E 1. 89M.4 Magadi 10.5 3O 60-90 B Magadi 1. 86M.4T Magadi 12.3 56 >100 B 1. 88M.4 Magadi NR NR NR B *Clusters of microorganisms are obtained by analysis according to the 2 82M.4 Magadi 12 48 87 B principles of numerical taxonomy using the Ss/UPGMA method (see 2 1OONt.4 Natron NR NR NR B 60 discussion below and FIG. 1). 2 98Nt.4T Natron 10-10.5 35 35 C NR = not tested 2 99Nt.4 Natron NR 45 NR C The letter codes given for the Isolation Media refer to Appendix A. 2 96LM.4 Little 11 37 >100 D Magadi Treatment of the Samples: Enrichment and 2 102INt.4 Natron NR NR NR B Isolation of Haloalkaliphilic Bacteria 2 104Nt.4 Natron 10-10.5 NR 45 B 65 p 3 83M.4 Magadi NR NR NR C A diversity of enrichment and isolation methods were applied. Some of the methods were specifically designed for US 6,420,147 B1 7 8 the enrichment and isolation of haloalkaliphilic bacteria which exhibit Specific types of enzyme activity at an alkaline TABLE 4-continued pH. Other techniques of a more general nature were applied for the isolation of diverse sorts of haloalkaliphilic bacteria. Haloalkaliphilic Reference Strain In Some cases, the Specific conditions prevailing in the lakes RSS Natronobacterium pharaonis" DSM 2160 (Table 2) were taken into account when experiments were RS6 Natronococcus Occitus' NCIMB 2.192 performed for the isolation of bacteria. abbreviations are as used in FIG. 1 and FIG. 2. The different nutrient media employed for the isolation of "denotes “Type Strain” the new haloalkaliphilic bacteria are designated Medium A-Medium F. The composition of the various media Analysis of Test Data employed is shown in Appendix A. The Estimation of Taxonomic Resemblance For the isolation of non-Specific haloalkaliphilic organ The phenetic data, consisting of 107 characters was otrophic bacteria, Soda lake water Samples, or dilutions 15 Scored for 123 two-state characters (presence-absence thereof were Streaked out on an alkaline, Saline nutrient agar, characters) using binary notation as indicated in Appendix pH 10-pH 10.5 (Medium A). Samples of a more solid C. Additive Scaling was used where appropriate (e.g. growth consistency, mud, Sediment, etc. were first Suspended in an on NaCl) and where necessary qualitative multistate char alkaline, Saline nutrient broth (Medium A) before spreading acters (e.g. colony color) were Sub-divided into Several on an alkaline, Saline nutrient agar (Medium A). The bacteria mutually exclusive character States. The data was set out in were cultivated in a heated incubator, preferably at 37 C. In the form of an “nxt matrix, whose t columns represent the Some cases, the Samples were Suspended in an alkaline, t bacterial Strains which are to be grouped on the basis of resemblances, and whose n rows are the unit characters. saline nutrient broth (Medium A) and the bacteria cultivated Taxonomic resemblance of the bacterial Strains was esti by shaking, preferably at 37 C. for 2-7 days before spread mated by means of a similarity coefficient (Sneath, P. H. A. ing the broth onto an alkaline, Saline nutrient agar (Medium 25 and Sokal, R. R., Numerical Taxonomy, supra, pp. 114-187). A) for the isolation of bacterial colonies. Although many different coefficients have been used for For the isolation of haloalkaliphilic bacteria exhibiting biological classification, only a few have found regular use Specific types of enzyme activity, Samples were spread onto in bacteriology. We have chosen to apply two association alkaline, Saline nutrient agar containing specific Substrates coefficients (Sneath, P. H. A. and Sokal, R. R., ibid, p. 129 Such as lactalbumin or casein. In Some instances, the bac et seq.), namely, the Simple Matching and Jaccard coeffi teria in the Sample may be enriched for 1 day or Several cients. These have been frequently applied to the analysis of Weeks in a non-Specific alkaline, Saline nutrient broth Such bacteriological data and have a wide acceptance by those skilled in the art since they have been shown to result in as Medium A before spreading the broth onto an alkaline, robust classifications. Saline nutrient agar Specific for the detection of bacteria 35 exhibiting enzyme activity Such as proteolytic activity. The coded data were analyzed using the TAXPAK pro gram package (Sackin, M.J., “Programmes for classification and identification”. In Methods in Microbiology, Volume 19 Taxonomic Analysis (eds. R. R. Colwell and R. Grigorova), pp. 459-494, Aca 40 demic Press, London, 1987) run on a DEC VAX computer Twenty-five strains of bacteria isolated from in an around system at the University of Leicester, U.K. In addition, the alkaline, hypersaline lakes were assigned to the category data were analyzed using the NTSYS-pc (version 1.50) haloalkaliphile on the basis of their ability to grow in a NaCl program package run on a IBM PS/2 desk top computer concentration of 15% or more and at greater than pH 10. (Rolf, F. J., Numerical taxonomy and multivariate analysis The 25 strains were tested for 107 characters. For prac 45 System, Applied Biostatistics Inc. and Exeter Publishing tical purposes the characters were divided into 123 character Ltd., Setauket, N.Y., 1988). States. The results were analyzed using the principles of A similarity matrix was constructed for all pairs of Strains numerical taxonomy (Sneath, P. H. A. and Sokal, R. R., in using the Simple Matching Coefficient (Ss), (Sneath, P. H. Numerical Taxonomy, W. H. Freeman & Co., San Francisco, A. and Sokal, R. R., Numerical Taxonomy, p. 132; W. H. 1973). The characters tested and manner of testing are 50 Freeman & Company, San Francisco, 1973) using the compiled in Appendix B. In addition, Appendix C records RTBNSIM program in TAXPAK. Cluster analysis of the how each character State was coded for taxonomic analysis. Similarity matrix was accomplished using the Unweighted AS controls, 5 known haloalkaliphilic archaeobacteria Pair Group Method with Arithmetic Averages (UPGMA) were Subjected to the same analysis using the same condi algorithm, also known as the Unweighted Average Linkage tions. These reference bacteria are the only available known 55 procedure by running the SMATCLST sub-routine in TAX haloalkaliphilic bacteria. These 5 known reference bacteria PAK are recorded in Table 4 from which it will be seen that the The result of the cluster analysis is a dendrogram which “Type Strain” of the known species has been used where is provided in FIG.1. The dendrogram illustrates the levels available. of Similarity between the bacterial Strains. The dendrogram 60 is obtained by using the DENDGR program in TAXPAK. TABLE 4 The phenetic data were re-analyzed using the Jaccard Coefficient (S) (Sneath, P. H. A. and Sokal, R. R., Numeri Haloalkaliphilic Reference Strain cal Taxonomy, p. 131; W. H. Freeman & Company, San RS2 Natronobacterium gregoryi' NCIMB 21.89 Francisco, 1973) by running the RTBNSIM program in RS3 Natronobacterium magadi" NCIMB 21.90 65 TAXPAK. A further dendrogram was obtained by using the RS4 Natronobacterium pharaonis NCIMB 2191 SMATCLST with UPGMA option and DENDGR sub routines in TAXPAK and is illustrated in FIG. 2. US 6,420,147 B1 9 10 Results of the Cluster Analysis program in TAXPAK. The centroid of a cluster of points S/UPGMA Method representing real organisms projected into hyperspace rep FIG. 1 illustrates the results of the cluster analysis, based resents a hypothetical average organism. The centroid rarely, on the Simple Matching coefficient and the UPGMA if ever, represents a real organism. Therefore, the Euclidean algorithm, of 25 new, haloalkaliphilic bacteria isolated from 5 distances of each of the members of the cluster from the in and around alkaline lakes, together with 5 known haloal centroid of the cluster were calculated in order to establish kaliphilic bacteria. which organism was closest to the hypothetical average Four natural clusters or phenons of haloalkaliphilic are organism. The organism closest to the centroid was desig generated at the 78.5% similarity level. These four clusters nated the “centrotype organism” (indicated with the Super include 22 of the 25 new, haloalkaliphilic bacteria isolated script “CT"). from alkaline lakes. Although the choice of 78.5% for the The centrotype organism can be thought of as the “Type level of delineation may seem arbitrary, it is in keeping with Strain' which most closely represents the essential and current practices in numerical taxonomy (Austin, B. and discriminating features of each particular cluster. The cen Priest, F., in Modern Bacterial Taxonomy, p. 37; Van Nos trotype strains are recorded in Table 5. trand Reinhold; Wokingham, U.K., (1986). Placing the 15 delineation at a lower percentage would combine groups of TABLE 5 clearly unrelated organisms whose definition is not Sup ported by the data, while a higher percentage would produce Centrotype Strains a multitude of less well defined clusters. Furthermore, an Mean Euclidean inspection of the original data, especially the colony Distance of Stand- Euclidean characteristics-and the antibiotic Sensitivity, indicates that Number of Strains ard Distance Cluster 1 and Cluster 2 contain exclusively archaeobacteria, Cluster Strains in from Devia- from and that Cluster 3 and Cluster 4 contain only eubacteria. This Number Cluster Centroid tion Strain Centroid conclusion is Supported by chemotaxonomic evidence (See 1. 7 3.14 0.72 86M.4 1.79 discussion below). The group membership of the clusters is 25 2 11 2.85 O.36 98Nt.4 2.14 further confirmed by the pattern of clusters obtained using 3 3 3.61 1.08 93dLM.4 1.53 the Jaccard coefficient (see below and FIG. 2). 4 6 2.55 O41 95LM.4 1.60 At the 78.5% level, 2 of the clusters (Cluster 3 and Cluster 4) exclusively contain novel haloalkaliphilic eubacteria rep resenting 9 of the newly isolated Strains, and these may Description of Centrotype Strains represent new taxa. Three of the new haloalkaliphilic Strains Strain 86M.4 fall outside the major clusters. These non-clustering Strains An aerobic, coccoid bacterium. No Spores observed. are 103Nt.4, 31 M.4 and 931 LM.4 and their inter Haloalkaliphile. Grows at pH 10 on a medium containing relationships are more difficult to define, but they probably 20% NaCl. Grows in the presence of 12–30% NaCl. represent new phenons presently not described. 35 On alkaline, Saline, nutrient-agar (Medium A) forms The distribution of positive characters in the clusters is Salmon-pink colored, circular colonies, about 2 mm in given in Appendix E. diameter, which have a conveX elevation and entire margin. S/UPGMA Method Temperature: grows optimally at 30-40 C. Growth at 20 The Jaccard coefficient is a useful adjunct to the Simple C. but not at 45° C. Matching coefficient as it can be used to detect phenons in 40 KOH Test: negative the latter due to undue weight being attached to negative Aminopeptidase Test: negative matching data. Consequently, the Jaccard coefficient is use Oxidase Reaction: negative ful for confirming the validity of clusters defined initially by Catalase Reaction: positive the use of the Simple Matching coefficient. The Jaccard Hydrolysis of Gelatin: negative coefficient is particularly useful in comparing biochemically 45 Hydrolysis of Starch: negative unreactive or slow growing organisms (Austin, B. and Growth is not inhibited by the antibiotics: gentamicin, Priest, F., supra, p. 37). ampicillin, penicillin G, chloramphenicol, Streptomycin, The 4 clusters generated by the S/UPGMA method are tetracycline, oleandomycin, polymixin, rifampicin, recovered fully in the dendrogram produced by the neomycin, Vancomycin and kanamycin. Growth is inhibited S/UPGMA method (FIG. 2). Although there is some rear 50 rangement in the pattern of the clusters, the group member by the antibiotics: erythromycin, novobiocin and bacitracin. ships remain the same in both dendrograms. However, in Chemoorganotroph. Grows on complex Substrates Such as this case the clusters are defined at the 59% (S) level (the yeast extracts, peptones and casamino acids. minimum required to define Cluster 1 (Ss) in which case The membrane lipids contain glycerol diether moieties, the (eubacterial) Clusters 3 and 4 are combined. Although 55 indicating the archaeobacteria nature of Strain 86M.4. this could indicate that these two clusters are differentiated Strain 98Nt.4 mainly on matching negative characters, an examination of An aerobic, coccoid bacterium. No Spores observed. both original dendrograms Suggests that a more likely expla Haloalkaliphile. Grows at pH 10 on a medium containing nation is the heterogeneity of Cluster 1. Strains 30M.4 and 20% NaCl. Grows in the presence of 12–30% NaCl. 87M.4 appear to form a sub-group within Cluster 1. 60 On alkaline, Saline, nutrient-agar (Medium A) forms When the phenetic data was examined using the NTSYS opaque, friable, pink colored, circular colonies, 1-2 mm in programmes exactly the Same result was achieved for the diameter, which have a conveX elevation and entire margin. clustering of the Strains. Temperature: grows optimally at 30-40 C. Growth at 20 C. but not at 45° C. Determination of Representative Strains 65 KOH Test: positive The centroid of each individual cluster generated by the Aminopeptidase Test: negative S/UPGMA method was computed using the RGROUPS Oxidase Reaction: positive US 6,420,147 B1 11 12 Catalase Reaction: positive been made So as to be able to distinguish these organisms Hydrolysis of Gelatin: positive from all other bacteria previously known and described. Strain 103Nt.4 Hydrolysis of Starch: positive An aerobic, Gram-negative, rod-shaped bacterium. No Growth is not inhibited by the antibiotics: gentamicin, Spores observed. ampicillin, penicillin G, chloramphenicol, Streptomycin, Haloalkaliphile, grows at pH 10 on a medium containing tetracycline, oleandomycin, polymixin, neomycin, Vanco 20% NaCl. Grows in presence of 0–30% NaCl. mycin and kanamycin. Growth is inhibited by the antibiot On alkaline, Saline, nutrient-agar (Medium A) forms ics: erythromycin, novobiocin, rifampicin and bacitracin. opaque orange colored, circular colonies, 2-3 mm in Chemoorganotroph. Grows on complex Substrates Such as diameter, which have a conveX elevation and entire margin. yeast extracts, peptones and casamino acids. Strain 93dLM.4 Temperature: grows optimally at 30-40 C. Growth at 20 An aerobic, Gram-negative, rod-shaped bacterium. No C. but not at 45° C. Spores observed. KOH test: positive Haloalkaliphile. Grows at pH 10 on a medium containing Aminopeptidase test: negative 20% NaCl. Grows in the presence of 15–30% NaCl. 15 Oxidase reaction: negative On alkaline, Saline, nutrient-agar (Medium A) forms Catalase reaction: positive opaque, mucoid, red colored, circular colonies, about 1 mm Hydrolysis of Gelatin: positive in diameter, which have a conveX elevation and entire Hydrolysis of Starch: positive margin. Growth is not inhibited by the antibiotics: gentamicin, Temperature: grows optimally at 30-40 C. Growth at 20 chloramphenicol, fusidic acid, erythromycin, methicillin, C. but not at 45° C. oleandomycin, rifampicin, Vancomycin, and bacitracin. Growth is inhibited by the antibiotics: nitrofurantoin, KOH Test: positive amipicillin, nalidixic acid, Sulpha methoxazole, Aminopeptidase Test: negative trimethoprim, penicillin G, novobiocin, Streptomycin, Oxidase Reaction: negative 25 tetracycline, polymixin, neomycin, and kanamycin. Catalase Reaction: positive Chemoorganotroph. Grows on complex Substrates Such as Hydrolysis of Gelatin: positive yeast extract, peptones and casamino acids. Growth is Hydrolysis of Starch: positive Stimulated by a variety of Simple Sugars, amino acids and Growth is not inhibited by the antibiotics: streptomycin, organic acids. tetracycline, polymixin, neomycin and kanamycin. Growth The membrane lipids are based on fatty acid esters is inhibited by the antibiotics: gentamicin, amplicillin, peni indicating the eubacterial nature of Strain 103Nt.4. cillin G, erythromycin, novobiocin, oleandomycin, Strain 31 M.4 rifampicin, Vancomycin and bacitracin. An aerobic, Gram-positive, rod-shaped bacterium. No Spores observed. Chemoorganotroph. Grows on complex Substrates Such as 35 yeast extracts, peptones and casamino acids. Obligate haloalkaliphile, grows at pH 10 on a medium Strain 95LM.4 containing 20% NaCl. Grows in the presence of 15-30% An aerobic, Gram-negative, rod-shaped bacterium. No NaCl. No growth in 12% NaCl. Spores observed. On alkaline, Saline, nutrient-agar (Medium A) forms cream colored, circular colonies, about 1 mm in diameter, Haloalkaliphile. Grows at pH 10 on a medium containing 40 20% NaCl. Grows in the presence of 8–30% NaCl. which have a conveX elevation and entire margin. On alkaline, Saline, nutrient-agar (Medium A) forms Temperature: grows optimally at 30-40 C. Grows at 20 opaque, yellow colored, circular colonies, about 2 mm in C. and 45 C., but not at 50° C. diameter, which have a conveX elevation and entire margin. KOH test: negative Temperature: grows optimally at 30-40 C. Growth at 20 45 Aminopeptidase test: negative C. but not at 45° C. Oxidase reaction: negative KOH Test: positive Catalase reaction: negative Aminopeptidase Test: negative Hydrolysis of Starch: positive Oxidase Reaction: negative Grows is not inhibited by the antibiotics: 50 Sulphamethoxazole, trimethoprim, polymixin, rifampicin, Catalase Reaction: positive and bacitracin. Growth is inhibited by the antibiotics: Hydrolysis of Gelatin: positive gentamicin, nitrofurantoin, amplicillin, nalidixic acid, peni Hydrolysis of Starch: negative cillin G, chloramphenicol, erythromycin, fusidic acid, Growth is not inhibited by the antibiotics: gentamicin, methicillin, novobiocin, Streptomycin, tetracycline, and ole Streptomycin, tetracycline, polymixin, neomycin and kana 55 andomycin. mycin. Growth is inhibited by the antibiotics: ampicillin, Chemoorganotroph. Grows on complex Substrates Such as penicillin G, chloramphenicol, erythromycin, novobiocin, yeast extract, peptones and casamino acids. oleandomycin, rifampicin, Vancomycin and bacitracin. The membrane lipids contain glycerol diether moieties Chemoorganotroph. Grows on complex Substrates Such as indicating the archaeobacterial nature of Strain 31 M.4. yeast extracts, peptones and casamino acids. 60 Strain 931 LM.4 An aerobic, Gram-variable, coccoid bacterium. No spores Non-clustering Strains observed. The strains which do not fall into the four clusters defined Haloalkaliphile, grows at pH 10 on a medium containing here are also novel bacteria not previously known or 20% NaCl. Grows in presence of 0–30% NaCl. described. These strains, coded 103Nt.4, 31 M.4 and 65 On alkaline, Saline, nutrient-agar (Medium A) forms pink 931LM.4 may represent rarer varieties of haloalkaliphilic colored, irregular colonies, 1-2 mm in diameter, which have bacteria. A description of these “non-clustering Strains has a raised elevation and entire margin. US 6,420,147 B1 13 14 KOH test: positive Oxidase reaction: negative TABLE 6-continued Hydrolysis of Starch: positive Core Lipids of Haloalkaliphilic Bacteria Growth is not inhibited by the antibiotics: nitrofurantoin and trimethoprim. CLUSTER STRAIN CORE LIPID Growth is inhibited by the antibiotics: gentamicin, 105Nt.4 FAME ampicillin, nalidixic acid, penicillin G. chloramphenicol, 101Nt.4 FAME erythromycin, fusidic acid, methicillin, novobiocin, 103Nt.4 FAME Streptomycin, tetracycline. 31M4 GDEM Chemoorganotroph. Grows on complex Substrates Such as 93ILM.4 GDEM yeast extract, peptones and casamino acids. GDEM = glycerol diether moieties The membrane lipids contain glycerol diether moieties FAME = fatty acid methyl esters indicating the archaeobacterial nature of Strain 931LM.4. Production and Application of Alkali- and Salt Chemotaxonomic Definition of the Clusters 15 tolerant Enzymes Chemotaxonomy is the Study of the chemical variations of The haloalkaliphilic microorganisms of the present inven cells in relation to Systematics. The analysis of chromosomal tion produce a variety of enzymes (c.f. Appendices D and E). DNA, ribosomal RNA, proteins, cell walls and membranes, These enzymes are capable of performing their functions at for example, can give valuable insights into taxonomic an extremely high pH and high Salt concentrations, making relationships and may be used as a further tool to classify or them uniquely Suited for their application in a variety of to verify the taxonomy of microorganisms (Goodfellow, M. processes requiring Such enzymatic activity in Such envi and Minnikin, D. E. in Chemical Methods in Bacterial ronments or reaction conditions. Systematics, (eds. Goodfellow, M. and Minnikin, D. E.), Examples of the various applications for enzymes having Academic Press, London and Orlando, Fla., (1985), pp. 25 alkali- and Salt-tolerance are in detergent compositions, 1-15). leather tanning, food treatment, waste treatment and in the Analysis of Core Lipids textile industry. These enzymes may also be used for All membrane lipids of the archaeobacteria identified to biotransformations, especially in the preparation of pure date are characterized by unusual Structural features, which enantiomers. can be considered to be specific taxonomic markers for this The haloalkaliphiles may easily be Screened for the pro group of microorganisms. While all living organisms So far duction of alkali- and Salt-tolerant enzymes having, for known have membrane lipids based on ester linkages, the example, lipolytic, proteolytic, Starch-degrading or other archaeobacteria have lipids based on ether linkages. (De activities using the methods described in Appendix B. Rosa, M. et al., (1986), Microbiological Reviews, 50, The broth in which haloalkaliphilic bacteria are cultured 70–80). 35 typically contains one or more types of enzymatic activity. The membrane lipids were extracted from bacteria and The broth containing the enzyme or enzymes may be used analyzed by this layer chromatography according to the directly in the desired process after the removal of the methods described by Ross, H. N. M. et al., (1981), Journal bacteria therefrom by means of centrifugation or filtration, of General Microbiology, 123, 75-80). 40 for example. The results of this analysis for representative strains of the If desired, the culture filtrate may be concentrated by haloalkaliphilic bacteria of the present invention are set out freeze drying, before or after dialysis, or by ultrafiltration. in Table 6. These show clearly that the strains of Cluster 1 The enzymes may also be recovered by precipitation and and Cluster 2 lack fatty acid methyl esters, but contain filtration. Alternatively, the enzyme or enzymes contained in glycerol diethyl moieties characteristic of the archaeobac the broth may be isolated and purified by chromatographic teria. The strains of Cluster 3 and Cluster 4 contain fatty acid 45 means or by gel electrophoresis, for example, before being methyl esters but no glycerol diethyl moieties, thus confirm applied to the desired process. ing their identity as eubacteria. These results further under The genes encoding these alkali- and Salt-tolerant line a fundamental difference between the bacteria of Clus enzymes may be isolated, cloned and brought to expression ters 1 and 2 and Clusters 3 and 4. 50 in compatible expression hosts to provide a Source of larger Volumes of enzyme products which may be, if desired, more TABLE 6 easily purified and used in a desired industrial application, should the wild-type Strain fail to produce Sufficient amounts Core Lipids of Haloalkaliphilic Bacteria of the desired enzyme, or does not ferment well. CLUSTER STRAIN CORE LIPID 55 In one embodiment, the enzymatic preparation may be 1. 3OM.4 GDEM used in wash tests to determine the efficacy of the enzymatic 87M.4 GDEM activity. 89M.4 GDEM Enzyme preparations from the haloalkaliphilic bacteria 86M.4T GDEM may be tested in a specially developed mini-wash test using 88M.4 GDEM 2 82M.4 GDEM 60 cotton Swatches Soiled, for example, with protein-, lipid 1OONt.4 GDEM and/or Starch-containing components. Prior to the wash test, 99Nt.4 GDEM the Swatches can be pre-treated with a Solution containing an 96LM.4 GDEM 102INt.4 GDEM anionic Surfactant, Sodium perborate and a bleach activator 104Nt.4 GDEM (TAED). After this treatment, the test Swatches are rinsed in 3 84M.4 FAME 65 running demineralized water and air-dried. This treatment 4 9OM.4 FAME results in the fixation of the Soil, making its removal more difficult. US 6,420,147 B1 15 16 The Washing tests may be performed using a defined detergent composition plus a specific amount of enzymatic APPENDIX A-continued activity in the presence of the test Swatches. After washing, the Swatches are rinsed in running demineralized water and Media Used in the Present Invention air-dried. The reflectance of the test Swatches is measured 5 KHPO, 1.O gl' MgSO4.7HO O.2 gl' with a photometer. NaCl 40.O gl' Na2CO 1.O.O gl' APPENDIX A Casein 2O.O gl' Agar 2O.O gl' Media Used in the Present Invention *(when required for a solid medium) MEDIUMA Yeast Extract (Difco: Detroit, MI, USA) 1O.O gl' Appendix B Casamino Acids (Difco) 7.5 gl' Trisodium citrate 3.0 gl' 15 Methods for Unit Tests KCI 2.O gl' 1. Character Numbers 1 to 12 MgSO4.7H2O 1.O gl' MnCl4HO O.OOO36 gl' Colony color. Size, form, elevation. margin FeSO.7HO O.05 gl' A Suspension of bacteria was spread over an alkaline, NaCl 2OO.O gl' Na2CO 18.5 gl' saline, nutrient agar (Medium A) and cultivated at 37 Agar 2O.O gl' C. Colonies were examined after 7 to 14 days. MEDIUMB 2. Character Numbers 13 to 15 Cell morphology. Gram’s Stain reaction Yeast Extract 1O.O gl' Casamino Acids 7.5 gl' Bacterial cells were cultivated in alkaline, Saline, nutrient Trisodium citrate 3.0 gl' broth (Medium A without agar) until adequate growth KCI 2.O gl' 25 was obtained. The cells were spun down in a centrifuge MgSO4.7H2O 1.O gl' MnCl4HO O.OOO36 gl' and resuspended in a Small amount of fresh medium. A FeSO.7HO O.05 gl' drop of the bacterial Suspension was allowed to air-dry NaCl 2OO.O gl' on a microScope slide. The Gram’s Staining test was Na2CO 18.5 gl' performed using the Dussault modification (Journal of Casein 2O.O gl' Agar 2O.O gl' Bacteriology, 70, 484-485, 1955) with safrainin as MEDIUM C counterstain. 3. Character Number 16 Yeast Extract 1O.O gl' KOH test Casamino Acids 7.5 gl' The test was performed using 3% KOH in 20% NaCl--1% Trisodium citrate 3.0 gl' 35 KCI 2.O gl' NaCO on 7 to 14 days old bacterial cultures grown on MgSO4.7H2O 1.O gl' alkaline, Saline, nutrient agar (Medium A) as described MnCl2.4H2O O.OOO36 gl' by Halebian et al., in Journal of Clinical Micro-biology, FeSO.7HO O.05 gl' NaCl 2OO.O gl' 13, 444-448, 1981 and compared with the reaction in Na2CO 18.5 gl' solution containing only 20% NaCl--1% NaCO. Lactalbumin 1O.O gl' 40 4. Character Number 17 Agar 2O.O gl' Aminoreptidase reaction MEDIUMD The test was performed using the diagnostic test Strips Yeast Extract O.2 gl' Bactident(R) Aminopeptidase (E. Merck, Darmstadt, Casamino Acids O.15 gl' Trisodium citrate 1.5 gl' 45 Germany). A yellow color within 30 minutes was KCI 2.O gl' recorded as a positive reaction. MgSO4.7H2O 1.O gl' 5. Character Number 18 MnCl2.4H2O O.OOO36 gl' Oxidase reaction FeSO.7HO O.05 gl' NaCl 1SO.O gl' Filter paper moistened with a 1% acqueous Solution of Na2CO 1SO.O gl' 50 N,N,N',N'-tetramethyl-p-phenylenediamine or, oxi Casein 2O.O gl' dase identification discs (bioMérieux: Charbonieres Agar 2O.O gl' les-Bains, France) were Smeared with a young bacterial MEDIUME culture from alkaline, Saline, nutrient agar. A purple Yeast Extract O.2 gl' color within 1 minute was recorded as a positive Casamino Acids O.15 gl' 55 reaction. E. coli was used as a negative control. Trisodium citrate 1.5 gl' 6. Character Number 19 KCI 2.O gl' Catalase reaction MgSO.7HO 1.O gl' MnCl2.4H2O O.OOO36 gl' Abacterial colony from alkaline, Saline, nutrient agar was FeSO.7HO O.05 gl' Suspended in a drop of 3% hydrogen peroxide Solution, NaCl 1SO.O gl' Na,CO. 1SO.O gl' 60 or “1D color catalase” reagent (bioMérieux). Bubbles Lactalbumin 1O.O gl' of oxygen released was recorded as a positive reaction. Agar 2O.O gl' 7. Character Number 20 MEDIUMF Gelatin hydrolysis Glucose O.2 gl' Charcoal-gelatin discs (bioMérieux) or “chargels” Peptone (Difco) O1 gl' 65 (Oxoid) were incubated at 37° C. in an alkaline, saline, Yeast Extract O1 gl' nutrient broth (Medium A) together with bacteria. A black Sediment indicated a positive reaction. US 6,420,147 B1 17 18 8. Character Number 21 and 39 15. Character Numbers 92–123 Skim milk and starch hydrolysis test Growth on carbon Substrates Bacteria were inoculated on to alkaline, Saline, nutrient agar (Medium A) Supplemented with 5.0 g/l skim Use was made of the commercially available test Strip powder or 2.0 g/l starch, and incubated at 37 C. Areas ATB 32 GN (API-bioMérieux: La Balme les Grottes, of clearing around bacterial colonies in an otherwise France). The Strips were used according to the manu opaque agar were recorded as a positive reaction. Zones facturer's instructions but with the addition of 1.0 ml of of Starch hydrolysis were confirmed by Staining with a solution containing 20% NaCl and 1% NaCO to the iodine Solution (lugol). vials of basal medium provided. The strips were incu bated at 37° C. for 48 hours. 9. Character Numbers 22–29 1O NaCl tolerance Two methods were applied. APPENDIX C (a) Bacterial strains were cultivated at 37° C. on an alkaline, nutrient agar (Medium A) containing 0%, Unit Tests for Analysis by Numerical Taxonomy 4%, 8%, 12%, 15%, 20%, 25% or 30% (w/v) NaCl. 15 CHAR- POSI The agar plates were examined for bacterial growth ACTER TIVE NEGA after 7-14 days. NUM- CHARACTER (pre- TIVE (b) Bacterial strains were cultivated at 37° C. in an TEST BER STATE sent) (absent) alkaline nutrient broth (Medium A) containing 0%, Colony 1 yellow O 4%, 8%, 12%, 15%, 20%, 25% or 30% (w/v) NaCl. color 2 cream/beige O Bacterial growth was monitored regularly up to 14 days 3 orange O by optical density measurements using a Klett meter (green 4 pink/red O filter). Colony 5 s1 mm. O 10. Bacterial Numbers 30 and 31 size 6 >1 mm O in (dia Growth temperature lete Bacterial Strains were inoculate in alkaline, Saline, nutri 25 mm) ent broth (Medium A) and incubated at 10° C., 15 C., Colony 7 circular O form 8 punctiform O 20° C., 45° C. or 50° C. Bacterial growth was moni 9 irregular O tored regularly up to 14 days by optical density mea Colony 10 convex O Surements using a Klett meter (green filter). elevation 11 raised O 11. Character Numbers 32–38 Colony 12 entire O Carbohydrate utilisation margin Cell 3 OC = COC A minimal medium composed (g/l distilled water) of yeast morph- cus = 0 extract, 1.0; KNO, 1.0; KC1, 2.0; MgSO.7H2O, 1.0; ology MnC14HO, 0.00036; FeSO.7HO, 0.05; NaCl, Grams 14 Gram positive O 200.0; NaCO, 18.5; agar, 20.0 was supplemented 35 stain 15 Gram negative O with 2.0 g/l of the carbohydrate under test and poured KOH test 16 O Amino- 17 O into Square Petri dishes. peptidase Bacteria were inoculated, using a 25 point multi-point reaction inoculator, from 1.0 ml of a bacterial Suspension in an Oxidase 18 O reaction alkaline, Saline, nutrient broth (Medium A). The agar 40 Catalase 19 O plates were incubated at 37 C. for up to 14 days. The reaction results were recorded by comparing bacterial growth on Gelatin 2O O minimal nutrient medium containing a carbohydrate hydrolysis Supplement with growth on a minimal medium without Skim milk 21 O test the carbohydrate under test. 45 NaCl 22 growth at 0% O 12. Character Numbers 40–51 tolerance 23 growth at 4% O Amino acids as carbon and nitrogen Source 24 growth at 8% O The same technique was employed as for tests 32–38. 25 growth at 12% O 13. Character Numbers 52–70 26 growth at 15% O 27 growth at 20% O Enzymatic activities 50 28 growth at 25% O Use was made of the commercially available test Strip 29 growth at 30% O Growth 30 growth at s2O C. O APIZYM (API-bioMérieux) which was used according temper- 31 growth at 245 C. O to the manufacturer's instructions, except that the ature haloalkaliphilic bacterial cells were Suspended in Carbo- 32 Fumerate O alkaline, saline, nutrient broth (Medium A). The strips 55 hydrate 33 Fructose O were incubated at 37 C. for 4 hours. utili- 34. Succinate O zation 35 Formate O 14. Character Numbers 71-91 36 Lactose O Antibiotic sensitivity 37 Galactose O 38 Xylose O A light Suspension of bacteria in alkaline, Saline, nutrient 39 Starch O broth was spread on the Surface of alkaline nutrient, 60 Amino 40 Serine O Saline agar (Medium A) and allowed to dry. Commer acids as 41 Proline O cially available antibiotic Susceptibility test disks carbon 42 Asparagine O and 43 Arginine O (Oxoid or Mast Laboratories: Merseyside, U.K.) were nitrogen 44 Alanine O applied to the agar Surface. The bacteria were cultivated SOCCS 45 Lysine O at 37 C. for up to 14 days. Clear Zones around the 65 46 Methionine O antibiotic disks indicated Sensitivity and were recorded 47 Phenylalanine O as positive. US 6,420,147 B1 19 20

APPENDIX C-continued APPENDIX C-continued Unit Tests for Analysis by Numerical Taxonomy Unit Tests for Analysis by Numerical Taxonomy

CHAR POSI CHAR POSI ACTER TIVE NEGA ACTER TIVE NEGA NUM- CHARACTER (pre TIVE NUM- CHARACTER (pre- TIVE TEST BER STATE sent) (absent) TEST BER STATE sent) (absent) 48 Glycine 118 3-hydroxybenzoate 1. O 49 Valine 119 Serine 1. O 50 Glutamate 51 Leucine 120 2-ketogluconate 1. O Enzymatic 52 Alkaline phosphatase 121 3-hydroxybenzoate 1. O activity 53 Esterase (C4) 122 4-hydroxybutyrate 1. O 54 Esterase lipase (C8) 123 Proline 1. O 55 Lipase (C14) 15 56 Leucine arylamidase 57 Valine arylamidase 58 Cystine arylamidase APPENDIX ID 59 Trypsin 60 Chymotrypsin Screening for proteolytic, Amylolytic and Lipolytic Activity 61 Acid phosphatase 62 Naphthol-AS-BI Proteolytic Activity phosphohydrolase 63 C-galactosidase STRAIN LACTALBUMIN CASEIN GELATIN 64 f-galactosidase 65 f-glucuronidase Cluster 1 66 C-glucosidase 67 f-glucosidase 25 3OM.4 .. n.t. 68 N-acetyl-f- 87M.4 .. -- glucosaminidase 85M.4 .. -- 69 C-mannosidase 89M.4 .. -- 70 C-fucosidase 86M.4T .. -- Antibiotic 71 Gentamicin 10 ug 88M.4 .. -- sensitivity 72 Nitrofurantoin 50 tug RS4 .. n.t. : (inhibition 73 Ampicillin 25 ug Cluster 2 of 74 Nalidixic Acid 30 tug growth = 75 Sulphmethoxazole 50 tug 82M.4 .. -- positive) 76 Trimethoprim 25 lug 1OONt.4 1.C. -- 77 Penicillin G 10 IU 98Nt4T -- n.t. 78 Chloramphenicol 25 ug 35 99Nt.4 -- n.t. 79 Erythromycin 5 lig RSS .. n.t. 8O Fusidic Acid 10 lig RS6 .. n.t. 81 Methicillin 10 lig RS2 .. n.t. 82 Novobiocin 5 lig RS3 .. n.t. 83 Streptomycin 10 lig 96LM.4 .. -- 84 102INt.4 .. -- Tetracycline 25 ug 40 85 Oleandomycin 5 lig 104Nt.4 .. -- 86 Polymixin 3OO IU Cluster 3 87 Rifampicin 2 lig 88 Neomycin 30 tug 83M.4 -- .. 89 Vancomycin 30 tug 93dLM.4T -- .. : 90 Kanamycin 30 tug 84M.4 n.t. -- 91 Bacitracin 10 IU 45 Cluster 4 Growth on 92 Rhamnose Carbon 93 N-acetylglucosamine 90M.4 -- .. Substrates 94 Ribose 95LM.4CT n.t. -- (ATB) 95 Inositol 97Nt.4 -- .. 96 Saccharose 1OSNt.4 n.t. -- 97 Maltose 50 102dNt.4 n.t. -- 98 Itaconate 101Nt.4 -- .. 99 Suberate Non-Clustering Strains OO Malonate O1 Acetate 103Nt.4 n.t. -- O2 Lactate 31M4 n.t. .. O3 Alanine 55 931L.M.4 -- .. O4 Mannitol 05 Glucose Amylolytic and Lipolytic Activity O6 Salicin O7 Melibiose STARCH ESTERASE O8 Fucose STRAIN HYDROLYSIS LPASE LPASE O9 Sorbitol 1O Arabinose 60 Cluster 1 11 Propionate 12 Caprate 3OM. 4 -- 13 Valerate 87M.4 -- -- 14 Citrate 85M.4 -- 15 Histidine 89M.4 -- -- 16 5-ketogluconate 65 86M.4T -- 17 Glycogen 88M.4 -- US 6,420,147 B1 21 22

APPENDIX D-continued APPENDIX E-continued Screening for proteolytic, Amylolytic and Lipolytic Activity Distribution of Positive Characters to Clusters of Haloalkaliphilic Bacteria Defined at the 78.5% Similarity Level (SSM RS4 -- 5 Cluster 2 CHARACTER 1. 2 3 4

82M.4 -- Growth at 30% NaCl 71 1OO 1OO 1OO 1OONt.4 -- Growth at s2O. C. 57 64 1OO 33 98Nt4T -- -- Growth at 2.45 C. 14 27 33 33 99Nt.4 -- -- 1O Fumarate 57 82 67 33 R55 -- Fructose 43 91 33 33 RS6 -- -- Succinate 57 82 33 33 RS2 -- Formate O 55 O O RS3 -- Lactose O O O O 96LM.4 -- -- Galactose 57 91 67 33 102INt.4 -- -- 15 Xylose 29 O O 17 104Nt.4 -- Starch 29 27 1OO O Cluster 3 Serine 14 9 33 O Proline 57 91 67 33 83M.4 -- -- Asparagine O 9 O O 93dIM.4T -- -- Arginine 57 91 67 33 84M.4 -- -- Alanine 57 91 67 33 Cluster 4 2O Lysine 43 82 67 O Methionine 29 18 33 O 9OM.4 -- Phenylalanine O O O 17 95LM.4CT -- Glycine O O O O 97Nt.4 -- Valine 43 91 33 O 105Nt.4 -- Glutamate 14 O 33 O 102dNt.4 -- 25 Leucine O 9 O O 101Nt.4 -- Alkaline phosphatase O 27 O O Non-clustering Strains Esterase (C4) 1OO 1OO 1OO 1OO Esterase Lipase (C8) 1OO 1OO 1OO 1OO 103Nt.4 -- -- Lipase (C14) O 18 O O 31M4 -- -- Leucine arylamidase 1OO 1OO 1OO 83 93ILM.4 ------3O Valine arylamidase 14 73 O O Cystine arylamidase 14 18 O O n.t. - not tested Trypsin 14 9 O O Proteolytic Activity determined on media B-F (Appendix A) and accord- Chymotrypsin 29 36 O 33 ing to character 20 (Appendix B). Acid phosphatase O 18 O 17 Starch Hydrolysis determined according to character 39 (Appendix B) Naphthol-AS-BI- O 27 O O Esterase Lipase Activity determined according to character 54 (Appendix 35 phosphohydrolase B) C-galactosidase O O O O Lipase Activity determined according to character 55 (Appendix B). f-galactosidase O O O O f-glucuronidase O O O O C-glucosidase 57 55 33 33 APPENDIX E f-glucosidase 29 O O O 40 N-acetyl-f-glucosaminidase O 9 O O Distribution of Positive Characters to Clusters of Haloalkaliphilic C-mannosidase O O O O Bacteria Defined at the 78.5% Similarity Level (SSM C-fucosidase O O O O Gentamycin O O 67 50 CHARACTER 1. 2 3 4 Nitrofurantoin 43 82 O O Ampicillin O O 1OO 67 Colony color yellow O O O 67 Nalidixic Acid O 18 O O Colony color cream/beige 14 9 33 17 45 Sulphmethoxazole 43 82 O 17 Colony color orange O O O 17 Trimethoprim 57 91 O 17 Colony color pink/red 86 91 67 O Penicillin B O O 1OO 1OO Colony size s 1 mm 57 91 1OO O Chloramphenicol O O 1OO 1OO Colony size 2.1 mm 43 9 O 1OO Erythromycin 86 73 1OO 1OO Colony circular 71 82 1OO 1OO Fusidic Acid 14 9 O 17 Colony punctiform 29 18 O O 50 Methicillin O O O O Colony irregular O O O O Novobiocin 1OO 82 33 1OO Colony elevation convex 1OO 1OO 1OO 1OO Streptomycin O 18 67 O Colony elevation raised O O O O Tetracycline 14 9 33 17 Colony margin entire 1OO 1OO 1OO 1OO Oleandomycin O O 1OO 1OO Cells rod-shaped 29 36 67 83 Polymixin O O O O Gram positive 29 27 33 17 55 Rifampicin 14 64 67 83 Gram negative 43 82 67 67 Neomycin O O O O KOH test 43 1OO 1OO 33 Vancomycin O O 1OO 83 Aminopeptidase reaction O O O O Kanamycin O O O 17 Oxidase reaction O 9 O 17 Bacitracin 1OO 82 1OO 1OO Catalase reaction 1OO 91 1OO 1OO Rhamnose 57 O 33 O Gelatin hydrolysis 71 82 67 1OO 60 N-acetylglucosamine 71 O 33 O Skim milk test O O O 17 Ribose O O 33 O Growth at 0% NaCl O O O 17 Inositol 71 9 67 O Growth at 4% NaCl O 9 33 33 Saccharose 71 9 33 33 Growth at 8% NaCl 29 18 33 83 Maltose 71 9 67 17 Growth at 12% NaCl 86 1OO 67 1OO Itaconate O O O O Growth at 15% NaCl 1OO 1OO 1OO 1OO Suberate 14 O 33 O Growth at 20% NaCl 1OO 1OO 1OO 1OO 65 Malonate 71 O 67 O Growth at 25% NaCl 1OO 1OO 1OO 1OO Acetate 71 O 67 O US 6,420,147 B1 23 24 4) esterase APPENDIX E-continued 5) esterase lipase, b) a negative response to the following tests: Distribution of Positive Characters to Clusters of Haloalkaliphilic Bacteria Defined at the 78.5% Similarity Level (SSM 1) colonies colored red or pink 5 2) starch hydrolysis CHARACTER 1. 2 3 4 3) alkaline phosphatase; Lactate 71 O 67 O c) growth is inhibited by the antibiotics penicillin G, Alanine 71 9 33 O chloramphenicol, novobiocin, oleandomycin, and baci Mannitol 57 O 33 O tracin. Glucose 71 O 33 17 1O Salicin 29 O O O 3. A pure bacterial culture useful for the production of Melibiose 57 O 67 O enzymes, wherein the bacteria consist of aerobic, non Fucose 43 O O O phototrophic, Gram-negative, rod Shaped, haloalkaliphilic Sorbitol 43 O 33 O bacteria having the following characteristics: Arabitol 43 O 33 O Propionate 57 O 67 O 15 a) on akaline, Saline, nutrient agar, forms opaque, orange Caprate 14 27 O 17 colored circular colonies, 2 to 3 millimeters in Valerate 57 O 67 O Citrate 71 O 67 17 diameter, which have a conveX elevation and entire Histidine 57 O 67 17 margin; 5-ketogluconate 14 O 33 O b) grows at 20° C.; Glycogen 71 9 67 O 3-hydroxybenzoate O O O O c) no growth at 45 C.; Serine 57 73 67 O d) KOH test is positive; 2-ketogluconate 43 73 67 33 3-hydroxybutyrate 71 O 67 17 e) aminopeptidase test is negative; 4-hydroxybenzoate 71 18 67 O f) oxidase test is negative; Proline 71 55 67 O 25 g) catalase test is positive; h) grows in presence of 0% to 30% NaCl; What is claimed is: i) hydrolysis of gelatin is positive; 1. A pure bacterial culture useful for the production of enzymes, wherein the bacteria consist of aerobic, non j) hydrolysis of Starch is positive; phototrophic, haloalkaliphilic bacteria which grow as colo k) growth is not inhibited by the antibiotics gentamicin, nies which are circular, convex, and entire, which bacteria chloramphenicol, fusidic acid, erythromycin, give: methicillin, oleandomycin, rifampicin, Vancomycin, or a) a positive response in the following tests: bacitracin; 1) KOH test 1) growth is inhibited by the antibiotics nitrofurantoin, 2) catalase 35 ampic illin, nalidixic acid, Sulfame thizole, 3) growth in 15% to 30% NaCl trimethoprim, penicillin G, novobiocin, Streptomycin, 4) growth at 20° C. or less tetracycline, polymyxin, neomycin, or kanamycin; 5) starch hydrolysis m) grows on simple Sugars; 6) esterase n) grows on amino acids; 7) esterase lipase 40 8) leucine arylamidase; O) grows on organic acids; b) a negative response to the following tests: p) grows on yeast extract and peptones, and 1) oxidase q) contains membrane lipids based on fatty acid esters. 2) growth in 0% NaCl 4. A method for the preparation of enzymes comprising: 3) alkaline phosphatase 45 a) culturing the bacteria of claim 3 in a culture medium: 4) acid phosphatase; b) separating the bacteria from the culture medium; and c) growth is inhibited by the antibiotics amplicillin, peni c) recovering enzyme activity from the culture medium. cillin G, chloramphenicol, oleandomycin, Vancomycin, 5. A method for the preparation of enzymes comprising: and bacitracin. a) culturing the bacteria of claim 4 in a culture medium: 2. A pure bacterial culture useful for the production of 50 enzymes, wherein the bacteria consist of aerobic, non b) separating the bacteria from the culture medium; and phototrophic, haloalkaliphilic bacteria which grow as colo c) recovering enzyme activity from the culture medium. nies which are circular, convex, and entire, which bacteria 6. A method for the preparation of enzymes comprising: give: a) culturing the bacteria of claim 5 in a culture medium: a) a positive response in the following tests: 55 b) separating the bacteria from the culture medium; and 1) catalase c) recovering enzyme activity from the culture medium. 2) gelatin hydrolysis 3) grows in 12% to 30% NaCl k k k k k